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Fragile X chromosome associated with familial sex-linked mental retardation : expression in fibroblast… Jacky, Peter Bruce 1980

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FRAGILE X CHROMOSOME ASSOCIATED WITH FAMILIAL SEX-LINKED MENTAL RETARDATION EXPRESSION IN FIBROBLAST CULTURE by PETER BRUCE JACKY B . S c , P o r t l a n d S t a t e U n i v e r s i t y , 1974 M.Sc, P o r t l a n d S t a t e U n i v e r s i t y , 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Me d i c a l G e n e t i c s We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1980 (c) Peter Bruce Jacky, 1980 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Medical Genetics Department o f , The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 July 19th, 1980 Date ABSTRACT A form of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n has been a s s o c i a t e d w i t h the e x p r e s s i o n of a f r a g i l e s i t e near the t e r m i n a l end of the long arm of the X chromosome. Pre-v i o u s r e p o r t s on the f r a g i l e X chromosome showed e x p r e s s i o n of the f r a g i l e s i t e to be l i m i t e d t o chromosome p r e p a r a t i o n s from p e r i p h e r a l blood lymphocytes of m e n t a l l y r e t a r d e d males and t h e i r female r e l a t i v e s i n f a m i l i e s i n which the d i s o r d e r was s e g r e g a t i n g . F r a g i l e s i t e e x p r e s s i o n has a l s o been shown to be a f u n c t i o n of the medium employed i n c e l l c u l t u r e . The f r a g i l e X chromosome c o u l d ; o n l y be demonstrated i n lymphocytes c u l t u r e d i n medium 199 or media d e p r i v e d of f o l i c a c i d . T h i s study was undertaken t o develop a method f o r demonstrating the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s . . F i b r o b l a s t c e l l l i n e s from f i v e p a t i e n t s (two me n t a l l y r e t a r d e d males, two o b l i g a t e c a r r i e r females, and a p o t e n t i a l c a r r i e r female) from a f a m i l y i n which f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n was known to be se g r e g a t i n g were e s t a b l i s h e d and r o u t i n e l y maintained i n a complete c u l t u r e medium. F o r t y - t h r e e hours p r i o r to chromosome har v e s t , c e l l s from each p a t i e n t were t r a n s f e r r e d t o media d e f i c i e n t i n f o l i c a c i d . Under c o n d i t i o n s of f o l i c a c i d d e p r i v a t i o n , i t was p o s s i b l e to e l i c i t e x p r e s s i o n of the f r a g i l e X chromosome i n s k i n f i b r o b l a s t s from a l l f i v e p a t i e n t s s t u d i e d . No f r a -g i l e X chromosomes were d e t e c t e d i n f i b r o b l a s t s from three normal c o n t r o l s u b j e c t s . In a p r e l i m i n a r y assessment of the r e l i a b i l i t y of the f i b r o b l a s t method, three p a t i e n t s (two m e n t a l l y r e t a r d e d males and a p o t e n t i a l c a r r i e r female) from a second u n r e l a t e d f a m i l y i n which the d i s o r d e r i s known to be s e g r e g a t i n g were s t u d i e d w i t h t h i s method. The f r a g i l e X chromosome c o u l d be demonstra-ted i n f i b r o b l a s t s from both of the r e t a r d e d male p a t i e n t s but cou l d not be. demonstrated i n f i b r o b l a s t chromosome prepara-t i o n s from the p o t e n t i a l . c a r r i e r female. Lymphocytes f o r a l l p a t i e n t s s t u d i e d were grown under s i m i l a r f o l a t e d e p r i v e d c o n d i t i o n s f o r the purpose of com-p a r i n g the e f f e c t i v e n e s s of f i b r o b l a s t c u l t u r e with lympho-cyte c u l t u r e i n demonstrating the e x p r e s s i o n of the f r a g i l e X chromosome. N e i t h e r t i s s u e was shown to c o n s i s t e n t l y p r o v i d e a higher frequency of e x p r e s s i o n of the f r a g i l e X chromosome. In a d d i t i o n to f o l a t e d e p r i v a t i o n , i t was shown t h a t two ot h e r f e a t u r e s of the f i b r o b l a s t method i n f l u e n c e d the frequency of e x p r e s s i o n of the f r a g i l e X chromosome. The f r a g i l e s i t e was expressed at a s i g n i f i c a n t l y h i g h e r f r e -quency i n chromosome p r e p a r a t i o n s i n which the chromosomes were not s e v e r e l y c o n t r a c t e d . The frequency of e x p r e s s i o n i n f i b r o b l a s t s was a l s o shown to be s i g n i f i c a n t l y higher w i t h a hypotonic treatment at chromosome h a r v e s t u s i n g 1% N a C i t r a t e r a t h e r than 0.075M KC1. Because f r a g i l e s i t e e x p r e s s i o n was shown to be a fun c -t i o n of the degree of chromosome condensation, two agents, 5-BrdU and actinomycin-D, were s t u d i e d t o examine t h e i r decondensation e f f e c t s on the frequency of e x p r e s s i o n . N e i t h e r BrdU nor actinomycin D proved e f f e c t i v e i n accentu-a t i n g the frequency of e x p r e s s i o n . Since f i b r o b l a s t s behave much l i k e amniocytes i n terms of c e l l c u l t u r e and chromosome h a r v e s t , the development of a method f o r demonstrating the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s i s a step toward the p r o s p e c t of r e l i a b l e a n t e n a t a l d i a g n o s i s of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n a s s o c i a t e d w i t h a f r a g i l e X chromosome. V TABLE OF CONTENTS ABSTRACT . . LIST OF TABLES. LIST OF FIGURES ABBREVIATIONS ACKNOWLEDGEMENTS CHAPTER 1: INTRODUCTION 1.1 F a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n 1.11 Prevalence. 1.12 A s s o c i a t i o n w i t h a f r a g i l e X chromosome. 1.13 C l i n i c a l , m a n i f e s t a t i o n s of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n a s s o c i a -t e d w i t h a f r a g i l e X chromosome. 1.14 F r a g i l e X e x p r e s s i o n i n m e n t a l l y r e -tarded males and t h e i r female r e l a t i v e s . 1.2 H e r i t a b l e f r a g i l e s i t e s 1.21 D i s t r i b u t i o n . 1.22 C y t o g e n e t i c s . 1.23 T i s s u e o r i g i n and c u l t u r e c o n d i t i o n s . 1.3 R a t i o n a l e f o r the present study CHAPTER 2: MATERIALS AND METHODS 2.1 Sources of t i s s u e samples and t i s s u e c u l t u r e media 2.11 S u b j e c t s . 2.12 T i s s u e c u l t u r e media.. 2.2 Demonstration of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s 2.21 R a t i o n a l e f o r the procedure. 2.22 Procedure f o r e s t a b l i s h i n g f i b r o b l a s t c e l l l i n e s . 2.2 3 Procedure f o r chromosome p r e p a r a t i o n from f i b r o b l a s t s . 2.3 Procedure f o r p e r i p h e r a l blood lymphocyte c u l t u r e and chromosome p r e p a r a t i o n 2.4 Examination of supplemental media f a c t o r s i n t i s s u e c u l t u r e 2.41 R a t i o n a l e f o r the procedure. 2.42 M o d i f i c a t i o n of the f i b r o b l a s t c u l t u r e procedure. Page i i v i i i i x x x i i 1 11 19 22 22 26 29 31 v i 2. 5 2.6 CHAPTER 3 3.1 3.2 3.3 CHAPTER 4 4.1 4.2 4. 3 2.43 M o d i f i c a t i o n of the lymphocyte c u l t u r e procedure. Chromosome s t a i n i n g procedure 3 5 Methods of o b s e r v a t i o n and t a b u l a t i o n of data 36 : RESULTS 38 Demonstration of the f r a g i l e X chromo-some i n c u l t u r e d s k i n f i b r o b l a s t s 3 8 3.11 E x p r e s s i o n i n f i b r o b l a s t s from p a t i e n t s from Family No. 1. 3.12 E x p r e s s i o n i n f i b r o b l a s t s from p a t i e n t s from Family No. 2. 3.13 Comparison of the frequency of ex p r e s s i o n i n f i b r o b l a s t s and lymphocytes. F a c t o r s a f f e c t i n g a r e l i a b l e d e t e r m i n a t i o n of the frequency of e x p r e s s i o n 45 3.21 Condensation c r i t e r i o n . . 3.22 V a r i a b l e appearance of the f r a g i l e X chromosome. 3.23 D e f i n i t i v e a s s o c i a t i o n of the f r a g i l e s i t e w i t h the X chromosome by G-banding. 3.2 4 Autosomal chromosome markers ex-pressed under f o l a t e d e p r i v a t i o n . Factors, t h a t i n f l u e n c e the frequency of expres-s i o n of the f r a g i l e X chromosome 54 3.31 C u l t u r i n g f a c t o r s : MEM-Ad, BrdU, and.actinomycin-D. 3.32 Hypotonic e f f e c t s a t chromosome harvest. DISCUSSION 63 F r a g i l e . X e x p r e s s i o n i n f i b r o b l a s t s from men-t a l l y r e t a r d e d males and t h e i r female r e l a t i v e s 63 E x p r e s s i o n of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s : c u l t u r i n g and chromosome h a r v e s t i n g f a c t o r s 65 4.21 F o l a t e d e p r i v a t i o n i n c e l l . . c u l t u r e . 4.2 2 Hypotonic e f f e c t s a t chromosome har-v e s t . R e l i a b l e d e t e r m i n a t i o n of the frequency of ex p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t s "71 4.31 V a r i a b l e appearance of the f r a g i l e X chromosome. 4.32 Condensation. 4.33 Autosomal t e l o m e r i c markers. v i i 4.4 Co n c l u s i o n s and f u t u r e p r o s p e c t s 7 9 REFERENCES... ... 82 APPENDICES 8 9 A Consent form and c e r t i f i c a t e of appr o v a l f o r c l i n i c a l r e s e a r c h and other s t u d i e s i n v o l v i n g human s u b j e c t s 89 B Formulations of t i s s u e c u l t u r e media 91 C F o l a t e d e t e r m i n a t i o n on v a r i o u s l a b o r a t o r y l o t s o f f e t a l serum and Medium 199 w i t h and without a serum supplement 93 V 1 X 1 LIST OF TABLES Table Page I E x p r e s s i o n of the. f r a g i l e X chromosome 7 i n members of f a m i l i e s r e p o r t e d i n the l i t e r a t u r e . I I P a t i e n t s s t u d i e d from Family No. 1 and 25 Family No. 2 and matched c o n t r o l s . I I I E x p r e s s i o n of the f r a g i l e . X chromosome i n 40 c u l t u r e d s k i n f i b r o b l a s t s from p a t i e n t s of Family No. 1 and normal c o n t r o l s : compari-son o f v a r i o u s growth media. IV E x p r e s s i o n of the f r a g i l e X chromosome i n 43 c u l t u r e d s k i n f i b r o b l a s t s from p a t i e n t s of Family No. 2 and a normal c o n t r o l . V E x p r e s s i o n of the f r a g i l e X chromosome i n 44 p e r i p h e r a l blood lymphocytes from p a t i e n t s of both f a m i l i e s and normal c o n t r o l s . VI E x p r e s s i o n of the f r a g i l e X chromosome as 47 a f u n c t i o n o f the degree of chromosome con-de n s a t i o n . VII E x p r e s s i o n of autosomal t e l o m e r i c markers 55 i n c u l t u r e d s k i n f i b r o b l a s t s from p a t i e n t s and normal c o n t r o l s . V I I I E f f e c t s of 5-BrdU and actinomycin-D on the 57 frequency o f e x p r e s s i o n of the f r a g i l e X chromosome i n lymphocytes and f i b r o b l a s t s . IX E f f e c t s of d i f f e r e n t hypotonic treatments 60 a t chromosome h a r v e s t on the frequency of e x p r e s s i o n o f the f r a g i l e X chromosome i n f i b r o b l a s t s . LIST OF FIGURES F i g u r e Page F r a g i l e X chromosome of a m e n t a l l y 3 r e t a r d e d male p a t i e n t from a f a m i l y i n w h i c h . f a m i l i a l s e x - l i n k e d mental r e -t a r d a t i o n i s s e g r e g a t i n g . Pedigrees of Family No. 1 ( P a t i e n t s A - E) 23 and Family No. 2 ( P a t i e n t s F. - H) showing s e g r e g a t i o n of f a m i l i a l s e x - l i n k e d mental retardation., e.g. t r a n s m i s s i o n of the d i s -order through normal heterozygous females to a f f e c t e d sons. The pedigree presented f o r Family No. 1 i s reduced i n p a r t from the o r i g i n a l r e p o r t on the f a m i l y (Dunn et a l . , 1963) . V a r i a b l e e x p r e s s i o n of the f r a g i l e X chromo- 4 9 some: a c e t o - o r c e i n s t a i n i n g . Detached s a t e l l i t e - l i k e p i e c e of chromatin .50 u n a s s o c i a t e d w i t h the X chromosome. V a r i a b l e e x p r e s s i o n of the f r a g i l e X chromo- 51 some: t r y p s i n G-banding. Comparison to the standard banding p a t t e r n of the X chromo-some ( P a r i s Conference, 1971). Autosomal t e l o m e r i c markers expressed under 53 f o l a t e d e p r i v a t i o n i n c e l l c u l t u r e . Extended chromosomes and gaps and breaks pro-„.58 duced by actinomycin-D in- combination w i t h f o l a t e d e p r i v a t i o n i n c e l l c u l t u r e . The chromo-some a t the u p p e r l e f t may be the f r a g i l e X. 8 E f f e c t s of d i f f e r e n t hypotonic treatments a t 62 chromosome h a r v e s t on chromosome morphology. ABBREVIATIONS T i s s u e culture- media: MEM -Eagle's Minimal E s s e n t i a l Medium. Complete t i s s u e c u l t u r e medium c o n t a i n i n g 15% f e t a l c a l f serum. M199 -Medium 199. T i s s u e c u l t u r e medium c o n t a i n i n g 5% f e t a l c a l f serum and low i n f o l i c a c i d (0.01 mg/L). MEM-FA -Eagle's Minimal E s s e n t i a l Medium without f o l i c a c i d . T i s s u e c u l t u r e medium c o n t a i n i n g 5% f e t a l c a l f serum and no f o l i c a c i d . MEM-Ad -Eagle's Minimal E s s e n t i a l Medium without adenine. T i s s u e c u l t u r e medium c o n t a i n i n g 5% f e t a l c a l f serum and d e f i c i e n t i n adenine. A d e t a i l e d d e s c r i p t i o n of the v a r i o u s t i s s u e c u l t u r e media i s presented i n s e c t i o n 2.12 and Appendix I I . Chromosome banding methods: AgNOR-banding C-banding G-banding R-banding -Sat-banding or s i l v e r n u c l e o l a r o r g a n i z e r -banding. Ammoniacal-silver s t a i n i n g of the s a t e l l i t e r e g i o n s of the D and G group a c r o -c e n t r i c chromosomes, and g e n e r a l l y thought to be s p e c i f i c f o r the rDNA sequences asso-c i a t e d w i t h n u c l e o l a r o r g a n i z e r a c t i v i t y . - C o n s t i t u t i v e heterochromatin banding. A chromosome banding method employing giemsa s t a i n i n g f o l l o w i n g d e n a t u r a t i o n p r e t r e a t -ments i n a c i d and a l k a l i n e and a p e r i o d of r e n a t u r a t i o n i n 2xSSC. S p e c i f i c a l l y s t a i n s c entromeric heterochromatin and the par a -centromeric heterochromatic r e g i o n s on chromosomes 1, 9, 16, and the long arm of the Y chromosome. -Giemsa banding. Chromosome banding accom-p l i s h e d w i t h a v a r i e t y of methods, bel o n g i n g to two major t e c h n i c a l c a t e g o r i e s . 1. GTG G-banding, giemsa s t a i n i n g a f t e r p r e t r e a t -ment wi t h a p r o t e o l y t i c enzyme (pronase or t r y p s i n ) . 2. ASG G-banding, giemsa s t a i n i n g a f t e r pretreatment with a c i d or a l k a l i n e f o l l o w e d by s a l i n e . -Reverse banding. A chromosome banding me-thod employing giemsa s t a i n i n g f o l l o w i n g d e n a t u r a t i o n pretreatment w i t h heat and weak a c i d , or a c r i d i n e orange s t a i n i n g f o l l o w i n g the i n c o r p o r a t i o n of BrdU d u r i n g c e l l c u l t u r e . Banding p a t t e r n i s e s s e n t i a l -l y the r e v e r s e of the G-banding p a t t e r n . continued. x i Other a b b r e v i a t i o n s : BrdU 5-Bromo-2 1-Deoxyuridine. A base analog of thymidine. EDTA E t h y l e n e - D i a m i n e - T e t r a - A c e t i c a c i d (disodium s a l t ) . A c h e l a t i n g agent of c a l c i u m (Ca +2) and magnesium (Mg+2). EGTA Ethylene G'lycol-bis- (B-amino-ethyl ether) N,N1 - T e t r a -A c e t i c a c i d . A c h e l a t i n g agent u s e f u l i n the d e t e r -mination of c a l c i u m ( C a + 2 ) i n the presense of magne-sium ( M g + 2 ) . FCS F e t a l C a l f Serum. HBSS Hank's Balanced S a l t S o l u t i o n . A standard s a l i n e s o l u t i o n . PHA Phytohemoglutinin (M Form). P l a n t l e c t i n used f o r mitogenic s t i m u l a t i o n of lymphycytes i n c e l l c u l t u r e . ACKNOWLEDGEMENTS I wish to thank the members of my t h e s i s committee f o r t h e i r a d v i c e and encouragement d u r i n g the course of t h i s pro-j e c t , P r o f e s s o r James R. M i l l e r , Chairman, and Dr. F r e d e r i c k J . D i l l , T h e s i s A d v i s o r , (Department o f Me d i c a l G e n e t i c s ) , Dr. Connie J . Eaves (B i o p h y s i c s D i v i s i o n of the B.C. Cancer Agency and Department of Me d i c a l G e n e t i c s ) , Dr. Dagmar K. Kalousek ( P a e d i a t r i c Pathology, Vancouver General H o s p i t a l and Department of Medical G e n e t i c s ) , Dr. David G. Holm (De-partment of Zoology), Dr. C l a y t o n 0. Person (Department of Botany), and Dr. David L. B a i l l i e (Department of B i o l o g i c a l S c i e n c e s , Simon F r a s e r - U n i v e r s i t y and Department of Med i c a l G e n e t i c s ) . I would a l s o l i k e to thank Dr. R. K e i t h Humphries f o r h i s h e l p i n o b t a i n i n g blood and s k i n specimens f o r t h i s study, Dr. Roque Monteleone f o r h i s a s s i s t a n c e i n the s t a t i s -t i c a l a n a l y s i s of the data, and Dr. F r e d e r i c k Hecht and Dr. Barbara Kaiser-McCaw f o r t h e i r c o n t i n u i n g encouragement. I g r a t e f u l l y acknowledge the p e r s o n a l f i n a n c i a l support of a Me d i c a l Research C o u n c i l of Canada Studentship from 1977 to 1980. I would a l s o l i k e t o thank other i n v e s t i g a t o r s of the f r a g i l e X chromosome whose work and correspondence pro-v i d e d support and i n s p i r a t i o n d u r i n g the course of t h i s study, and I would l i k e to express my a p p r e c i a t i o n t o the p a r t i c i p a t i n g f a m i l i e s f o r t h i e r i n t e r e s t and f u l l coopera-x i i i t i o n . In p a r t i c u l a r , I would l i k e to acknowledge my wife, Lee, for her assistance i n every aspect of the r e a l i z a t i o n of t h i s manuscript. CHAPTER 1 INTRODUCTION 1.1 F a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n . Mental r e t a r d a t i o n i s i n c l u d e d i n the d i a g n o s i s of some 40 d i f f e r e n t s e x - l i n k e d g e n e t i c d i s e a s e s (McKusick, 1978; Jennings e t a l . , 1980). Many of these d i s o r d e r s are d i s -t i n g u i s h a b l e on the b a s i s of p h y s i c a l and b i o c h e m i c a l ab-n o r m a l i t i e s . However, u n t i l r e c e n t l y , one of the most common forms of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n , the M a r t i n -B e l l syndrome (Martin and B e l l , 1943), has l a c k e d any c l e a r l y d e f i n a b l e p h y s i c a l or b i o c h e m i c a l abnormality. Since 1943, s e v e r a l l a r g e p edigrees have been r e p o r t e d documenting the s e g r e g a t i o n of t h i s n o n - s p e c i f i c form of f a m i l i a l s e x - l i n k e d s e x - l i n k e d mental r e t a r d a t i o n (Renpenning e t a l . , 1962; Dunn e t a l . , 1963; O p i t z e t a l . , 1965; Lehrke, 1974; Deroover et a l . , 1977; Ruvalcaba e t a l . , 1977). 1.11 P r e v a l e n c e . N o n - s p e c i f i c s e x - l i n k e d mental r e -t a r d a t i o n r e p r e s e n t s about 70% of a l l diagnosed s e x - l i n k e d mental r e t a r d a t i o n and i s thought to.be r e s p o n s i b l e f o r the documented excess of males.in the m e n t a l l y r e t a r d e d popula-t i o n (reviewed by Herbst, 1980). Turner and Turner (1974) proposed t h a t t h i s n o n - s p e c i f i c form of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n probably accounts f o r one i n f i v e i n s t i -t u t i o n a l i z e d m a n t a l l y r e t a r d e d males with IQ 1s between 30 and 55. On an o v e r a l l p o p u l a t i o n base, they c a l c u l a t e d a frequency of 0.74/1000 males. A r e c e n t study has i n d i c a t e d t h a t the p r e v a l e n c e of t h i s d i s o r d e r may be higher; using 2 data from the B r i t i s h Columbia H e a l t h S u r v e i l l a n c e R e g i s t r y , Herbst (1980) c a l c u l a t e d a prev a l e n c e of 1.83/1000 males. 1.12 A s s o c i a t i o n with a f r a g i l e X chromosome. Lubs (1969) f i r s t r e p o r t e d the presence o f a f r a g i l e X chromosome i n a f a m i l y 1 i n which n o n - s p e c i f i c f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n was s e g r e g a t i n g . He observed the f r a g i l e X chromosome i n f o u r m e n t a l l y r e t a r d e d males and two normal f e -males i n th r e e g e n e r a t i o n s of the f a m i l y . The f r a g i l e X chromosome appeared to have a c o n s t r i c t i o n , or f r a g i l e s i t e , near the t e r m i n a l end of the long arm of the chromosome. (F i g . 1) and was expressed i n only a p o r t i o n of the c e l l s exa-mined from a p a t i e n t . Since the f i r s t r e p o r t , the a s s o c i a t i o n of a f r a g i l e X chromosome wit h f a m i l i a l s e x - l i n k e d mental r e -t a r d a t i o n has been documented by a number of other i n v e s t i g a -t o r s (Giraud e t a l . , 1976; Harvey et a l . , 1977; Howard-Peebles et a l . , 1979; Suth e r l a n d and A s h f o r t h , 1979; Turner et a l . , 1980). In 1977, Sutherland showed t h a t e x p r e s s i o n of the f r a g i l e s i t e on the X chromosome i n p e r i p h e r a l blood lymphocytes was dependent on the medium used i n c e l l c u l t u r e . Only i n Medium 199, of the t i s s u e c u l t u r e media examined, was the demonstra-t i o n of the f r a g i l e X chromosome p o s s i b l e . T h i s f i n d i n g prompted a number of i n v e s t i g a t o r s t o re - s t u d y the chromosomes of members, of s e v e r a l f a m i l i e s i n which the d i s o r d e r was known t o be s e g r e g a t i n g i n order to look f o r the presence o f the f r a g i l e X chromosome. I n s e v e r a l of these f a m i l i e s the f r a g i l e X chromosome was found (Dunn e t a l . , 1963, r e -3 F I G U R E 1 F r a g i l e X c h r o m o s o m e ( a r r o w ) o f a m e n t a l l y r e t a r d e d m a l e p a t i e n t f r o m a f a m i l y i n w h i c h f a m i l i a l s e x -l i n k e d m e n t a l r e t a r d a t i o n i s s e g r e g a t i n g . 4 -s t u d i e d by Dunn e t a l . , 1980, and Jacobs et a l . , 1980; Ruvalcaba e t a l . , 1977, r e s t u d i e d by Jennings e t a l . , 1980; Bowen et a l . , 1978, r e s t u d i e d by M a r t i n e t a l . , 1980). Sutherland's d i s c o v e r y a l s o encouraged a number of i n v e s t i g a t o r s t o re-examine t h e i r c l i n i c a l f i l e s f o r case r e p o r t s of n o n - s p e c i f i c f a m i l i a l s e x - l i n k e d mental r e t a r d a -t i o n , and to study the chromosomes of these f a m i l i e s u s i n g the a p p r o p r i a t e c u l t u r i n g c o n d i t i o n s . Soudek et a l . (1980) found t h a t 10 of 22 f a m i l i e s r e s t u d i e d e x h i b i t e d the f r a g i l e X chromosome. Turner e t a l . (1980) found t h e . f r a g i l e : X chrom-osome to' be se g r e g a t i n g i n seven of." 23 families-, and, i n another r e c e n t study, Jacobs et a l . (1980) found the f r a -g i l e X i n s i x of seven f a m i l i e s . The one f a m i l y ^ h a t d i d not e x h i b i t the marker i n the Jacobs et a l . study was the; f a m i l y o r i g i n a l l y r e p o r t e d by Renpenning et a l . (1962). Because n o n - s p e c i f i c f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n has f r e -q u e n t l y been r e f e r r e d t o as "Renpenning syndrome" and taken as synonymous w i t h " M a r t i n - B e l l syndrome", i t i s important now t h a t the d i s t i n c t i o n be made between Renpenning syndrome and " f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n a s s o c i a t e d w i t h the f r a g i l e X chromosome". I.13 C l i n i c a l m a n i f e s t a t i o n s of f a m i l i a l s e x - l i n k e d  mental r e t a r d a t i o n . The need f o r a d i s t i n c t i o n between f a m i l -i a l s e x - l i n k e d mental r e t a r d a t i o n a s s o c i a t e d w i t h the f r a g i l e X chromosome and other forms of n o n - s p e c i f i c s e x - l i n k e d men-t a l r e t a r d a t i o n has been underscored by the. r e c e n t a s s o c i a t i o n of some phenotypic a b n o r m a l i t i e s w i t h the f r a g i l e X chromo-some. In g e n e r a l , these phenotypic a b n o r m a l i t i e s occur 5 i r r e g u l a r l y among p a t i e n t s . While no: c l i n i c a l f e a t u r e can as y e t be c o n s i d e r e d pathognomonic f o r s e x - l i n k e d mental r e t a r d a t i o n a s s o c i a t e d w i t h the f r a g i l e X chromosome, some c h a r a c t e r i s t i c s have been noted by an i n c r e a s i n g number of i n v e s t i g a t o r s and are c l i n i c a l l y s i g n i f i c a n t . Among them are : .macrofirchidism, prognathism and prominent ears, high arched p a l a t e , and speech impairment incompatible w i t h the degree of i n t e l l e c t u a l d e f i c i t (Herbst, 198 0; Jacobs et a l . , 1980; Jennings et a l . , 1980; Soudek et a l . , 1980). Other f e a t u r e s , o c c u r r i n g l e s s f r e q u e n t l y , i n c l u d e dermatoglyphic and CNS i r r e g u l a r i t i e s and forms of p s y c h o s i s . "Macroorchidism, or megalotestes, a s s o c i a t e d w i t h the s e g r e g a t i o n of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n has r e c e i v e d the a t t e n t i o n of a number of i n v e s t i g a t o r s (Ruvalcaba et a l . , 1977; Bowen et a l . , 1978; Cantu e t a l . , 1978; Turner e t a l . , 1978; Howard-Peebles and Stoddard, 1979, 1980; Jacobs et-al,.1979,198 0; Sutherland and A s h f o r t h , 1979). While i t appears t h a t p a t i e n t s s u f f e r i n g from f a m i l i a l s e x - l i n k e d mental r e -t a r d a t i o n a s s o c i a t e d w i t h the f r a g i l e X chromosome may or may not e x h i b i t rimacroorchidism, very few, i f any, m e n t a l l y r e -tarded p a t i e n t s w i t h : macroorchidism have been r e p o r t e d i n whom the f r a g i l e X chromosome i s not p r e s e n t (Sutherland and A s h f o r t h , 197 9; Howard-Peebles and Stoddard, 198 0; Jacobs e t a l . , 1980; Jennings e t a l . , 1980). F u r t h e r documentation w i l l be necessary before macroorchidism can be used as a c o n s i s -t e n t m o r p h o l o g i c a l i n d i c a t o r f o r chromosome s t u d i e s , but a t p r e s e n t i t i s being used s u c c e s s f u l l y as a p r e - s c r e e n i n g t e s t 6 f o r re—examining i n s t i t u t i o n a l i z e d m e n t a l l y r e t a r d e d males f o r the f r a g i l e X chromosome (Howard-Peebles, p e r s o n a l com-munication; Soudek and P a r t i n g t o n , p e r s o n a l communication). On the b a s i s of the re-examination of c l i n i c a l case f i l e s and p r e v i o u s l y r e p o r t e d f a m i l i e s , w i t h f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n , f o r the presence of the f r a g i l e X chromo-some, 52 of 8 6 f a m i l i e s have demonstrated the marker X chromo-some (Table I ) . Although t h i s frequency may r e f l e c t some ascertainment b i a s , i t appears t h a t . t h e " f r a g i l e X chromosome is.'.- s e g r e g a t i n g , i n a t l e a s t . 50% of cases o r i g i n a l l y -•categorized ..as.nor.Aspecif i c f a m i l i a l s e x - l i n k e d mental, retardation.-• T h i s frequency suggests t h a t f a m i l i a l s e x - l i n k e d mental r e t a r -d a t i o n a s s o c i a t e d w i t h the f r a g i l e X chromosome may be second onl y to Down syndrome as a form of mental r e t a r d a t i o n a s s o c i -ated w i t h a s p e c i f i c chromosome abnormality. 1.14 F r a g i l e X e x p r e s s i o n i n m e n t a l l y r e t a r d e d males  and t h e i r female r e l a t i v e s . Table I shows the number of m e n t a l l y r e t a r d e d males, o b l i g a t e c a r r i e r females, and po-t e n t i a l c a r r i e r females, having a 50% r i s k of c a r r y i n g the d i s o r d e r , who have been r e p o r t e d i n the l i t e r a t u r e as demonstrating the f r a g i l e X chromosome i n chromosome s t u d i e s of c u l t u r e d p e r i p h e r a l blood lymphocytes. While i t appears t h a t a n a l y s i s of p e r i p h e r a l blood lymphocytes c u l -t u r e d i n M199 p r o v i d e s a r e l i a b l e and s a t i s f a c t o r y method f o r d e t e r m i n i n g . i f a m e n t a l l y r e t a r d e d male i s c a r r y i n g the f r a g i l e X chromosome, i t has proven l e s s s a t i s f a c t o r y i n determining female c a r r i e r s t a t u s . In the o r i g i n a l r e p o r t TABLE I Exp r e s s i o n of the f r a g i l e X chromo-some i n members of f a m i l i e s r e p o r t e d i n the l i t e r a t u r e . No. F a m i l i e s Marker P o s i t i v e No. F a m i l i e s Studied No. P a t i e n t s Marker P o s i t i v e No. P a t i e n t s S t u d i e d Reference (Frequency Range) M e n t a l l y Retarded Males O b l i g a t e C a r r i e r Females P o t e n t i a l C a r r i e r Females (50% r i s k ) 6/6 5/5 2/2 1/1 Giraud e t a l . (18-50%) (?) (20%) (1976) 4/4 14/14 4/6 2/2 Harvey e t a l . (8-41%) (2-24%) (7%,17%) (1977) 3/6 9/9 1/2 — Howard-Peebles (4-33%) (15%) e t a l . (1979) Howard-Peebles & Stoddard (1979, 1980) 6/7 36/36 4/17 6/20 Jacobs et a l . (7-50%) (3-12.9%) (1.5-21.4%) (1980) 2/3 8/8 1437./6 2/3?+ Jennings e t a l . (1.5-18%) (0.5-4.5%) (1.5%,8.5%) (1980) continued... No. F a m i l i e s Marker P o s i t i v e No. P a t i e n t s Marker P o s i t i v e Reference No. F a m i l i e s Studied No. P a t i e n t s S t u d i e d (Frequency Range) M e n t a l l y Retarded Males O b l i g a t e C a r r i e r Females P o t e n t i a l C a r r i e r Females (50% r i s k ) 1/1 4/4 1/2 1/5 Lubs (1969) (12-33%) (28%) (3%) 1/1 2/2 3/3 3/3 M a r t i n e t a l . (3.0%,15.5%) (0.5-1.5%) (0.5-6.5%) (1980) 1/1 1/1 0/1 0/1 T u r l e a u e t a l . (7%) (1979) 7/23 25/25 4/12 8/20 Turner e t a l . (24-45%) (? ) * ( ? ) * (1980) 9/22 27/29 2/4 2/5 Soudek e t a l . (2-29%) (?)* ( ? ) * (1980) 12/1,2 21/21 5/13 9/17 Sutherland (4-44%) (2.5-19.5%) (6-26%) (1979c) 52/86 152/154 29/68 34/77 TOTALS + u n c l e a r s t a t u s i n manuscript * f r e q u e n c i e s not given i n manuscript 9 o f t h e f r a g i l e X chromosome by Lubs (1969), a l l f o u r a f f e c t e d males e x p r e s s e d t h e f r a g i l e X chromosome, a t f r e -q u e n c i e s o f 12 - 33% o f t h e c e l l s examined. Lymphocytes from a 59 y e a r o l d o b l i g a t e c a r r i e r female d i d not e x h i b i t the f r a g i l e X chromosome, w h i l e t h o s e from a n o t h e r c a r r i e r , aged 24, e x h i b i t e d t h e marker i n 28% o f t h e c e l l s . I n t h e 22 y e a r o l d s i s t e r o f t h e l a t t e r p a t i e n t , t h e f r a g i l e X chromosome was p r e s e n t i n o n l y 3% o f t h e c e l l s . I n t h e s t u d y o f lymphocytes from s i x o b l i g a t e c a r r i e r f e m a les from t h r e e f a m i l i e s , Harvey e t a l . , (1977)' were a b l e t o demonstrate the marker i n a l l but two a t f r e q u e n c i e s r a n g i n g from 2% t o 24%. A l l 14 m e n t a l l y r e t a r d e d males s t u d i e d demonstrated t h e f r a g i l e X a t f r e q u e n c i e s between 8 and 41%. I n t h e 11 s t u d i e s o f 8 6 f a m i l i e s r e p o r t e d i n T a b l e I , v i r t u a l l y a l l o f t h e a f f e c t e d males s t u d i e d demonstrated th e f r a g i l e X chromosome. The f r e q u e n c y o f e x p r e s s i o n f e l l w i t h i n a wide range: 1.5 - 50%. The f r a g i l e X chromosome has n o t been demonstrated i n any normal r e l a t e d male c o n t r o l s ( b r o t h e r s and u n c l e s ) when t h e y have been s t u d i e d (Jacobs e t a l . , 1980; Soudek e t a l . , 1980; Turner e t a l . , 1980). However, i t seems t h a t r e l i a b l e d e t e r m i n a t i o n o f female c a r r i e r s t a t u s by t h e use o f t h e f r a g i l e X chromosome i s not p o s s i b l e . L e ss t h a n h a l f o f t h e o l d e r age o b l i g a t e c a r r i e r f e m a l e s demonstrated t h e f r a g i l e X chromosome i n t h e s t u d i e s r e p o r t e d i n T a b l e I . About h a l f o f the p o t e n t i a l c a r r i e r f e m a l e s demonstrated i t . The l a t t e r o b s e r v a t i o n i s c o n s i s -10 t e n t w i t h the p r o p o r t i o n expected from Mendeliah t r a n s m i s s i o n of a sex'-linked abnormality to daughters of o b l i g a t e c a r r i e r females. The frequencies, of e x p r e s s i o n of the f r a g i l e X chromosome i n o b l i g a t e and p o t e n t i a l c a r r i e r females are g e n e r a l l y much lower than those obtained i n r e l a t e d m e n t a l l y r e t a r d e d males. F r a g i l e X e x p r e s s i o n i n females a l s o appears t o show an age dependency. In h i s study of 30 c a r r i e r f e -males, Sutherland (1979c) concluded t h a t the frequency of marker e x p r e s s i o n i n c a r r i e r s r a p i d l y d e c l i n e s i n females over the age of 25 y r s . , and t h a t the marker i s f r e q u e n t l y not observed i n o l d e r o b l i g a t e c a r r i e r females (30+'yrs.). One i n t e r e s t i n g aspect of these f a m i l i e s i s t h a t oc-c a s i o n a l l y the p e d i g r e e s w i l l c o n t a i n a female who i s of low normal i n t e l l i g e n c e , a n d who a l s o c a r r i e s the f r a g i l e X chromo-some (Giraud e t a l . , 1976; Howard-Peebles et a l . , 1979; S u t h e r l a n d , 1979c; Jacobs et a l . , 1980). Only a few of these females have been s t u d i e d i n d e t a i l , but the. l i m i t e d -i •:. evidence suggests t h a t t h e r e may be a p o s i t i v e c o r r e l a t i o n between the frequency of e x p r e s s i o n of the marker chromosome and the degree of t h e i r i n t e l l e c t u a l d e f i c i t ; , the lower the i n t e l l i g e n c e of the heterozygous female, the g r e a t e r i s the frequency of e x p r e s s i o n of the f r a g i l e X chromosome ob-served (Jacobs et a l . , 198 0; Soudek e t a l , , p e r s o n a l communi-ca t i o n ) . An e x p l a n a t i o n f o r the occurrence of heterozygous females w i t h low i n t e l l i g e n c e may be t h a t these p a r t i c u l a r i n d i v i d u a l s r e p r e s e n t an extreme i n the d i s t r i b u t i o n of 11 p a t e r n a l and maternal X chromosome i n a c t i v a t i o n . In these females, the X chromosome c a r r y i n g the f r a g i l e s i t e , and t h e r e f o r e the:the abnormal genes r e s p o n s i b l e f o r -mental r e -'tardation, *ay be- a c t i v e i n a high ^ p r o p o r t i o n of c e l l s . In an attempt to determine i f the p a t t e r n of X chromo-some i n a c t i v a t i o n i n such females was unusual, Uchida (person-a l communication) examined the l a t e r e p l i c a t i o n p a t t e r n s of X chromosomes i n two c a r r i e r females w i t h normal i n t e l l i g e n c e who e x h i b i t e d the marker and compared them to the p a t t e r n s i n two low i n t e l l i g e n c e c a r r i e r females who a l s o e x h i b i t e d the marker. She determined t h a t both the normal X and the f r a g i l e X were l a t e r e p l i c a t i n g i n an equal p r o p o r t i o n of c e l l s i n the normal female c a r r i e r s . [This p a t t e r n of random i n a c t i v a -t i o n has a l s o been r e p o r t e d by M a r t i n e t a l . (198 0)\] In the two d u l l c a r r i e r females, the normal X was p r e f e r e n t i a l l y i n a c t i v a t e d about t h r e e times more f r e q u e n t l y than the f r a g i l e X. While low i n t e l l i g e n c e c a r r i e r females have been r e p o r t e d i n f r e q u e n t l y i n the l i t e r a t u r e , and the p r e c i s e e t i o l o g y of t h e i r low i n t e l l i g e n c e remains obscure, f u r t h e r s t u d i e s may c l a r i f y the r e l a t i o n between the e x p r e s s i o n of the f r a g i l e - X chromosome i n c a r r i e r females, X chromosome i n a c t i v a t i o n , and the i n f r e q u e n t m a n i f e s t a t i o n i n some female p a t i e n t s of a degree of mental d e f i c i e n c y . 1.2 H e r i t a b l e f r a g i l e s i t e s . 1.21 D i s t r i b u t i o n . In a d d i t i o n t o the f r a g i l e X chromo-12 some, h e r i t a b l e f r a g i l e s i t e s on human chromosomes 2, 9, 10, 11, 12, 16, 17, and 20 have been r e p o r t e d (reviewed by Sutherland, 1979b). The f r a g i l e s i t e s on chromosome No.'s 2, 10, 16, and 20 have been shown t o be i n h e r i t e d i n simple Mendelian f a s h i o n . The autosomal f r a g i l e s i t e s have been demonstrated i n p h e n o t y p i c a l l y normal i n d i v i d u a l s as w e l l as abnormal i n d i v i d u a l s , but they have not been a s s o c i a t e d s o l e l y w i t h any s p e c i f i c phenotypic d i s o r d e r (Schmid a n d r V i s c h e r ; 1969, Btihler et a l . , 1970; Magenis et a l . , 1970; Reeves and Lawler, 1970; F r a c c a r o e t a l . , 1972; Ferguson-Smith, 1973; O l i v e r e t a l . , 1978; SjzSrensen e t a l . , 1979). T h e r e f o r e , they have g e n e r a l l y been regarded as normal chromosome v a r i a n t s . The estimated frequency o f occurrence o f h e r i t a b l e f r a g i l e s i t e s i n the p o p u l a t i o n i s 1 i n 444 or 0.2% (Hecht and Kaiser-McCaw, 1979). These f r a g i l e s i t e s u s u a l l y appear as achromatic d i s c o n -t i n u i t i e s t r a v e r s i n g both chromatids of one of the arms of a metaphase chromosome. They are g e n e r a l l y l o c a t e d a t a median i n t e r s t i t i a l p o s i t i o n on the chromosome arm and appear very much, l i k e secondary c o n s t r i c t i o n s t h a t have been a s s o c i a t e d w i t h n u c l e o l a r o r g a n i z e r a c t i v i t y i n c l a s s i c a l p l a n t and a n i -mal c y t o g e n e t i c s . F r a g i l e s i t e s , as d i s t i n c t from secondary c o n s t r i c t i o n s , have not been d e s c r i b e d i n any other mammalian s p e c i e s (Sutherland, 1979b) .' 1.22 C y t o g e n e t i c s • While r e s e a r c h on h e r i t a b l e f r a g i l e s i t e s other than the one on the X have not y i e l d e d any i n -formation o f d i r e c t p r a c t i c a l a p p l i c a b i l i t y i n g e n e t i c or r e p r o d u c t i v e c o u n s e l i n g , i t has r e s u l t e d i n b e t t e r under-13 standing of the f r a g i l e s i t e i n r e l a t i o n t o i r r e g u l a r chromo-some s t r u c t u r e and behavior. H e r i t a b l e f r a g i l e s i t e s occur., not randomly, but a t p a r t i c u l a r r e g i o n s on chromosomes (Giraud. et a l . , 1976; Sutherland, 197 9b). Chromosome banding s t u d i e s have p e r m i t t e d assignment of each of the f r a g i l e s i t e s t o a p a r t i c u l a r r e -g i o n on a chromosome arm. The r e p o r t e d s i t e s a r e : 2 q l l , 9ql2, 10q23, l l q 2 3 , 12ql3, 16q22, 17pl2, 2 0 p l l , and Xq27-28. The b e s t documented of these are the s i t e s at 2 q l l , 10q23,16q22, and Xq27-28. (Sutherland, 1979b). Minor d i s c r e p a n c i e s i n the p r e c i s e l o c a t i o n of some f r a g i l e s i t e s have been r e p o r t e d . For i n s t a n c e , Sutherland (197 9b) r e p o r t e d the l o c u s of the f r a g i l e s i t e on chromo-some No. 10 as 10q23 on the b a s i s of G-banding. Using R-banding, Gi r a u d et a l . (1976) r e p o r t e d the l o c u s as 10q242. D i s c r e p a n c i e s i n the l o c a t i o n of a d i s c o n t i n u i t y seem to be a f u n c t i o n of the d i f f e r e n t banding methods employed (Savage, 1977). P r e c i s e l o c a t i o n of the f r a g i l e s i t e on the X chromosome has been impeded by the f a c t t h a t the f r a g i l e s i t e i s l o c a t e d v e r y near the t e r m i n a l end of the long arm of the chromosome, and c o n v e n t i o n a l G-banding does not c l e a r l y d i s t i n g u i s h the G-negative band a t Xq28 from the G - p o s i t i v e band at Xq27. R-banding r e s u l t s seem to i n d i c a t e t h a t the f r a g i l e s i t e i s l o c a t e d i n the proximal p o r t i o n of band q28 (Turleau e t a l . , 1979). There i s evidence to suggest t h a t e x p r e s s i o n of the 14 f r a g i l e s i t e on the X chromosome i s due i n p a r t t o an abnormality i n the chromosome condensation process a t m i t o s i s and not due to a s t r u c t u r a l a l t e r a t i o n : . of the X chromosome such as a t r a n s l o c a t i o n . Banding s t u d i e s of chromosomes wit h f r a g i l e s i t e s have shown t h a t the banding p a t t e r n s of the chromosome arms ad j a c e n t to the f r a g i l e s i t e s are con-s i s t e n t w i t h the normal banding p a t t e r n s f o r t h a t chromo-some. R and G-banding s t u d i e s have shown t h a t the banding p a t t e r n of the f r a g i l e X chromosome i s c o n s i s t e n t w i t h the banding p a t t e r n of the normal X (Harvey et a l . , 1977; S u t h e r l a n d , 1979a; T u r l e a u e t a l . , 1979; Howard-Peebles and Stoddard, 1980). Because of the s t r i k i n g s i m i l a r i t y i n appearance between the s a t e l l i t e s on the t e r m i n a l end of the f r a g i l e X chromosome and the s h o r t arm s a t e l l i t e s on the D and G group chromosomes, i t c o u l d be suggested t h a t the s a t e l l i t e i s t r a n s l o c a t e d from one of these chromosomes. However, a study by T u r l e a u et a l . (197 9) showed the Xq27-28 f r a g i l e s i t e t o be C-band n e g a t i v e , or l a c k i n g any t r a n s -l o c a t e d centromeric heterochromatin. In a d d i t i o n , s t u d i e s using, s i l v e r s t a i n i n g , which i s s p e c i f i c f o r n u c l e o l a r r e g i o n s (AgNOR banding), d i d not r e v e a l any n u c l e o l a r o r g a n i z e r ac-t i v i t y a t the Xg27-28 f r a g i l e s i t e (Sutherland and Leonard, 1979; Howard-Peebles and Howell, 1979; T u r l e a u et a l . , 1979). The f a c t t h a t s a t e l l i t e s t a l k s from a c r o c e n t r i c chromosomes known to be i n v o l v e d i n t r a n s l o c a t i o n are s t i l l s t a i n a b l e with t h i s technique (Neu et a l . , 197 6), suggests t h a t i t i s un-l i k e l y t h a t the f r a g i l e X i n c l u d e s m a t e r i a l t r a n s l o c a t e d from 15 one of the D or G group chromosomes. DNA r e p l i c a t i o n s t u d i e s have p r o v i d e d more d i r e c t e v i -dence t h a t the c o n s t r i c t i o n i n the chromosome arm c h a r a c t e r -i s t i c of a f r a g i l e s i t e may indeed be the r e s u l t of abnormal chromosome condensation. A l l of the known h e r i t a b l e f r a g i l e s i t e s occur i n r e g i o n s t h a t are normally l a t e r e p l i c a t i n g , i . e . , r e p l i c a t i o n occurs w i t h i n the l a s t f i v e hours of DNA s y n t h e s i s (Kondra and Ray, 1978). Abnormally l a t e r e p l i c a -t i v e behavior has been r e p o r t e d i n connection w i t h the e x p r e s s i o n of the 2ql2 f r a g i l e s i t e . In experiments u s i n g t r i t i a t e d thymidine, Buhler e t a l . (1970) found t h a t the 2ql2 f r a g i l e r e g i o n was r e p l i c a t e d l a t e r than normal. The 9ql2 f r a g i l e r e g i o n has also, been shown to be abnormally l a t e r e p l i c a t i n g (Schmid and V i s h e r , 1969). Since incomplete condensation has been shown to p a r a l l e l l a t e r e p l i c a t i o n (Stubb.lef.ield., 1964; Zakharov and E g o l i n a , 1968), i t can be concluded t h a t d e f e c t i v e condensation, i s r e s p o n s i b l e , a t l e a s t i n p a r t , f o r the e x p r e s s i o n of some of the h e r i t a b l e f r a g i l e s i t e s . Recent work by Sutherland (1979a) on s p e c i f i c t i s s u e c u l t u r e requirements f o r h e r i t a b l e f r a g i l e s i t e e x p r e s s i o n suggests t h a t the commitment to- f r a g i l e s i t e e x p r e s s i o n i s more l i k e l y due to. c e l l u l a r events p r i o r t o , or e a r l y i n , the. r e p l i c a t i v e p e r i o d of the c e l l c y c l e . T h i s w i l l be d i s c u s s e d further; i n the f o l l o w i n g s e c t i o n . l.:2 3 T i s s u e o r i g i n and c u l t u r e c o n d i t i o n s . V i r t u a l l y a l l o b s e r v a t i o n s of h e r i t a b l e f r a g i l e s i t e s have been c o n f i n e d 16 to s t u d i e s of p e r i p h e r a l blood lymphocytes. At the i n c e p t i o n of t h i s study, none of the h e r i t a b l e f r a g i l e s i t e s had been demonstrated i n c u l t u r e d s k i n f i b r o b l a s t s , with the e x c e p t i o n of the f r a g i l e s i t e a t 2ql2 (Fraccaro e t a l . , 1972; Ferguson-Smith, 1973; Sutherland, 1979b). In the few i n s t a n c e s where bone marrow c e l l s have been studied., f r a g i l e s i t e s were de-monstrable o n l y a t 16g22 and Xq27-28 (Magenis e t a l . , 1970; Sutherland, 197 9b). However, the f r e q u e n c i e s of e x p r e s s i o n of these f r a g i l e s i t e s i n f i b r o b l a s t s and bone marrow p r e p a r a t i o n s were f r e q u e n t l y lower than those found i n lympho-cyte: i c u l t u r e f o r the same p a t i e n t . (Sutherland, 197 9b) . Some s i g n i f i c a n t work on the i n f l u e n c e of d i f f e r e n t t i s s u e c u l t u r e media on the e x p r e s s i o n . o f f r a g i l e s i t e s i n p e r i p h e r a l blood, lymphocytes has r e c e n t l y been r e p o r t e d by S u t h e r l a n d (1977, 1979a). P r e l i m i n a r y s t u d i e s (Sutherland, 1977) had shown t h a t the frequency of e x p r e s s i o n of the f r a g i l e s i t e s at 2q21, 10q23, and Xq27-28 was s u b s t a n t i a l l y h i g h e r i f the c e l l s were c u l t u r e d i n Medium 199 r a t h e r than other t i s s u e c u l t u r e media such as Eagle's MEM, RPMI 164 0, Ham's F10, or CMRL 1969. The Xq27-28 f r a g i l e s i t e c o u l d o n l y be demonstrated i n M199. More r e c e n t l y , Sutherland (1979a) suggested t h a t i t i s the r e l a t i v e l y low l e v e l (0.01 mg/L) of f o l i c a c i d i n Medium.19 9 t h a t i s r e s p o n s i b l e f o r the i n -crease i n the frequency of e x p r e s s i o n of some f r a g i l e s i t e s . He showed t h a t i f p a r a l l e l lymphocyte c u l t u r e s were es-t a b l i s h e d i n media c o n t a i n i n g graduated c o n c e n t r a t i o n s of f o l i c a c i d (0.0 to 0.5 mg/L f o l i c a c i d ) , the frequency of 17 e x p r e s s i o n r a p i d l y d e c r e a s e d as t h e c o n c e n t r a t i o n o f f o l i c a c i d i n c r e a s e d . Some f r a g i l e s i t e s , namely 2 q l 2 , 10q23, and Xq27-28, were p a r t i c u l a r l y s e n s i t i v e t o a l t e r e d c o n c e n t r a t i o n s o f f o l a t e , w h i l e t h e f r e q u e n c y o f e x p r e s s i o n o f t h e f r a g i l e s i t e a t 16q22 was not a f f e c t e d a t a l l . I n c r e a s i n g c o n c e n t r a t i o n s o f t h y m i d i n e o r 5-BrdU, a base a n a l o g o f t h y m i d i n e , (0.0 t o 3.0 mg/L) produced r e s u l t s compar-a b l e t o t h o s e observed w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f f o - i l a t e f o r t h e f r a g i l e s i t e s a t 2 q l 2 , 10q23, and Xq27-28. Thymidine and BrdU were,, however, l e s s e f f e c t i v e t h a n f o l i c a c i d a t i n h i b i t i n g e x p r e s s i o n o f t h e f r a g i l e s i t e s . F u r t h e r e v i d e n c e i n s u p p o r t o f a h y p o t h e s i s t h a t i t i s th e low f o l a t e l e v e l i n M199 t h a t i s r e s p o n s i b l e f o r the i n -c r e a s e d f r e q u e n c y o f f r a g i l e s i t e e x p r e s s i o n came from ex-p e r i m e n t s i n w h i c h lymphocytes were c u l t u r e d i n media con-t a i n i n g normal l e v e l s o f f o l a t e p l u s t h e f o l i c a c i d a n t a -g o n i s t , m e t h o t r e x a t e ( S u t h e r l a n d , 1979a). Under t h e s e c u l t u r e c o n d i t i o n s t h e f r e q u e n c y o f e x p r e s s i o n i n c r e a s e d . S u t h e r l a n d p o s t u l a t e d t h a t t h e m e t a b o l i c mode of a c t i o n o f f o l a t e d e p r i v a t i o n on f r a g i l e s i t e e x p r e s s i o n was t h r o u g h p y r i m i d i n e b i o s y n t h e s i s , s p e c i f i c a l l y i n t h e c o n v e r -s i o n o f dUMP to. dTMP medi a t e d by t h e coenzyme 5,10-meTHFA: u r i d i n e monophosphate (dUMP>t5,10-methylene t e t r a h y d r o f o l a t e (5,10-meTHFA)—^thymidine monophosphate (dTMP) +d>ihydrof o l a t e (DHFA). I f t h i s r e a c t i o n i s i n h i b i t e d m i l d l y , t h e r e s u l t i n g d e f i c i e n c y i n dTMP, t h y m i d y l a t e , would r e s t r i c t DNA s y n t h e s i s and p o t e n t i a l l y l e a d t o l e s i o n s i n t h e chromosomes. That t h i s 18 r e a c t i o n may be the area of metabolism i n v o l v e d i n f r a g i l e s i t e e x p r e s s i o n has been supported by the e x p r e s s i o n enhancing e f f e c t s of the f o l a t e a n t a g o n i s t methotrexate (Sutherland, 1979a). Methotrexate b l o c k s the c o n v e r s i o n r e a c t i o n of DHFA to THFA-by i n h i b i t i o n of d i h y d r o f o l a t e . r e d u c t a s e (Erbe, 1975). T h i s r e a c t i o n i s t i g h t l y i n t e g r a t e d w i t h the p r o d u c t i o n of the coenzyme, 5,10-meTHFA. Suthe r l a n d (1979a) showed t h a t the time of adding e i t h e r f o l i c a c i d or thymidine to the lymphocyte c u l t u r e s a l s o a f f e c t e d the frequency of f r a g i l e s i t e e x p r e s s i o n . Both f o l i c a c i d and thymidine were most e f f e c t i v e i n i n h i b i t i n g expres-s i o n i f they were added at l e a s t 24 h r s . p r i o r t o chromosome ha r v e s t . The i n h i b i t o r y e f f e c t s s u b s t a n t i a l l y decreased when e i t h e r f a c t o r was added c l o s e r t o chromosome h a r v e s t . These f i n d i n g s seem to i n d i c a t e t h a t c u l t u r e media i n f l u e n c e s on f r a g i l e s i t e e x p r e s s i o n are most e f f e c t i v e p r i o r t o , or e a r l y i n , the S-phase of the c e l l c y c l e preceeding chromosome harvest. T h i s t i m i n g e f f e c t i s c o n s i s t e n t w i t h the p o s t u l a t e d mode of a c t i o n of f o l a t e d e p r i v a t i o n , namely, reduced p y r i m i d i n e b i o s y n t h e s i s . Low f o l a t e l e v e l s c o u l d l e a d to s u b s t a n t i a l l y reduced l e v e l s of t h y m i d y l a t e a v a i l a b l e f o r DNA s y n t h e s i s . S u t h e r l a n d f u r t h e r suggested t h a t extending the c u l t u r e p e r i o d i n media low i n f o l i c a c i d from 72 t o 96 h r s . f o r those f r a g i l e s i t e s ' s e n s i t i v e : to f o l a t e d e p r i v a t i o n (2ql2, 10q23, Xq27-28) improves the frequency of e x p r e s s i o n of the marker chromosomes, and t h a t ' r e d u c i n g the c o n c e n t r a t i o n of f e t a l c a l f serum (FCS) to 5% from the 10 - 20% FCS l e v e l s used 19 c o n v e n t i o n a l l y i n lymphocyte c u l t u r e , a l s o improved the f r e -quency of marker chromosome e x p r e s s i o n . With p a r t i c u l a r r e -f e r e n c e to the Xq27-28 f r a g i l e s i t e , o ther i n v e s t i g a t o r s (Howard-Peebles and Pryor, 197 9; Gerrard and Fox, p e r s o n a l communication) have a l s o suggested t h a t reduced l e v e l s (0 -5%) of FCS improve the frequency of e x p r e s s i o n . Some i n v e s t i -g a t o r s have cautioned, however, t h a t extremely low l e v e l s of FCS can s u b s t a n t i a l l y impair c e l l growth and l e a d to a v i r t u a l l y unscorable m i t o t i c index (Turleau e t a l . , 1979). Sutherland a l s o showed t h a t the f r e q u e n c i e s of e x p r e s s i o n of the f r a g i l e s i t e s a t 2ql2 and Xq27-28 were s e n s i t i v e to the pH of the c u l t u r e , media with o p t i m a l e x p r e s s i o n between pH 7.4 and 7.6. T h i s i s s l i g h t l y more a l k a l i n e than the normal c u l t u r i n g pH of 6.8 to 7.2. F r a g i l e s i t e s at 10q23 and 16q22 were not s e n s i t i v e t o a l t e r a t i o n s i n pH. Based on response by the v a r i o u s f r a g i l e s i t e s to f o l a t e , and thymidine, d e p r i v e d c u l t u r i n g c o n d i t i o n s , as w e l l as an optimal e x p r e s s i o n of some f r a g i l e s i t e s a t a p a r t i c u l a r pH, Sutherland (1979a) proposed a c l a s s i f i c a t i o n of f r a g i l e s i t e s : those t h a t are s e n s i t i v e t o f o l a t e d e p r i v a t i o n (e.g. 2q21, 10q23, and Xq27-28), those t h a t are r e s i s t a n t t o f o l a t e d e p r i v a t i o n . (16q22) ,. those t h a t are pH dependent (2q21 and Xq27-28), and those t h a t are not pH dependent (10q23 and 16q22). A c c o r d i n g l y , the f r a g i l e s i t e a t Xq27-28 would be c l a s s i f i e d as both f o l a t e and pH dependent. 1.3 R a t i o n a l e f o r the p r e s e n t study. While e a r l i e r s t u d i e s of the f r a g i l e . X chromosome i n 20 f a m i l i e s i n which f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n was s e g r e g a t i n g e s t a b l i s h e d t h a t the prevalence of the d i s o r d e r was high, they a l s o d e f i n e d what appeared to be severe l i m i t a -t i o n s to the d i a g n o s t i c u s e f u l n e s s of the f r a g i l e X chromo-some. For i n s t a n c e , the f r a g i l e X chromosome, while s p e c i f i c f o r the d i s o r d e r , was found to be g e n e r a l l y expressed o n l y i n a p o r t i o n of the metaphases scored from c u l t u r e d p e r i p h e r a l blood lymphocytes from a f f e c t e d males. The presence of the marker chromosome i n ; many o b l i g a t e or p o t e n t i a l c a r r i e r f e -males, e s p e c i a l l y those over 25 yrs. of age, f r e q u e n t l y proved d i f f i c u l t or i m p o s s i b l e to show. Furthermore, the e x p r e s s i o n of the f r a g i l e X chromosome i n lymphocytes c o u l d be seen o n l y under c e r t a i n c u l t u r i n g c o n d i t i o n s . F i n a l l y , e x p r e s s i o n of the marker X chromosome c o u l d not be demonstrated i n s k i n f i -b r o b l a s t metaphases (Hecht and Kaiser-McCaw, 1979; Sutherland, 1979c) . The f a i l u r e to f i n d the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s i m p l i e d t h a t i t s demonstration i n c u l t u r e d amniocytes would u n l i k e l y , and the f r a g i l e X chromosome would t h e r e f o r e not be a u s e f u l chromosome marker i n a n t e n a t a l d i a g -n o s i s of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n . T h i s study was undertaken t o : 1) develop a method f o r demonstrating the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o -b l a s t s , examining some of the f a c t o r s t h a t i n f l u e n c e a r e l i a -b l e d e t e r m i n a t i o n of the frequency of e x p r e s s i o n i n t h i s t i s s u e , and 2) examine the e f f e c t s i n both c u l t u r e d lympho-21 c y t e s and f i b r o b l a s t s o f s p e c i f i c s u p p l e m e n t a l media f a c t o r s w h i c h , because o f t h e i r a b i l i t i e s t o i n t e r a c t w i t h chromo-some components, might be u s e f u l i n a c c e n t u a t i n g t h e f r e -quency o f e x p r e s s i o n o f t h e f r a g i l e X chromosome i n t i s s u e c u l t u r e . I n a d d i t i o n , i t was hoped t h a t t h e r e s u l t s from t h i s s t u d y would p r o v i d e f u r t h e r i n f o r m a t i o n on t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f f r a g i l e chromosomes. CHAPTER 2 MATERIALS AND METHODS 2.1 Sources of t i s s u e samples and t i s s u e c u l t u r e media. 2.11 S u b j e c t s . Blood and s k i n biopsy specimens were obtained from e i g h t i n d i v i d u a l s from two u n r e l a t e d f a m i l i e s i n both of which there i s c l i n i c a l and g e n e t i c s evidence f o r f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n . For convenience these i n d i v i d u a l s are r e f e r r e d t o as P a t i e n t s A, B, C, D, E, F, G, and H. Family No. 1 i s p a r t of a l a r g e pedigree o r i g i n a l l y r e -por t e d by Dunn e t a l . , (1963). Family No. 2 i s a case on f i l e w i t h the Department of M e d i c a l G e n e t i c s , U n i v e r s i t y of B r i t i s h Columbia ( f i l e #02438). Pedigrees of F a m i l i e s No. 1 and 2, i n c l u d i n g the i d e n t i t i e s of the members s t u d i e d are presented i n F i g u r e 2. Family No. 1 i s of, German C a t h o l i c background. The parents of. P a t i e n t A immigrated t o North America from the Ukraine i n the l a t e 19th century. She i s co n s i d e r e d t o be an o b l i g a t e c a r r i e r female of normal i n t e l l i g e n c e , who has had three f-mentally r e t a r d e d sons and f i v e normal daughters, three o whom gave b i r t h . t o r e t a r d e d sons. P a t i e n t C i s one of the men t a l l y r e t a r d e d sons o f P a t i e n t A, and P a t i e n t B i s one of her. o b l i g a t e c a r r i e r daughters. P a t i e n t D i s a me n t a l l y r e -tarded son of P a t i e n t B. Patient. E i s a daughter of P a t i e n t B and has a 50% r i s k of being a c a r r i e r female. Family No. 2 i s of Dutch Mennonite background and i s a l s o descended from immigrants from the Ukraine. P a t i e n t s F and G 23 Famiy No.1 IV D E . / Y £61, P 9 Family No.2 IV f •o«: 6VD J3rO I T T 00 L i ? T n m n i O O D O O • = Mentally retarded male. 0) = Obligate c a r r i e r female. ®.® = Low normal i n t e l l i g e n c e . * = Normal male, f r a g i l e X negative; studied by Dunn. et a l . (1980). / = Propositus. FIGURE 2 Pedigrees of Family No. 1 ( P a t i e n t s A - E) and Family No. 2 ( P a t i e n t s F - H) showing s e g r e g a t i o n of f a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n , e.g. t r a n s m i s s i o n of the d i s o r d e r through normal heterozygous females to a f f e c t e d sons. The pedigree presented f o r Family No. 1 i s reduced i n p a r t from the o r i g i n a l r e p o r t on the f a m i l y (Dunn et a l . , 1963). 24 a r e m e n t a l l y r e t a r d e d b r o t h e r s , and P a t i e n t H i s t h e i r s i s t e r . She i s a p o t e n t i a l c a r r i e r female who i s p r o b a b l y o f low n o r -mal i n t e l l i g e n c e ( p e r s o n a l communication from mother o f Pa-\ t i e n t H and case f i l e ) . C o n t r o l specimens were o b t a i n e d from, t h r e e i n d i v i d u a l s u n r e l a t e d to. e i t h e r f a m i l y s t u d i e d . They were matched f o r sex and age t o t h e two younger a f f e c t e d males ( P a t i e n t s D and G) and t o a p o t e n t i a l c a r r i e r female ( P a t i e n t E ) . The i n d i -v i d u a l ages, o f p a t i e n t s and'controls a r e p r e s e n t e d i n T a b l e I I . B l o o d samples were o b t a i n e d by venepucture, and s k i n specimens were o b t a i n e d by s c i s s o r s n i p b i o p s y i n a l l cases w i t h i n f o r m e d c o n s e n t (Appendix A ) . 2.12 T i s s u e c u l t u r e media. Four d i f f e r e n t t i s s u e c u l -t u r e media were used i n " - e x p e r i m e n t s t o be r e p o r t e d on p e r i p h e r a l b l o o d lymphocytes and e s t a b l i s h e d f i b r o b l a s t c e l l l i n e s . These were: MEM: Eagle's. M i n i m a l E s s e n t i a l Medium w i t h E a r l e ' s S a l t s & L - g l u t a m i n e (Gibco, L o t No. R892106) supplemented w i t h 15% FCS b u f f e r e d w i t h sodium b i c a r b o n a t e : ( f o l i c a c i d a t 0.01 mg/L). M199: Medium 199 w i t h 25 mM HEPES b u f f e r , Hank's S a l t s & L - g l u t a m i n e (Gibco, L o t No. A891012), supplemented w i t h 5% FCS and a d j u s t e d t o pH 7.6 w i t h 7.5% NaHCC>3 ( f o l i c a c i d a t 0.01 mg/L). MEM-FA: MEM w i t h o u t f o l i c a c i d supplemented w i t h 5% FCS and a d j u s t e d t o pH 7.6 w i t h 7.5% NaHC0 3 ( f o l i c a c i d a t 0.00 mg/L). MEM-Ad: MEM d e f i c i e n t i n a d e n i n e , made by s u p p l e m e n t i n g MEM w i t h 0.3 mg/L guanine and a d d i n g 0.2 mg/L a z a s e r i n e t o b l o c k p u r i n e b i o s y n t h e s i s , sup-plemented w i t h 5% FCS and a d j u s t e d t o pH 7.6 w i t h 7.5% NaHC03 ( f o l i c a c i d a t 0.01 mg/L). MEM and M199 were pu r c h a s e d d i r e c t l y from t h e Grand I s l a n d TABLE I I P a t i e n t s s t u d i e d from Family No. 1 and Family No. 2 and matched c o n t r o l s . Family No. 1 P a t i e n t Status Age A O b l i g a t e C a r r i e r Female 74 B O b l i g a t e C a r r i e r Female 55 C A f f e c t e d Male 49 D A f f e c t e d Male 31 E P o t e n t i a l C a r r i e r Female 25 Pedigree Number I I - 10 I I I - 58 11-63 IV- 144 IV-145 Family No. 2 P a t i e n t F A f f e c t e d Male 33 I I I - 3 G A f f e c t e d Male 29 II I - 7 H P o t e n t i a l C a r r i e r Female 26 I I I - 9 C o n t r o l s I J K Normal Male Normal Male Normal Female 29 31 25 B i o l o g i c a l Company (Gibco). MEM-FA and MEM-Ad were made up i n the l a b o r a t o r y . A d e t a i l e d l i s t i n g of components of each of the f o u r media i s giv e n i n Appendix B. A l i q u o t s of 100 ml of each medium were supplemented w i t h 1 ml of an a n t i b i o t i c - a n t i -mycotic s o l u t i o n (100X). c o n t a i n i n g 10, 000 U/ml p e n i c i l l i n , 25 mcg/ml F u n g i z o n e ^ , and 10, 000 mcg/ml streptomycin (Gibco Lot No.. A990311) . Any media used to c u l t u r e lymphocytes was a l s o supplemented w i t h PHA (Gibco), 0.2 ml per c u l t u r e , f o r mito-genic s t i m u l a t i o n at c u l t u r e i n i t i a t i o n . A l l F e t a l C a l f Serum (FCS) media supplements were taken from Lot No. 29101111, Flow L a b o r a t o r i e s , which co n t a i n e d a low l e v e l of f o l i c a c i d r e l a -t i v e t o s e v e r a l other l a b o r a t o r y l o t s of serum (Appendix C). 2.2 Demonstration of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s . 2.21 R a t i o n a l e f o r the procedure. The work of Sutherland (1979a) had suggested t h a t the r e p o r t e d l a c k of e x p r e s s i o n of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s might be overcome by a p p r o p r i a t e m a n i p u l a t i o n of the c u l t u r e c o n d i -t i o n s , p a r t i c u l a r l y those a f f e c t i n g f o l a t e metabolism. Since i t was determined t h a t i t would be d i f f i c u l t i f not im p o s s i b l e to o b t a i n chromosome p r e p a r a t i o n s from, f i b r o b l a s t s main-t a i n e d f o r a long term i n media low. i n , or e n t i r e l y l a c k i n g f o l i c a c i d (Erbe., 1975, 1979), a method was adopted f o r t h i s study whereby s k i n f i b r o b l a s t c e l l l i n e s would be e s t a b l i s h e d and r o u t i n e l y maintained i n medium c o n t a i n i n g normal l e v e l s of f o l a t e , and then the c e l l s would be t r a n s f e r r e d to media 27 d e f i c i e n t i n f o l i c a c i d a b r i e f p e r i o d p r i o r to chromosome ha r v e s t . -..To f a c i l i t a t e t h i s t r a n s f e r to f o l a t e d e f i c i e n t c u l t u r i n g c o n d i t i o n s , a f i b r o b l a s t c u l t u r i n g and h a r v e s t p r o t o c o l o r i g i n a l l y d e s c r i b e d by K a j i i e t a l . (1973) was adopted w i t h some m o d i f i c a t i o n . At the time of t r a n s f e r to f o l a t e d e p r i v a t i o n , f i b r o b l a s t s would be removed from f l a s k c u l t u r e i n medium c o n t a i n i n g normal l e v e l s of f o l a t e , r i n s e d of any r e s i d u a l medium, and then t r a n s f e r r e d to medium low i n , or e n t i r e l y l a c k i n g , f o l i c a c i d . From the time of t r a n s f e r to f o l a t e d e f i c i e n t media, c e l l s would be grown d i r e c t l y on s l i d e s t h a t would be s t u d i e d on the microscope. T h i s method would have two advantages t h a t c o u l d be im-p o r t a n t i n e l i c i t i n g f r a g i l e s i t e e x p r e s s i o n i n f i b r o b l a s t s . F i r s t , f i b r o b l a s t c e l l s from a s i n g l e t i s s u e c u l t u r e f l a s k c o u l d be s p l i t and t r a n s f e r r e d to d i f f e r e n t t i s s u e c u l t u r e media wi t h v a r y i n g c o n c e n t r a t i o n s of f o l i c a c i d . T h i s would permit comparison of the e f f e c t s of d i f f e r e n t . l e v e l s of f o l a t e on the frequency of e x p r e s s i o n i n a r e l a t i v e l y uniform popu-l a t i o n of c e l l s . Second, the hypotonic and f i x a t i o n t r e a t -ments a t chromosome h a r v e s t would be done on c e l l s adhering t o a s l i d e . T h i s would p r o v i d e a r e l a t i v e l y g e n t l e h a r v e s t procedure and e l i m i n a t e any s a t e l l i t e l o s s t h a t might occur with J m o r e r i g o r o u s h a r v e s t techniques. . : > . 2.22 Procedure f o r e s t a b l i s h i n g f i b r o b l a s t c e l l l i n e s . F i b r o b l a s t c e l l l i n e s were i n i t i a t e d from s k i n b i o p s i e s by the standard e x p l a n t procedure (Fraccaro e t al.j., 1960; Hamerton, 1971) and r o u t i n e l y maintained in. Eagle's MEM (Gibco) supple-mented with 15% FCS. A 3mm, s k i n s n i p was taken from the f o r e -arm from p a t i e n t s and c o n t r o l s and t r a n s p o r t e d t o the l a b o r a -t o r y i n c o l d s a l i n e . The s k i n biopsy was minced, and the p i e c e s were e q u a l l y d i s t r i b u t e d i n t o t hree 35 x 10 mm (Falcon) p e t r i d i s h e s . The e x p l a n t s were covered w i t h a No. 2: 22 mm square s t e r i l e cover s l i p , and 3 ml of f i b r o b l a s t maintenance medium (MEM) was added to each d i s h . The e x p l a n t s were then c u l t u r e d a t 37°C i n a 5% CO,, atmosphere. The medium was changed weekly, and c o n f l u e n t f i b r o b l a s t c u l t u r e s were r o u t i n e l y o b tained w i t h i n t h r e e t o f i v e weeks. At con-f l u e n c e , the f i b r o b l a s t s were t r y p s i n i z e d (1:20 D i f c o t r y p -s i n : HBSS) from each c u l t u r e d i s h and grown to confluence i n T75 f l a s k s (Lux). A l i q u o t s of f i b r o b l a s t s from a l l i n d i v i d u -a l s were f r o z e n a f t e r the t h i r d passage and s t o r e d i n l i q u i d n i t r o g e n . 2.23 Procedure for, chromosome p r e p a r a t i o n from f i b r o -b l a s t s . A l l f i b r o b l a s t experiments were done on c e l l s i n the t h i r d to n i n t h passage i n f l a s k c u l t u r e ; each passage r e p r e -sented a 1:4 s p l i t of a c o n f l u e n t T75 (Lux) f l a s k , or approx-i m a t e l y 2.3 c e l l g e n e r a t i o n s . F o r t y - t h r e e hours p r i o r to chromosome h a r v e s t , c e l l s grown to co n f l u e n c e i n T7 5 (Lux) f l a s k s i n medium c o n t a i n i n g normal l e v e l s of f o l i c a c i d were removed f o l l o w i n g incuba-t i o n f o r 15-25 minutes a t 37°C wi t h 2 mis of 1:20 t r y p s i n ( D i f c o ) . They were resuspended i n MEM-FA, 3 mis per f l a s k . A 1 ml a l i q u o t of t h i s c e l l suspension was. d i l u t e d 1:10 i n medium low i n (M199) or e n t i r e l y lacking. (MEM-FA) f o l i c a c i d . At t h i s step i n the procedure, 1 ml a l i q u o t s of f i b r o b l a s t s from three p a t i e n t s , P a t i e n t s B, C, and D, were a l s o r e s u s -pended i n MEM as w e l l as MEM-FA and M199. A l i q u o t s of 1.0 -1.5 mis of these i n d i v i d u a l suspensions were s e p a r a t e l y l a y e r e d onto s t e r i l e microscope, s l i d e s h e l d i n a 100 x 15 mm square p e t r i d i s h (3 s l i d e s per dish), and then incubated 30 to 45 min. a t 37°C. A f t e r the c e l l s had s e t t l e d , the p e t r i d i s h was f i l l e d w ith 15 ml of warm medium and allowed to i n -cubate 24 hrs., a t which time the medium was changed, and the c e l l s were incubated an a d d i t i o n a l 19 h r s . Three h r s . p r i o r t o chromosome har v e s t , 0.3 ml Colcemid (10 mcg/ml) was added to each c u l t u r e d i s h . Chromosome h a r v e s t was i n i t i a t e d by g e n t l y a s p i r a t i n g o f f the c u l t u r e medium and then c o v e r i n g the s l i d e s w i t h 15 mis of warm.1% N a C i t r a t e . C e l l s were exposed to t h i s hypo-t o n i c c o n d i t i o n f o r 15 min. a t 37°C, a f t e r which 15 ml of c o l d 3:1 m e t h a n o l :acetic a c i d was g e n t l y p i p e t t e d over the N a C i t r a t e and allowed t o f i x the c e l l s f o r 5 min. The s l i d e s were, removed from.the p e t r i d i s h e s and taken through two a d d i t i o n a l 5 min. f i x e s i n C o p l i n j a r s . The s l i d e s were removed from the C o p l i n j a r , b r i s k l y blown on, and b r i e f l y warmed over a bunsen burner. T h i s was. immediately f o l l o w e d by a 10 to 15 second d i p i n 1:50 ethanol:75% a c e t i c a c i d . Each s l i d e was f i n a l l y rewarmed b r i e f l y and a i r - d r i e d . 2.3 Procedure f o r p e r i p h e r a l blood lymphocyte c u l t u r e and chromosome p r e p a r a t i o n . Lymphocyte c u l t u r e s were e s t a b l i s h e d a c c o r d i n g to the recommended p r o t o c o l of S u t h e r l a n d (1979c) w i t h minor m o d i f i -30 c a t i o n i n o r d e r t o p r o v i d e a standard frequency of marker e x p r e s s i o n f o r each p a t i e n t , a g a i n s t which r e s u l t s from f i b r o -b l a s t c u l t u r e s and r e s u l t s from f u r t h e r m o d i f i c a t i o n of the lymphocyte c u l t u r i n g procedure c o u l d be compared. A s i n g l e sample of blood was obtained from each i n d i -v i d u a l with the e x c e p t i o n of P a t i e n t D from whom two samples were obtained on separate o c c a s i o n s . Lymphocyte c u l t u r e s were i n i t i a t e d w i t h i n 3 to 5 h r s . of o b t a i n i n g the blood sample. A lymphocyte enrichment method (Moorhead e t a l . , 1960) was adopted, r a t h e r than the whole blood technique c u r r e n t l y recommended for.lymphocyte c u l t u r e , i n a n t i c i p a t i o n of the p o s s i b i l i t y t h a t the use of low l e v e l s of F e t a l C a l f Serum (5%) i n the c u l t u r e media might lower the m i t o t i c index (Turleau e t a l . , 1979). P r i o r t o i n i t i a t i n g lymphocyte c u l t u r e s , a 10 ml blood sample was allowed to separate f o r one t o two hours a t room temperature. The l e u k o c y t e - r i c h plasma f r a c t i o n , i n c l u d i n g b u f f y c o a t and 5 to 6 drops of the red c e l l f r a c t i o n , was then c a r e f u l l y drawn o f f and mixed thoroughly. A 0.25 to 0.5 ml volume of t h i s suspen-3 s i o n (approximately 2 X 10 c e l l s ) was added to a l i q u o t s of 2 c u l t u r e medium which were p l a c e d i n 25 cm F a l c o n t i s s u e c u l -t u r e f l a s k s . C u l t u r e media c o n s i s t e d of M199 b u f f e r e d w i t h 25 mM HEPES., a d j u s t e d to pH 7.6 w i t h 7.5% NaHC0 3 and supple-mented wi t h 5% FCS. A 0.2 ml a l i q u o t of PHA (Gibco) was added to each f l a s k j u s t p r i o r t o i n c u b a t i o n , and the c u l t u r e s were capped t i g h t l y and maintained at 37°C f o r 96 h r s . The c u l t u r e medium and the p e r i o d of i n c u b a t i o n f o l l o w e d the recommended p r o t o c o l of Sutherland (1979c) f o r e x p r e s s i o n of the f r a g i l e X chromosome i n p e r i p h e r a l blood lymphocytes. Colcemid (Gibco, 10 mcg/ml), 0.075 ml per 5 ml c u l t u r e , was added 5 0 min. p r i o r to chromosome h a r v e s t . At chromosome h a r v e s t , the lymphocyte suspension c u l t u r e s were t r a n s f e r r e d t o s i l i c o n i z e d 15 ml g l a s s c o n i c a l c e n t r i -fuge tubes and spun.for 9 min. a t 150xg. The c e l l s were ' washed once i n HBSS, and the supernatant was d i s c a r d e d . For hypotonic treatment, the c e l l p e l l e t was then g e n t l y r e s u s -pended i n pre-warmed 0.075 M KC1, and incubated f o r 15 min. at 37°C. The suspension was again c e n t r i f u g e d , 9 min. a t 150xg, and a l l but 1 ml of supernatant over the p e l l e t was a s p i r a t e d -o f f . For the purposes of f i x a t i o n , 1 to 2 ml of c o l d 3:1 me-t h a n o l : a c e t i c a c i d was g e n t l y l a y e r e d over the remaining SU-J pernatant. The p e l l e t was then, g e n t l y resuspended, and more c o l d f i x a t i v e was added i n 2 ml a l i q u o t s t o a f i n a l volume of 7 ml w i t h g e n t l e a g i t a t i o n . There were 3 a d d i t i o n a l washes i n f r e s h c o l d f i x . The p e l l e t was f i n a l l y resuspended i n 1 ml of f r e s h c o l d f i x , and the c e l l s were dropped on c o l d , wet s l i d e s . The s l i d e s were then b r i e f l y warmed over a bunsen burner and allowed t o a i r - d r y . 2.4 Examination of supplemental media f a c t o r s i n t i s s u e c u l -t u r e . 2.41 Rationale f o r the procedure. Evidence from the 32 study o f h e r i t a b l e f r a g i l e s i t e s has suggested t h a t f r a g i l e s i t e e x p r e s s i o n i s a s s o c i a t e d w i t h the chromosome r e p l i c a t i o n p r o c e s s . While the evidence from the study of the r e p l i c a t i v e behavior of h e r i t a b l e f r a g i l e s i t e s i s somewhat c o n t r a d i c -t o r y w i t h r e s p e c t to p r e c i s e l y when i n DNA r e p l i c a t i o n t h e r e i s a commitment to f r a g i l e s i t e e x p r e s s i o n , i . e . the abnormal-l y l a t e r e p l i c a t i v e behavior of the 2gl2 f r a g i l e s i t e r e p o r t e d by Buhler e t a l . (1970), while the e x p r e s s i o n of the 2ql2 and Xq27-28 f r a g i l e s i t e s were r e p o r t e d by Sutherland (1979a) t o r e q u i r e a commitment p r i o r to, or i n the e a r l i e r stages o f , DNA r e p l i c a t i o n , t here i s agreement t h a t e x p r e s s i o n i s a t l e a s t i n p a r t a f u n c t i o n of the r e p l i c a t i v e p r o c e s s . S u t h e r l a n d (197 9a) proposed t h a t the mode of a c t i o n of a f o l a t e d e f i c i e n c y on f r a g i l e s i t e e x p r e s s i o n was mediated through p y r i m i d i n e b i o s y n t h e s i s , s p e c i f i c a l l y i n the reduced l e v e l s of thymidylate a v a i l a b l e f o r de novo DNA s y n t h e s i s . I f p y r i m i d i n e b i o s y n t h e s i s . i s indeed the me t a b o l i c area a s s o c i a t e d w i t h the e x p r e s s i o n of the f r a g i l e s i t e on the X chromosome, the Xq27-28 r e g i o n may be an A T - r i c h DNA sequence very s e n s i t i v e t o a l t e r e d c o n c e n t r a t i o n s of thymi-d y l a t e . In an attempt to t e s t the n o t i o n t h a t both a v a i l a b l e t hymidylate and DNA r e p l i c a t i o n are a t l e a s t i n p a r t r e s p o n s i -b l e f o r e x p r e s s i o n of the Xq27-28 f r a g i l e s i t e , and t h a t the Xq27-28 f r a g i l e s i t e i s an A T - r i c h DNA sequence p a r t i c u l a r l y s e n s i t i v e to a l t e r e d c o n c e n t r a t i o n s of n u c l e o t i d e s , t h i s study examined the e f f e c t s of a medium d e f i c i e n t i n thymi-33 dine's DNA base p a i r , adenine (MEM-Ad). A d e f i c i e n c y i n ade-nine should e f f e c t i v e l y mimic the e f f e c t s of a thymidine de-f i c i e n c y on f r a g i l e s i t e e x p r e s s i o n . The r e l a t i o n s h i p between impaired, chromosome r e p l i c a t i o n and the a c t u a l e x p r e s s i o n of a chromosome l e s i o n or f r a g i l e s i t e a t m i t o s i s i s not known. The l a c k of chromosome conden-s a t i o n , e v i d e n t when a f r a g i l e s i t e i s expressed, suggests t h a t agents t h a t are known t o i n t e r f e r e with the normal condensation process might be u s e f u l i n e l i c i t i n g the ex-p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t s or, p er-haps, i n improving the frequency of e x p r e s s i o n i n e i t h e r lymphocyte or f i b r o b l a s t c u l t r e . The chromosome decondensa-t i o n e f f e c t s of 5-BrdU (Palmer, 1970; Zakharov e t a l . , 1974) and actinomycin-D (Yunis and Chandler, 1977) have been w e l l documented. BrdU has a l s o been shown to s p e c i f i c a l l y e f f e c t t e l o m e r i c decondensation on some chromosomes (Hsu and Somers, 1961). Sutherland et a l . (1980) have a l s o r e c e n t l y d e f i n e d a new c a t e g o r y of f r a g i l e s i t e s t h a t r e q u i r e BrdU f o r ex-p r e s s i o n . BrdU and actinomycin-D may t h e r e f o r e be u s e f u l i n a c c e n t u a t i n g the.frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n t i s s u e c u l t u r e . 2.42 M o d i f i c a t i o n of the f i b r o b l a s t c u l t u r e procedure. At the time of t r a n s f e r to f o l a t e d e f i c i e n t media, f i b r o -b l a s t s from P a t i e n t D were a l s o t r a n s f e r r e d and maintained to chromosome h a r v e s t i n MEM-Ad. To examine the i n f l u e n c e of 5-BrdU and actinomycin-D on the frequency of e x p r e s s i o n , -5 BrdU (10 M) was added 3h h r s . p r i o r to chromosome ha r v e s t to p a r a l l e l c u l t u r e s of M E M T F A f o r two of the c a r r i e r females studied. ( P a t i e n t s B and E ) , and actinomycin-D (Cosmegen, Merck Sharp & Dohme) was added a t a f i n a l c o n c e n t r a t i o n of 5 mcg/ml t o a p a r a l l e l c u l t u r e of MEM-FA f o r P a t i e n t E, 45 min. p r i o r t o chromosome h a r v e s t . Chromosome h a r v e s t f o r c e l l s c u l t u r e d i n MEM-Ad and those i n c o r p o r a t i n g BrdU and actinomycin-D f o l l o w e d the. standard p r o t o c o l o u t l i n e d i n s e c t i o n 2.23. 2.43 M o d i f i c a t i o n of the lymphocyte c u l t u r e procedure. Lymphocyte c u l t u r e s f o r P a t i e n t s B and D were e s t a b l i s h e d i n MEM-Ad. These p a r a l l e l c u l t u r e s f o l l o w e d the g e n e r a l c u l t u r e i n i t i a t i o n and chromosome ha r v e s t p r o t o c o l o u t l i n e d f o r Ml99. To examine the i n f l u e n c e of 5-BrdU and actinomycin-D on the frequency of e x p r e s s i o n Of the f r a g i l e X chromosome i n lymphocytes, m o d i f i c a t i o n s t o the standard lymphocyte t e c h -nique were as f o l l o w s . In experiments u s i n g BrdU, p a r a l l e l lymphocyte c u l t u r e s f o r P a t i e n t s B and E were e s t a b l i s h e d i n MEM-FA and MEM-FA p l u s 5-BrdU (10 _ 5M) added 3 t o 5 h r s . p r i o r to chromosome har v e s t . In experiments with actinomycin-D, lymphocytes from P a t i e n t s B and D were c u l t u r e d i n i t i a l l y i n M199 a t 37°C as u s u a l . A f t e r 72 h r s . i n c u b a t i o n , methotrexate 1 -7 (Lederle) was added t o g i v e a f i n a l c o n c e n t r a t i o n of 10 M. J-Methotrexate i s . a f o l a t e a n t a g o n i s t and a t low c o n c e n t r a t i o n s w i l l e f f e c t i v e l y s y n c h r o n i z e the c e l l p o p u l a t i o n i n c u l t u r e (Yunis, 1976), so t h a t many of the metaphase c e l l s at chromo-some h a r v e s t w i l l be a t the same r e l a t i v e degree of chromo-some condensation. 35 A f t e r l l h h r s . a d d i t i o n a l i n c u b a t i o n , the c e l l s were r e l e a s e d from the methotrexate b l o c k by t r a n s f e r to f r e s h medium and allowed to continue i n c u b a t i n g 5 h r s . to chromosome h a r v e s t . Actinomycin-D was added 4 5 min. p r i o r to chromosome h a r v e s t to g i v e a f i n a l c o n c e n t r a t i o n of 5 mcg/ml. F i f t e e n minutes p r i o r t o chromosome ha r v e s t 0.075 c c . Colcemid (10 mcg/ml) was added t o the c u l t u r e . 2.5 Chromosome s t a i n i n g procedure. On a r o u t i n e b a s i s , s l i d e s t h a t had aged a t l e a s t one week were s t a i n e d w i t h 2.0% a c e t o - o r c e i n (Gibco) according': to the f o l l o w i n g p r o t o c o l . Three drops of s t a i n were put on the s l i d e which was then covered with a cover s l i p . The cover s l i p was allowed t o s i t f o r 10 min. and was then r e -moved. The s l i d e was taken through t h r e e changes of 100% e t h a n o l and then a i r - d r i e d . For permanent mounting, s l i d e s were dipped b r i e f l y i n xylene, and. a cover s l i p was mounted wi t h E u k i t t . G-banding of the chromosome p r e p a r a t i o n s f o l l o w e d the technique o r i g i n a l l y d e s c r i b e d by Seabright (1971) with some m o d i f i c a t i o n . P r i o r to s t a i n i n g , a s l i d e was heated 3 to 5 h r s . a t 60°C. The slide.was t r e a t e d i n t r y p s i n (3cc of r e c o n s t i t u t e d D i f c o t r y p s i n in. 60 mis of 0.9% NaCl) f o r 20 to 3 0 sec. ; the treatment was extended f o r o l d e r s l i d e mater-i a l . The s l i d e was then.passed through 1% C a C ^ (1 min.), 2 washes i n d i s t i l l e d water, and s t a i n e d 45-60 sec. i n Giemsa (2 cc Gurr R66 Giemsa s t a i n , i n 2 0 cc phosphate b u f f e r , 36 pH 6.8, p l u s 30 cc d i s t i l l e d water). The s l i d e was r i n s e d i n d i s t i l l e d water, l i g h t l y b l o t t e d , and allowed t o a i r dry. G-banded s l i d e s were permanently mounted by the method de-s c r i b e d above. 2.6 Methods of o b s e r v a t i o n and. t a b u l a t i o n of data. A l l metaphases scored f e l l w i t h i n a condensation c r i t e r i o n i n which the No. 2 chromosome used as an index measured 9 to 15 ym i n l e n g t h . The s t u d i e s which l e d to the a p p l i c a t i o n of t h i s condensation c r i t e r i o n w i l l be d i s c u s s e d more comprehen-s i v e l y i n the R e s u l t s , s e c t i o n 3.21. These r e v e a l e d c l e a r l y t h a t the f r a g i l e X chromosome was more f r e q u e n t l y d e t e c t a b l e i n metaphases where the chromosomes were not s e v e r e l y c o n t r a c t e d . W e l l spread metaphases t h a t met the e s t a b l i s h e d condensa-t i o n c r i t e r i o n were scored f o r the f r a g i l e X chromosome and recorded as e i t h e r p o s i t i v e or negative and, o c c a s i o n a l l y , as q u e s t i o n a b l e . In t h i s q u e s t i o n a b l e category were those meta-phases i n :'• e i t h e r a s a t e l l i t e - l i k e p i e c e of chromatin was detached and not c l e a r l y a s s o c i a t e d with the long arm of the X chromosome. A l s o i n c l u d e d i n t h i s category were metaphases where th e r e was some ambiguity i n the e x p r e s s i o n o f the s a t e l -l i t e , f o r example chromosome o v e r l a p or the p o s s i b l e i n v o l v e -ment of a D or G group s a t e l l i t e . C e l l s scored i n t h i s ques-t i o n a b l e category were not used i n the d e t e r m i n a t i o n of the f r e -quency of t h e e x p r e s s i o n of the f r a g i l e X chromosome. The f r e -quency of e x p r e s s i o n of the f r a g i l e X chromosome f o r any gi v e n p a t i e n t under a p a r t i c u l a r c u l t u r i n g c o n d i t i o n was expressed as the number o f c e l l s e x p r e s s i n g t h e f r a g i l e X chromosome as a p r o p o r t i o n o f t h e t o t a l number o f c e l l s s c o r e d . S c o r i n g f o r t h e f r a g i l e X chromosome was done d i r e c t l y under t h e m i c r o s c o p e . A l l o b s e r v a t i o n s o f t h e f r a g i l e X chromosome were made on a Z e i s s P h o t o m i c r o s c o p e under o i l -immersion p h a s e - c o n t r a s t a t 1250X. Photographs were t a k e n on Kodak High. C o n t r a s t Copy F i l m 1276. To d e t e r m i n e i f d i f f e r e n c e s between any two f r e q u e n c i e s o f e x p r e s s i o n were s t a t i s t i c a l l y s i g n i f i c a n t , C h i square 2 ( X ) a n a l y s i s f o r independence o f t h e two no m i n a l v a r i a b l e s was employed u s i n g a c o n t i n g e n c y t a b l e . F o r e x p e c t e d v a l u e s o f t h e f r e q u e n c y o f e x p r e s s i o n o f t h e f r a g i l e X chromosome t h a t were low (< 5) and t h e r e f o r e i n a p p r o p r i a t e f o r a n a l y s i s u s i n g t h e C h i square t e s t , F i s h e r ' s E x a c t P r o b a b i l i t y T e s t was a p p l i e d ( S o k a l and R o h l f , 1969). 38 CHAPTER 3 RESULTS 3.1' Demonstration of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s . I n i t i a l o b s e r v a t i o n s of f i b r o b l a s t metaphases u t i l i z i n g the c u l t u r i n g and h a r v e s t i n g procedure developed f o r t h i s study i n d i c a t e d t h a t the f r a g i l e X chromosome was expressed. I t was a l s o e v i d e n t t h a t there was a c o n s i d e r a b l e amount of v a r i a t i o n i n the frequency of e x p r e s s i o n of the f r a g i l e X chromosome, and t h a t t h i s v a r i a b i l i t y was i n p a r t r e l a t e d to the degree of chromosome condensation i n metaphases being scored. T h e r e f o r e , an a n a l y s i s of the frequency of expres-s i o n of the f r a g i l e X chromosome wit h r e l a t i o n to the degree of condensation was conducted. The r e s u l t s of t h i s a n a l y s i s are presented below i n s e c t i o n 3.21, Table VI,and w i l l be d e s c r i b e d i n d e t a i l i n t h a t s e c t i o n . Since comparisons of f r e q u e n c i e s of e x p r e s s i o n be-tween s u b j e c t s were to be made, a s c o r i n g c r i t e r i o n based on t h i s a n a l y s i s was e s t a b l i s h e d and imposed on c e l l s to be scored. 3.11 E x p r e s s i o n i n f i b r o b l a s t s from p a t i e n t s from  Family No. 1. The f r a g i l e X chromosome was demonstrated i n c u l t u r e d s k i n f i b r o b l a s t s from a l l f i v e p a t i e n t s (A-E) from Family No. 1. C e l l l i n e s from three p a t i e n t s (B,C, and D) were t r a n s f e r r e d and maintained to chromosome h a r v e s t i n medium c o n t a i n i n g normal l e v e l s of f o l i c a c i d (MEM) as w e l l 39 as i n media d e f i c i e n t i n f o l i c a c i d (M19 9 and MEM-FA). The f r a g i l e X chromosome was expressed i n f i b r o b l a s t s t r a n s f e r r e d to MEM, a l b e i t a t very low f r e q u e n c i e s (1.2 -, 6.3%). There were s i g n i f i c a n t l y h i g h e r f r e q u e n c i e s of e x p r e s s i o n i n f i b r o -b l a s t s t r a n s f e r r e d to media d e f i c i e n t i n f o l i c a c i d ( T a b l e l l l ) . In Family No. 1, the h i g h e s t f r e q u e n c i e s of e x p r e s s i o n of a f r a g i l e X chromosome (5.9 - 35.0%) were found i n f i b r o -b l a s t s , c u l t u r e d i n media d e f i c i e n t i n f o l i c a c i d , from the two m e n t a l l y r e t a r d e d males,. .Patients C andiD. Frequencies of e x p r e s s i o n f o r the o b l i g a t e and p o t e n t i a l c a r r i e r females, P a t i e n t s A,B, and E, ranged from 9.2 to 19.3% i n the two media d e f i c i e n t i n f o l i c a c i d . P a r a l l e l b l i n d s t u d i e s on f i b r o b l a s t s from two p a t i e n t s (D and E) and normal c o n t r o l s (J and K) matched f o r age, sex, and f i b r o b l a s t passage number were done to determine whether f o l a t e d e p r i v a t i o n c o u l d e l i c i t e x p r e s s i o n of the f r a g i l e X chromosome i n c o n t r o l f i b r o b l a s t s . F o l a t e d e p r i v a -t i o n d i d not l e a d to d e t e c t a b l e f r a g i l e X chromosomes i n e i t h e r male or female c o n t r o l f i b r o b l a s t s . Table I I I d e t a i l s the f r e q u e n c i e s of e x p r e s s i o n of a f r a g i l e X chromosome i n f i b r o b l a s t metaphases f o r P a t i e n t s A-E and normal c o n t r o l s , J and K, f o r medium c o n t a i n i n g nor-mal l e v e l s of f o l i c a c i d (MEM) and media low i n (M199) or e n t i r e l y l a c k i n g (MEM-FA) f o l i c a c i d . The data i n Table I I I i n d i c a t e t h a t , w h i l e there was a s i g n i f i c a n t l y higher f r e -quency of e x p r e s s i o n of the f r a g i l e X chromosome i n the f o l a t e d e f i c i e n t media than i n the medium c o n t a i n i n g normal 40 TABLE I I I Ex p r e s s i o n of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s from p a t i e n t s from Family No. 1 and normal c o n t r o l s : comparison of v a r i o u s growth media. P a t i e n t F i b r o b l a s t Media No. C e l l s Ex- Frequency Passage No. p r e s s i n g Marker i n % No. C e l l s Scored*" A 3 M199 9/93 9.7 O b l i g a t e 3 MEM-FA — C a r r i e r , 74 y r s . 5 MEM-FA • - -B 3 MEM 5/115 4.3 O b l i g a t e 3 Ml 9 9 25/129+ 19. 3 C a r r i e r , 55 y r s . 3 MEM-FA 9/49 18. 3 C 3 MEM 2/173 1.2 A f f e c t e d Male 3 M199 11/179+ 5.9 4 9 y r s . 3 MEM-FA 43/168* 25. 6 5 35/155 22.6 D 3 MEM 9/143 6.3 A f f e c t e d Male 3 Ml 9 9 39/202+ 19.3 31 y r s . 3 MEM-FA 43/168 25. 6 5 19/54* 35. 0 _ 9 M199 19/109 17.4 9 MEM-FA 36/155 23. 2 C o n t r o l J 9 M199 IP/133 <1 Normal Male, 31 yrs. 9 MEM-FA 0/103 <1 . E 3 M199 19/107 17. 8 P o t e n t i a l 3 MEM-FA 11/119* 9.2 C a r r i e r , 25 y r s . C o n t r o l K 3 Ml 9 9 0/65 <1 Normal Female 3 MEM-FA 0/8 5 <1 25 y r s . + a l l metaphases scored f e l l w i t h i n the condensation c r i t e r -i o n d e s c r i b e d i n the t e x t . + s i g n i f i c a n t l y d i f f e r e n t when compared t o the frequency of ex p r e s s i o n i n MEM; B3 : x2]_=12.!74 , p< .001; . C3 : ' x 2l=6.16 , P<.05; D 3: x 2 1 = H - 8 5 ' p<.001., * s i g n i f i c a n t l y d i f f e r e n t when compared to.the frequency of ex p r e s s i o n in.M199.; C 3: x 2 i = 2 4 • 95, p<.001; E>5: x i = 6 . 1 3 , p<.05; E^: x2j=3•55, p<.05. 41 l e v e l s of f o l i c a c i d , there was no c o n s i s t e n c y i n the compar-a t i v e f r e q u e n c i e s of e x p r e s s i o n between.the two f o l a t e d e f i -c i e n t media, M199 and MEM-FA. For P a t i e n t C, the frequency of e x p r e s s i o n of the f r a -g i l e X chromosome was s i g n i f i c a n t l y higher i n MEM-FA than M199; f o r P a t i e n t E the op p o s i t e was t r u e . P a t i e n t B showed no s i g n i f i c a n t d i f f e r e n c e i n the frequency of expres-s i o n between M199 and MEM-FA, while P a t i e n t D showed a s i g -n i f i c a n t l y h i g h e r frequency o f e x p r e s s i o n o f the f r a g i l e X chromosome i n MEM-FA than i n M199 i n f i b r o b l a s t s a t passage 5, but no d i f f e r e n c e i n f r e q u e n c i e s between the two f o l a t e d e f i c i e n t media a t passage 3 and passage 9. The data i n Table I I I a l s o i n d i c a t e - t h a t d u p l i c a t e experiments, u s i n g the same c u l t u r e medium c o n d i t i o n s but l a t e r passage f i b r o -b l a s t c e l l l i n e s on a p a t i e n t , showed no s i g n i f i c a n t change i n the frequency of e x p r e s s i o n between e a r l y and l a t e r pas-sage c e l l s ( P a t i e n t C, MEM-FA; P a t i e n t D, M199 and MEM-FA). 3.12 E x p r e s s i o n i n f i b r o b l a s t s from p a t i e n t s from Family  No. 2. In order t o determine i f the success i n demonstrating the f r a g i l e X chromosome i n f i b r o b l a s t s from i n d i v i d u a l s i n Family No. 1 was due to behavior unique to the f r a g i l e X chromosome i n t h a t f a m i l y , the f i b r o b l a s t method was a p p l i e d to e s t a b l i s h e d c e l l l i n e s from three i n d i v i d u a l s froima second u n r e l a t e d f a m i l y . The f r a g i l e X chromosome was demonstrated i n f i b r o b l a s t metaphases from both of the me n t a l l y r e t a r d e d males s t u d i e d i n Family No. 2. The f r e q u e n c i e s of e x p r e s s i o n of a f r a g i l e X chromosome i n f i b r o b l a s t s c u l t u r e d i n M199 42 f o r P a t i e n t s F, G, and H and C o n t r o l I are d e t a i l e d i n Table IV. The f r e q u e n c i e s observed f o r P a t i e n t s F and G were 17.2% and 14.5% r e s p e c t i v e l y . Thus f a r , a f r a g i l e X chromosome has not been seen i n f i b r o b l a s t p r e p a r a t i o n s from P a t i e n t H, a p o t e n t i a l c a r r i e r female of low normal i n t e l l i -gence who does e x h i b i t the f r a g i l e X chromosome i n lympho-c y t e s ( f o l l o w i n g s e c t i o n ) . Two separate experiments have proved u n s u c c e s s f u l i n demonstrating the f r a g i l e X chromo-some i n f i b r o b l a s t s from t h i s p a t i e n t ; the f i b r o b l a s t c e l l l i n e s e s t a b l i s h e d f o r P a t i e n t H are slow growing, and good chromosome p r e p a r a t i o n s have been d i f f i c u l t to achieve. C u l t u r i n g i n M199 d i d not demonstrate the f r a g i l e X chromosome i n f i b r o b l a s t s from C o n t r o l I. 3.13 Comparison of the frequency of e x p r e s s i o n of  f i b r o b l a s t s and lymphocytes. A l l p a t i e n t s demonstrated the f r a g i l e X chromosome i n p e r i p h e r a l blood lymphocytes. The frequency of e x p r e s s i o n of a f r a g i l e X chromosome i n PHA s t i m u l a t e d lymphocytes c u l t u r e d i n f o l a t e d e f i c i e n t media f o r the e i g h t p a t i e n t s and three normal c o n t r o l s are presented i n Table V. In both f a m i l i e s t h e r e was a d i f f e r e n c e i n the frequen-c i e s of e x p r e s s i o n between d i f f e r e n t m e n t a l l y r e t a r d e d males. In Family No. 1, P a t i e n t C e x h i b i t e d the f r a g i l e X chromosome i n 6.5% of the lymphocyte metaphases analyzed, whereas v a l u e s of 15.9 - 19.3% were obtained f o r P a t i e n t D. In Family No. 2, P a t i e n t F demonstrated a frequency of e x p r e s s i o n of 13% com-pared to 32.6% f o r P a t i e n t G. The frequency of e x p r e s s i o n of 43 TABLE IV E x p r e s s i o n of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s from p a t i e n t s of Family No. 2 and a normal c o n t r o l . P a t i e n t F i b r o b l a s t Media No.. C e l l s Ex- Frequency Passage No. p r e s s i n g Marker i n % No. C e l l s Scored+ F A f f e c t e d Male, 33 y r s . 3 M199 5/29 17. 2 G A f f e c t e d Male, 2 9 y r s . 3 Ml 9 9 8/55 14. 5 H P o t e n t i a l C a r r i e r , 26 y r s . 3 5 M199 M199 — — C o n t r o l I Normal Male 29 y r s . 3 M199 0/50 <1 +" a l l metaphases scored f e l l w i t h i n the condensation c r i t e r i o n d e s c r i b e d i n the t e x t . 44 TABLE V E x p r e s s i o n o f t h e f r a g i l e X chromosome i n p e r i p h e r a l b l o o d lymphocytes from p a t i e n t s o f b o t h f a m i l i e s and normal c o n t r o l s . P a t i e n t Media No. C e l l s Expres- Frequency s i n g Marker i n % No. C e l l s Scored* Family No. 1 A O b l i g a t e C a r r i e r , 74 y r s . M199 MEM-FA 4/3 9 2-3/19 10. 3 11-16 B O b l i g a t e C a r r i e r , 55 y r s . M199 MEM-FA 9/175 4/98 5.1 4.3 C A f f e c t e d Male, 49 y r s . M199 MEM-FA 9/139 6.5 D A f f e c t e d Male, 31 y r s . M199 M199 MEM-FA 21/133 25/130 9/39 15. 9 19. 3 23.0 E P o t e n t i a l C a r r i e r , 25 y r s . M199 MEM-FA 29/89 16/105* 32.2 15. 2 Family No. 2 F A f f e c t e d Male, 33 y r s . M199 6/46 13. 0 G A f f e c t e d Male, 2 9 y r s . M199 14/43 32.6 H P o t e n t i a l C a r r i e r , 26 y r s . M199 5/43 11. 6 C o n t r o l I Normal Male, 29 y r s . M199 0/93 |1 C o n t r o l J Normal Male, 31 y r s . M199 0/102 <1 C o n t r o l K Ml 9 9 0/100 <1 * a l l metaphases scored f e l l w i t h i n the condensation c r i t e r -i o n d e s c r i b e d i n t h e t e x t . o s i g n i f i c a n t l y d i f f e r e n t , re:M199. x =8.14, .001<p<.01. 45 the f r a g i l e X chromosome ranged from 4.3% to 32.2% f o r c a r -r i e r females from both f a m i l i e s . Lymphocytes from f o u r of f i v e p a t i e n t s from Family No. 1 were s u c c e s s f u l l y c u l t u r e d i n MEM-FA and M199. For P a t i e n t s A, B, and D the r e was no d i f f e r e n c e i n frequency of expres-s i o n of the f r a g i l e X chromosome between M199 and MEM-FA, while P a t i e n t E showed a s i g n i f i c a n t l y lower frequency of ex p r e s s i o n of the f r a g i l e X chromosome i n MEM-FA than i n M199 (p'<'. 01). T h i s i s a p a t t e r n s i m i l a r t o t h a t observed i n t h i s p a t i e n t ' s f i b r o b l a s t s . In comparing the f r e q u e n c i e s of e x p r e s s i o n i n lympho-c y t e s and f i b r o b l a s t s f o r a p a t i e n t , there was no d i f f e r e n c e i n the frequency of e x p r e s s i o n i n these two c e l l types f o r three of the f o u r m e n t a l l y r e t a r d e d males s t u d i e d . One a f f e c t e d male, P a t i e n t G, e x h i b i t e d the f r a g i l e X chromo-some a t a lower frequency i n f i b r o b l a s t s than i n lymphocytes. One of the thr e e c a r r i e r females, P a t i e n t B, showed a higher frequency of e x p r e s s i o n i n f i b r o b l a s t s than i n lymphocytes. Another c a r r i e r female, P a t i e n t E, showed a lower frequency of e x p r e s s i o n i n f i b r o b l a s t s than i n lymphocytes. The t h i r d c a r r i e r female showed no d i f f e r e n c e i n frequency of e x p r e s s i o n between f i b r o b l a s t s and lymphocytes. 3.2 F a c t o r s a f f e c t i n g a r e l i a b l e d e t e r m i n a t i o n of the f r e -quency of e x p r e s s i o n . 3.21 Condensation c r i t e r i o n . P r e l i m i n a r y o b s e r v a t i o n s suggested t h a t the e x p r e s s i o n of the f r a g i l e X chromosome was 46 i n p a r t a f u n c t i o n of the degree of condensation of the meta-phase chromosomes; the f r a g i l e X chromosome being more f r e -q u e n t l y d e t e c t a b l e i n metaphases where the chromosomes were not s e v e r e l y c o n t r a c t e d . An a c t u a l study of t h i s was t h e r e -f o r e undertaken and the r e s u l t s are presented i n Table VI. The frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t metaphases where the No. 2.chromosome measured 4 - 7ym i n l e n g t h was 5.0%, whereas at 8 - 1 1 um the f r e -quency of e x p r e s s i o n i n c r e a s e d to 20.5%, and a t 12 - 15ym the frequency of e x p r e s s i o n was even hi g h e r , 37.1%. These d i f f e r e n c e s i n frequency of e x p r e s s i o n w i t h longer chromo-somes were s t a t i s t i c a l l y s i g n i f i c a n t (Table VI). The d e c l i n e i n the t o t a l number of c e l l s scored over the t h r e e l e n g t h ranges of the No. 2 chromosome r e f l e c t s d i f f i c u l t i e s encoun-t e r e d i n s c o r i n g more extended chromosome p r e p a r a t i o n s : s c o r i n g d i f f i c u l t i e s due t o o v e r l a p p i n g chromosomes and poor metaphase spreading. On the b a s i s of these r e s u l t s , i t was decided to r e s t r i c t a n a l y s i s t o metaphases i n which the No. 2 chromosome used as an index measured 9 - 15ym i n l e n g t h . A l l metaphases scored i n t h i s study f e l l w i t h i n t h i s condensation c r i t e r i o n with the e x c e p t i o n of the data shown i n Table V I . 3.22 V a r i a b l e appearance of the f r a g i l e X chromosome. There was a l s o c o n s i d e r a b l e v a r i a t i o n i n the appearance of the s a t e l l i t e on the t e r m i n a l end of the long arm of the X chromo-some independent of the c u l t u r i n g media c o n d i t i o n s imposed on c e l l s . In most cases the s a t e l l i t e appeared as a d i s c r e t e 47 TABLE VI E x p r e s s i o n of the f r a g i l e X chromosome as a f u n c t i o n o f the degree of chromo-some condensation. F i b r o b l a s t s from P a t i e n t D (MEM-FA). Chromosome No. 2 Length '4-7 ym 8-11 ym 12-15 ym T o t a l No. C e l l s E x p r e s s i n g F r a g i l e X 9/180 24/117+ 26/70* 59/367 No. C e l l s Scored Frequency i n % 5.0 20.5 37.1 16.1 + s i g n i f i c a n t l y d i f f e r e n t when compared t o 4-7 condensation c l a s s : x 2 1 = 1 7 . 2 8 , p<.001. y m * s i g n i f i c a n t l y d i f f e r e n t when compared t o 8-11 condensa-t i o n c l a s s : x 2-i =6.18, p<.05. y m 48 b i p a r t i t e p i e c e o f chromatin detached from the d i s t a l end of the long arm, f r e q u e n t l y showing t h r e a d - l i k e connections t o the t e r m i n a l end of the long arm ( F i g . 3, a-e). O c c a s i o n a l l y , the s a t e l l i t e s were t i g h t l y juxtaposed, perhaps o v e r l a p p i n g , and they appeared as a s i n g l e chromatin mass but s t i l l sepa-r a t e from the long arms (3d, 3e). A s i n g l e s a t e l l i t e d chroma-t i d i s a v a r i a t i o n of e x p r e s s i o n t h a t was o c c a s i o n a l l y ob-served ( F i g . 3 f ) . Example g i n F i g u r e 3 shows an a p p a r e n t l y b i - s a t e l l i t e d X chromosome. Another v a r i a t i o n i n the e x p r e s s i o n of the f r a g i l e X chromosome was a complete detachment or d i s s o c i a t i o n of the s a t e l l i t e fragment from the r e s t of the X chromosome ( F i g . 4). Thesedetached s a t e l l i t e s were not i n c l u d e d i n any determina-t i o n of a frequency of e x p r e s s i o n of the f r a g i l e X chromosome because of ambiguity w i t h r e s p e c t to the o r i g i n of the chromatin m a t e r i a l . 3.23 D e f i n i t i v e a s s o c i a t i o n of the f r a g i l e s i t e w i t h  the X chromosome by G-banding. Examples of the v a r i a b l e e x p r e s s i o n of the f r a g i l e X chromosome i n F i g u r e 3 were from metaphase chromosomes- s t a i n e d with a c e t o - o r c e i n . Aceto-o r c e i n r a t h e r t h a n G-banding was used f o r most of the s t u d i e s d e s c r i b e d ( r e s u l t s i n Tables I I I - VII) because i t gave a h i g h e r i n c i d e n c e of metaphases i n which the s a t e l l i t e s c o u l d be c l e a r l y d i s t i n g u i s h e d as d i s t i n c t from the t e r m i n a l end of the long arm of the X chromosome. Examples i n F i g u r e 5, a-e, are t y p i c a l o f the appearance of the f r a g i l e X chromosome a f t e r G-banding. From these 49 a . b. c . d . e. f. g . F I G U R E 3 V a r i a b l e e x p r e s s i o n o f t h e f r a g i l e X chromosome: a c e t o - o r c e i n s t a i n i n g . 50 D e t a c h e d s a t e l l i t e - l i k e p i e c e o f c h r o m a t i n ( a r r o w ) u n a s s o c i a t e d w i t h t h e X c h r o m o s o m e ( a s t e r i s k ) . a . b. c . d . e. FIGURE 5 V a r i a b l e e x p r e s s i o n of the f r a g i l e X chromosome: t r y p s i G-banding. Comparison t o the standard banding p a t t e r n of the X chromosome ( P a r i s Conference, 1971). 52 s t u d i e s , the i d e n t i f i c a t i o n of the X chromosome as d i s t i n c t from the o t h e r human C group cromosomes was d e f i n i t i v e l y e s t a b l i s h e d . In a d d i t i o n , the few w e l l G-banded p r e p a r a t i o n s d i d permit more p r e c i s e assignment of the p o s i t i o n of the f r a g i l e s i t e on the t e r m i n a l end of the long arm. I f the banding p a t t e r n of the f r a g i l e X chromosome i s compared wi t h the normal X chromosome ( F i g . 5, d, e, and i n s e r t ) , i t ap-pears t h a t the f r a g i l e s i t e begins i n the d i s t a l p o r t i o n of band Xq27 or a t the j u n c t i o n between bands q27 and q2 8. 3.24 Autosomal chromosome markers expressed under f o l a t e  d e p r i v a t i o n . T erminal markers were expressed on some of the autosomal chromosomes i n both lymphocytes and f i b r o b l a s t s c u l -t u r e d under f o l a t e d e p r i v a t i o n from a f f e c t e d males and c a r r i e r females. These t e l o m e r i c markers u s u a l l y appeared as an i s o -chromatid gap or break most o f t e n near the t e r m i n a l end of the long arm. They f r e q u e n t l y appeared to i n v o l v e s u b s t a n t i a l l y more chromatin m a t e r i a l than the s a t e l l i t e s on the X chromosome (F i g . 6). These t e l o m e r i c markers were expressed i n a l l of the major chromosome groups w i t h the e x c e p t i o n of the G-group, and o c c u r r e d most f r e q u e n t l y on chromosomes No. 's 1, 3, two of the C-group chromosomes (probably 6 and 12), one of the D-group chromosomes, (probably 13), and e i t h e r 19 or 2 0 of the F group chromosomes. Autosomal t e l o m e r i c markers were a l s o expressed i n normal c o n t r o l s u b j e c t s i n c e l l s c u l t u r e d under f o l a t e > d e p r i v a t i o n . The frequency o f e x p r e s s i o n o f t e l o m e r i c a l l y marked autosomal chromosomes i n a f f e c t e d males and c a r r i e r females 53 A . C . D. C h r o m o s o m e G r o u p F I G U R E 6 A u t o s o m a l t e l o m e r i c m a r k e r s e x p r e s s e d u n d e r f o l a t e d e p r i v a t i o n i n c e l l c u l t u r e . was extremely low when, compared to the e x p r e s s i o n of the f r a g i l e X chromosome i n these, p a t i e n t s (Table V i i ' ) . . While c o n t r o l s u b j e c t s d i d not e x h i b i t the f r a g i l e X chromosome, there, was no s i g n i f i c a n t d i f f e r e n c e between p a t i e n t s , and c o n t r o l s . i n the.frequency of e x p r e s s i o n of t e l o r a e r i c a l l y . marked autosomal.,.chromosomes (Table ' VII) . 3.3 F a c t o r s t h a t i n f l u e n c e the frequency of e x p r e s s i o n of the f r a g i l e X chromosome. 3.31 C u l t u r i n g f a c t o r s : MEM-Ad, BrdU, !and actinomycin-D. R e s u l t s presented i n the f i r s t two s e c t i o n s demonstrated t h a t f o l a t e d e p r i v a t i o n and the degree of condensation of the metaphase chromosomes appeared t o be important i n the expres-s i o n o f the f r a g i l e X chromosome i n c u l t u r e d f i b r o b l a s t s . F u r t h e r experimentation was undertaken to t e s t the mode of a c t i o n of f o l a t e d e p r i v a t i o n and to examine the e f f e c t s of p a r t i c u l a r agents, known t o a f f e c t chromosome condensation, on the frequency of e x p r e s s i o n of the f r a g i l e X chromosome. In an attempt t o t e s t the n o t i o n t h a t both the a v a i l a b i l i t y of thymidylate and DNA r e p l i c a t i o n were at l e a s t i n p a r t r e s p o n s i b l e f o r e x p r e s s i o n of the Xq27-28 f r a g i l e s i t e , c e l l s from two p a t i e n t s were c u l t u r e d i n a medium d e f i c i e n t i n adenine (MEM-Ad). U n f o r t u n a t e l y c u l t u r e s c o u l d not be su s t a i n e d i n t h i s medium, and no metaphases were observed a t chromosome h a r v e s t . T h e r e f o r e t h i s medium was not examined f u r t h e r . TABLE VII Expression of autosomal telomeric markers i n cultured skin f i b r o b l a s t s from patients and normal controls (MEM-FA). P a t i e n t s No. of T e l o m e r i c a l l y Marked Chromosomes (Frequency i n %) Group A Group B Group C Group D Group E Group F Total Xq27-28 Total-Cells Scored C and D 13 7 13 5 3 1 42* 112 589 A f f e c t e d Males (2.2) (1.2) (2.2) (0.8) (0.5) (0.2) (7.1) (19.0) I and J 8 9 3 6 26 1? 235 C o n t r o l Males (3.4) (3.8) (1.3) (2.6) (11.1) (0.4) B and E 23 12 11 5 3 4 58* 124 955 C a r r i e r Females (2.4) (1.3) (1.2) (0.5) (0.3) (0.4) (6.1) (13.0) K 1 2 4 4 1 3 15 193 C o n t r o l Female (0.5) (1. 0) (2.1) (2.1) (0.5) (1.6) (7.8) * not s i g n i f i c a n t l y d i f f e r e n t when compared to matched c o n t r o l . Males: x ^=3.43, p>.05. Females: y^- =Q.11, p>.05. Ln Ln 56 The a d d i t i o n of 5-BrdU and actinomycin-D to f o l a t e d e p r i v e d c u l t u r e media was a l s o i n v e s t i g a t e d s i n c e these agents are both known, to i n t e r f e r e , w i t h the chromosome con-d e n s a t i o n p r o c e s s . To examine the e f f e c t s of 5-BrdU on the frequency of e x p r e s s i o n of the f r a g i l e X chromosome, (1 0 " % ) BrdU was added to f o l a t e d e p r i v e d media 3 - 5 h r s . p r i o r t o chromosome h a r v e s t i n p a r a l l e l c u l t u r e s of lympho-c y t e s from P a t i e n t s B and E and a p a r a l l e l c u l t u r e of f i b r o -b l a s t s from P a t i e n t B. While the a d d i t i o n of BrdU d i d r e s u l t i n more extended chromosomes, w i t h p a r t i c u l a r chromosome r e g i o n s showing pronounced decondensation e f f e c t s , namely the paracentromeric heterochromatic r e g i o n s on chromosomes 1, 9, and 16, and the sho r t arm s a t e l l i t e s on the D and G group chromosomes, the a d d i t i o n of BrdU d i d not produce a s i g n i f i -c ant change i n the frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n e i t h e r of the two p a t i e n t s s t u d i e d (Table V I I I ) . The i n c o r p o r a t i o n of actinomycin-D (5mcg/ml) i n t o the c u l t u r e medium was examined u s i n g p e r i p h e r a l blood lympho-c y t e s from two p a t i e n t s and s k i n f i b r o b l a s t s from one p a t i e n t from Family No. 1 (Table V I I I ) . Actinomycin-D d i d produce more extended chromosome p r e p a r a t i o n s , and, while i t gave the im-p r e s s i o n of s i g n i f i c a n t l y improving the frequency of marker e x p r e s s i o n f o r P a t i e n t B, the frequency of chromosome breaks and gaps i n c e l l s grown i n f o l a t e d e p r i v e d media supplemented w i t h actinomycin-D was so e x t e n s i v e t h a t a r e l i a b l e determina-t i o n of the frequency of e x p r e s s i o n was not p o s s i b l e . An ^ example of t h i s breakage phenomenon i s g i v e n i n F i g u r e 7. TABLE VIII E f f e c t s of 5-BrdU and actinomycin-D on the frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n lympho-c y t e s and f i b r o b l a s t s . P a t i e n t T i s s u e Media No. C e l l s E x pressing Marker No. C e l l s Scored Frequency i n % B O b l i g a t e C a r r i e r , 55 y r s . Lymphocytes M199 s t d . Ml 99 + ac t i n - D MEM-FA MEM-FA + BrdU 9/175* ?9/65 4/93* 9/105 5.1 13. 9? 4.3 8.6 F i b r o b l a s t s MEM-FA MEM-FA + BrdU 9/49* 26/104 18.3 25.0 D A f f e c t e d Male, 31 y r s . Lymphocytes M199 s t d . M199 + acti n - D 21/133* ?2/13 15. 9 13. 3? E P o t e n t i a l C a r r i e r , 25 y r s . Lymphocytes MEM-FA MEM-FA + BrdU 16/105* 22/99 15. 2 22.2 F i b r o b l a s t s MEM-FA MEM-FA + acti n - D 11/119* ?3/15 9.2 20.0? p r e v i o u s l y r e p o r t e d f r e q u e n c i e s (Tables I I I and V). FIGURE 7 Extended chromosomes and gaps(G) and breaks(B) produced by actinomycin-D i n combination w i t h f o l a t e d e p r i v a t i o n i n c e l l c u l t u r e . The chromosome a t the upper l e f t may be the f r a g i l e X. 59 3.32 Hypotonic e f f e c t s at chromosome h a r v e s t . R e s u l t s presented i n the f i r s t s e c t i o n demonstrated the f r a g i l e X chromosome i n s k i n f i b r o b l a s t s from three p a t i e n t s (B, C, and D) c u l t u r e d i n medium c o n t a i n i n g normal, l e v e l s of f o l i c a c i d (MEM). Because t h i s r e s u l t 1 suggested t h a t aspects of the f i b r o b l a s t method other than f o l a t e d e p r i v a t i o n were i n f l u -e ncing f r a g i l e s i t e e x p r e s s i o n , experiments were undertaken t o determine the e f f e c t s of d i f f e r e n t hypotonic treatments at chromosome h a r v e s t on the frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t c u l t u r e . P a r a l l e l c u l -t u r e s of c e l l s from an a f f e c t e d male (P a t i e n t D) were esta b -l i s h e d and maintained i n accordance w i t h the p r o t o c o l de-s c r i b e d above i n media d e f i c i e n t i n f o l i c a c i d (M199 and MEM-FA) and medium c o n t a i n i n g normal l e v e l s of f o l i c a c i d (MEM). At chromosome har v e s t , f i b r o b l a s t s c u l t u r e d i n each of the d i f f e r e n t media were f u r t h e r s u b d i v i d e d and t r e a t e d s e p a r a t e l y w i t h one of th r e e hypotonics, 1% N a C i t r a t e , 0.075 M KC1, or d i s t i l l e d H 20. Under c u l t u r i n g c o n d i t i o n s of f o l a t e d e p r i v a t i o n , the e x p r e s s i o n of the f r a g i l e X chromosome was s i g n i f i c a n t l y h i g h e r w i t h a hypotonic treatment i n 1% N a C i t r a t e than when the hypotonic treatment employed was 0.075 M KC1 (Table IX)!/ The use of d i s t i l l e d water as hypotonic w i t h t h i s c u l t u r i n g method proved u n s a t i s f a c t o r y under a l l media c o n d i t i o n s be-cause i t r e s u l t e d i n a very low, v i r t u a l l y u n s corable, m i t o t i c index. As p r e v i o u s l y noted, the frequency of e x p r e s s i o n of a f r a g i l e X chromosome i n media c o n t a i n i n g normal l e v e l s of TABLE IX Effects of d i f f e r e n t hypotonic treatments at chromosome harvest on the frequency of expression of the f r a g i l e X chromosome i n fib r o b l a s t s . P a t i e n t Media Hypotonic No. C e l l s E x p r e s s i n g Marker No. C e l l s Scored Frequency i n % D A f f e c t e d Male MEM MEM . 1% .NaCitrate 0.07 5M KC1 4/93+ 0/89 4.3 <1 Ml 9 9 M199 1% N a C i t r a t e 0.075 M KC1 15/79* 2/99 19.0 2.0 MEM-FA MEM-FA 1% N a C i t r a t e 0.075 M KC1 46/202* 7/239 22.9 2.9 + not s i g n i f i c a n t l y d i f f e r e n t when compared t o a hypotonic treatment i n 0.075 M KC1 f o r the same medium. F i s h e r ' s Exact T e s t : p=0.12. * s i g n i f i c a n t l y d i f f e r e n t when compared to a hypotonic treatment, i n 0.075 M KC1 f o r the same medium. M199: x 2 1=7.32, p<.01. MEM-FA: x 2 n=20.39, p<.001. f o l i c acid (MEM) with a hypotonic treatment i n NaCitrate was about 5% for t h i s patient. Using a KC1 hypotonic, the frequency of expression was even lower, less than 1%. This difference i n frequency was not s t a t i s t i c a l l y s i g n i f i c a n t (p=0.12) . It was apparent that chromosome preparations treated with NaCitrate as a hypotonic were d i f f e r e n t i n appearance from those treated with KC1. NaCitrate appeared to sub-s t a n t i a l l y interact with, or disrupt, the st r u c t u r a l i n t e -g r i t y of the chromatin. This was p a r t i c u l a r l y evident along the periphery of the chromosomes where the chromatin appeared loose and poorly organized. With a hypotonic treatment i n KC1 the i n t e g r i t y of the chromatin along the periphery of the chromosome appeared more compact and undisrupted (Fig. 8). 62 F I G U R E 8 E f f e c t s o f d i f f e r e n t h y p o t o n i c t r e a t m e n t s a t c h r o m o s o m e h a r v e s t o n c h r o m o s o m e m o r p h o l o g y . CHAPTER 4 DISCUSSION 4,1 F r a g i l e X e x p r e s s i o n i n f i b r o b l a s t s from m e n t a l l y r e -tarded males and t h e i r female r e l a t i v e s . Using the f i b r o b l a s t method developed f o r t h i s study, i t was p o s s i b l e to e l i c i t the e x p r e s s i o n of the f r a g i l e X chromosome.in t h i s t i s s u e type. The f r a g i l e X chromosome was demonstrated i n s k i n f i b r o b l a s t c e l l l i n e s e s t a b l i s h e d from a l l f i v e p a t i e n t s from Family No. 1. The h i g h e s t f r e -quencies of e x p r e s s i o n obtained were f o r the two m e n t a l l y r e t a r d e d males from t h i s f a m i l y , a t f r e q u e n c i e s i n excess of 25% i n f i b r o b l a s t s c u l t u r e d i n medium without f o l i c a c i d . A h i g h e r frequency of e x p r e s s i o n i n m e n t a l l y r e t a r d e d males i n f i b r o b l a s t s i s c o n s i s t e n t w i t h the g e n e r a l l y higher f r e -quencies of e x p r e s s i o n r e p o r t e d i n lymphocytes f o r m e n t a l l y r e t a r d e d males (see Table I, s e c t i o n 1.13). The data on f r e q u e n c i e s of marker e x p r e s s i o n i n c a r r i e r females f o r f i b r o b l a s t s (as w e l l as lymphocytes) show n e i t h e r a c o r r e l a t i o n w i t h female age nor a l a c k of e x p r e s s i o n i n o l d e r o b l i g a t e c a r r i e r s . For example, i n Family No. 1, a 25 y r . o l d p o t e n t i a l c a r r i e r demonstrated the f r a g i l e X i n 17.8% of f i b r o b l a s t c e l l s c u l t u r e d i n M199, a 55 y r . o l d o b l i g a t e c a r r i e r , showed a frequency of 19.3% i n f i b r o b l a s t s i n M199, and a 74 y r . o l d o b l i g a t e c a r r i e r demonstrated i t i n 9.7% of f i b r o b l a s t c e l l s c u l t u r e d i n M199.. These r e s u l t s are not c o n s i s t e n t w i t h r e s u l t s i n lymphocytes f o r f r a g i l e X 64 e x p r e s s i o n i n c a r r i e r females r e c e n t l y r e p o r t e d by Sutherland (1979c), i n d i c a t i n g t h a t the frequency of e x p r e s s i o n i n c a r -r i e r females decreases w i t h age and t h a t f r a g i l e X chromosome e x p r e s s i o n i s f r e q u e n t l y d i f f i c u l t i f not i m p o s s i b l e t o e l i c i t i n females over the age of 3 0 y r s . The study of l a t e r passage f i b r o b l a s t s from two of the p a t i e n t s from Family No, 1 ( P a t i e n t s C and D) d i d not show a s i g n i f i c a n t d i f f e r e n c e i n the frequency of e x p r e s s i o n from t h a t obtained i n e a r l i e r passage c e l l s . T h i s suggests t h a t the method i s r e p r o d u c i b l e . Furthermore, the s u c c e s s f u l a p p l i c a t i o n of the f i b r o b l a s t method to e s t a b l i s h e d c e l l l i n e s from two p a t i e n t s from a second u n r e l a t e d f a m i l y , i n an a t r : tempt t o assess the r e l i a b i l i t y of the f i b r o b l a s t method, sug-g e s t s t h a t the method should be a p p l i c a b l e t o other f a m i l i e s i n which the d i s o r d e r i s .segregating. In comparing the f r e q u e n c i e s of e x p r e s s i o n i n f i b r o b l a s t s and lymphocytes f o r a l l p a t i e n t s s t u d i e d , who demonstrated the f r a g i l e X i n both t i s s u e s , no g e n e r a l statement can be made about the g r e a t e r e f f e c t i v e n e s s of e i t h e r f i b r o b l a s t c u l t u r e or lymphocyte c u l t u r e i n e l i c i t i n g the e x p r e s s i o n of the f r a -g i l e X chromosome. A l l e i g h t p a t i e n t s s t u d i e d showed the f r a -g i l e X i n lymphocytes c u l t u r e d i n f o l a t e d e f i c i e n t media over a wide range of frequencies(4.3 to 32.6%). Seven of the e i g h t p a t i e n t s showed the f r a g i l e X chromosome i n f i b r o b l a s t s c u l -t u r e d i n f o l a t e d e f i c i e n t media a t comparable f r e q u e n c i e s (5.9 t o 35.01.)., While f o u r of the e i g h t p a t i e n t s s t u d i e d showed no d i f f e r e n c e i n the frequency of e x p r e s s i o n between f i b r o b l a s t s and lymphocytes, two p a t i e n t s showed a s i g n i f i -c a n t l y lower frequency o f , e x p r e s s i o n i n f i b r o b l a s t s , and one p a t i e n t showed a higher frequency of e x p r e s s i o n i n f i b r o -b l a s t s . Thus, there appeared t o be no c o n s i s t e n t p a t t e r n of e i t h e r t i s s u e being more e f f e c t i v e i n demonstrating the f r a -g i l e X chromosome when the comparison was p o s s i b l e . The demonstration of the f r a g i l e X chromosome i n c u l -t u r e d s k i n f i b r o b l a s t s , h a s not s o l v e d the d i a g n o s t i c dilemma of demonstrating the marker chromosome r e l i a b l y i n c a r r i e r f e -males. None o f the females s t u d i e d i n t h i s p r o j e c t f a i l e d t o show the marker i n lymphocytes, so i t remains t o be seen whe-the r f i b r o b l a s t s w i l l be s u c c e s s f u l i n demonstrating the f r a -g i l e X chromosome i n female p a t i e n t s who do not express i t i n lymphocytes.. However, those females who do e x h i b i t the marker i n even a s m a l l percentage of c e l l s i n e i t h e r f i b r o b l a s t s o r lymphocytes can be c o n s i d e r e d c a r r i e r s . On the b a s i s of data c o l l e c t e d i n t h i s study, two p o t e n t i a l c a r r i e r females, Patients E and H, can now be c o n s i d e r e d heterozygous c a r r i e r s of the d i s o r d e r . 4.2 E x p r e s s i o n of the f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s : c u l t u r i n g and. chromosome h a r v e s t i n g f a c t o r s . While i t i s d i f f i c u l t t o say p r e c i s e l y why p r e v i o u s i n v e s t i g a t o r s have not been s u c c e s s f u l i n demonstrating the f r a g i l e X chromosome-.in f i b r o b l a s t s , the f a c t i s t h a t few a t -tempts have been made to study f i b r o b l a s t chromosomes from pa-t i e n t s f r o m . f a m i l i e s i n which the d i s o r d e r i s known to be se g r e g a t i n g (Lubs, 1969; Sutherland, 1979b; Turner e t a l . , 1980). 66 Probably the most important.. features,,.which, d i f f e r e n t i a t e the method developed i n this, study from e a r l i e r attempts to de-monstrate the f r a g i l e X chromosome i n fibroblasts, are the transfer of c e l l s t o : f o l a t e d e f i c i e n t media aft e r routine maintenance i n medium containing normal lev e l s of f o l i c acid and a hypotonic treatment at chromosome harvest i n NaCitrate rather than KCi. 4.21 Folate deprivation i n c e l l culture. The feature of the f i b r o b l a s t method which appeared to have the greatest influence on the frequency of expression of the f r a g i l e X chromosome was fola t e deprivation i n c e l l culture. While i t was possible to e l i c i t marker expression i n c e l l s cultured i n MEM, there was a s i g n i f i c a n t l y higher frequency of expression in c e l l s transferred to either M199 or MEM-FA. The f r a g i l e X chromosome was expressed at comparable frequencies i n f i b r o -blasts cultured i n M199, which i s low i n f o l i c acid, and MEM-FA, a complete medium from which f o l i c acid was deleted. The lack of any consistent difference between M19 9 and MEM-FA in demonstrating the f r a g i l e X chromosome i n either lymphocyte or f i b r o b l a s t culture p a r a l l e l s r e s u l t s reported by Sutherland (1979a) on the effectiveness of fola t e d e f i c i e n t media i n e l i c i t i n g the expression of the f r a g i l e X chromosome i n lympho-cyte culture. A very recent report of the expression of the f r a g i l e X chromosome i n f i b r o b l a s t s , from a mentally retarded male patient, cultured i n medium containing normal lev e l s of fo l a t e plus the addition of the f o l i c acid antagonist, methotrexate during the l a s t 48 hrs. of culture i s 67 c o n s i s t e n t w i t h r e s u l t s r e p o r t e d i n t h i s study on the e f f e c t -i v e n e s s of f o l a t e d e p r i v a t i o n f o r e l i c i t i n g f r a g i l e X ex-p r e s s i o n i n c u l t u r e d s k i n f i b r o b l a s t s (Jennings et a l . , 1979). While r e s u l t s from, t h i s study do support the hypothesis t h a t f o l a t e . d e p r i v a t i o n i s indeed of s i g n i f i c a n c e i n f r a g i l e s i t e expression., they do not c l a r i f y Sutherland's (1979a) pro-p o s a l f o r the mode of i t s a c t i o n , which suggested t h a t f o l a t e d e p r i v a t i o n leads t o reduced p y r i m i d i n e b i o s y n t h e s i s , s p e c i -f i c a l l y , r e d u c i n g the l e v e l of thymidylate a v a i l a b l e f o r de novo DNA s y n t h e s i s . Experiments u s i n g MEM-Ad i n c e l l c u l -t u r e were not u s e f u l i n t e s t i n g t h i s h y p o thesis s i n c e the medium would not support c e l l growth. However, experiments which were undertaken t o examine the chromosome decondensation e f f e c t s of BrdU d i d o f f e r . i n d i r e c t evidence i n support of Sutherland's, p r o p o s a l . In i t s c a p a c i t y to a c t as a thymidine analog, BrdU, when added to the c u l t u r e medium l a t e i n the r e p l i c a t i v e c y c l e , had no e f f e c t on the frequency of f r a g i l e X e x p r e s s i o n . T h i s r e s u l t . was c o n s i s t e n t w i t h Sutherland's (1979a) f i n d i n g s t h a t the a d d i t i o n of thymidine or BrdU r e -s u l t e d i n a lower frequency of f r a g i l e X e x p r e s s i o n only i f added p r i o r t o or. d u r i n g , the e a r l i e r stages of- DNA r e p l i c a -t i o n . The l a t e i n c o r p o r a t i o n of BrdU had l i t t l e or no e f f e c t on the frequency of. e x p r e s s i o n of the f r a g i l e X chromosome. 4.22 Hypotonic e f f e c t s a t chromosome, h a r v e s t . The e x p r e s s i o n of. the f r a g i l e X chromosome i n MEM, c o n t a i n i n g nor-mal l e v e l s , of f o l a t e , suggested t h a t f e a t u r e s of the f i b r o b l a s t method, other than f o l a t e d e p r i v a t i o n i n c e l l c u l t u r e , were 6 8 important f o r the e x p r e s s i o n of the f r a g i l e s i t e . The com-p a r i s o n of d i f f e r e n t hypotonic treatments of f i b r o b l a s t s c u l t u r e d i n media d e f i c i e n t i n f o l i c a c i d r e v e a l e d t h a t N a C i t r a t e p r o v i d e d a s i g n i f i c a n t l y h i g h e r frequency of ex-p r e s s i o n than KC1. That the data d i d not. show a s i g n i f i c a n t d i f f e r e n c e between, these two hypotonic treatments f o r f i b r o -b l a s t s c u l t u r e d i n medium c o n t a i n i n g normal l e v e l s of f o l a t e i s more l i k e l y due t o the.very low f r e q u e n c i e s of marker e x p r e s s i o n u s i n g t h i s medium than to a l a c k of e f f e c t of the N a C i t r a t e i n the absence of f o l a t e d e f i c i e n c y . However, the data do show the h i g h e s t f r e q u e n c i e s of f r a g i l e X e x p r e s s i o n when these two f e a t u r e s are combined: f o l a t e de-p r i v e d c u l t u r i n g c o n d i t i o n s and hypotonic treatment a t chromo-some ha r v e s t w i t h N a C i t r a t e . These r e s u l t s , do not e x p l a i n , the d i s c r e p a n c y between the frequencies, of marker e x p r e s s i o n i n f i b r o b l a s t s and i n lymphocytes.using KC1 as a hypotonic. For the same c u l t u r i n g c o n d i t i o n s , lymphocytes t h a t were treated, w i t h KC1 a t h a r v e s t showed f r e q u e n c i e s of e x p r e s s i o n comparable t o those o b t a i n e d i n f i b r o b l a s t s u s i n g N a C i t r a t e as a hypotonic. The e f f e c t s of d i f f e r e n t hypotonic treatments a t chromo-some ha r v e s t have not been e x t e n s i v e l y s t u d i e d by other i n v e s t i g a t o r s of h e r i t a b l e f r a g i l e s i t e s . The a n a l y s i s of h a r v e s t methods, when r e p o r t e d i n s t u d i e s of the f r a g i l e X chromosome by p r e v i o u s i n v e s t i g a t o r s , has i n d i c a t e d t h a t a l l p r i o r s t u d i e s used KC1. The o n l y i n d i c a t i o n i n the l i t e r a t u r e t h a t h e r i t a b l e . f r a g i l e ' .site's -are-.sensitive -to-idiffer-ent' >hypo-t o n i c treatments i s i n a r e p o r t by Buhler e t a l . (197 0) which showed t h a t the 2ql2 f r a g i l e s i t e i s expressed a t a h i g h e r frequency when the lymphocyte chromosome p r e p a r a t i o n s are t r e a t e d w i t h a hypotonic of N a C i t r a t e r a t h e r than KC1. A l -though the e f f e c t s of a NaCitrate. hypotonic treatment on lymphocytes was. not examined here, i t s a p p l i c a t i o n might r e s u l t i n a h i g h e r frequency of e x p r e s s i o n of the Xq27-28 f r a g i l e s i t e i n t h a t t i s s u e and may be p a r t i c u l a r l y u s e f u l i n determining the c a r r i e r s t a t u s of p o t e n t i a l female h e t e r o -zygotes . One c o u l d s p e c u l a t e t h a t the mechanism u n d e r l y i n g the improved frequency of e x p r e s s i o n w i t h N a C i t r a t e i n v o l v e s the c h e l a t i o n of d i v a l e n t c a t i o n s by the c i t r a t e moiety of the hypotonic. In d i s c u s s i n g the nature and o r i g i n of achromatic l e s i o n s i n chromosomes, Chaudhuri (1972) suggested t h a t , i n a d d i t i o n t o d i s t u r b a n c e s caused i n the DNA i t s e l f , a l t e r e d c o n c e n t r a t i o n s of d i v a l e n t c a t i o n s can produce decondensation i r r e g u l a r i t i e s . E a r l i e r work by Kabat (1967) and S t e f f e n s e n (1961) a l s o s t r e s s e d the importance of d i v a l e n t metal i o n s i n the o r g a n i z a t i o n and s t r u c t u r a l maintenance of the n u c l e o p r o t e i n complex of the chromosome. Golomb and Bahr (1974) showed t h a t the c h e l a t i o n of d i v a l e n t c a t i o n s , +2 p a r t i c u l a r l y Ca , can i n t e r f e r e w i t h the normal condensation +2 process, and, f u r t h e r , t h a t excess Ca can l e a d t o severe chromosome c o n t r a c t i o n under some circumstances. The marked d i f f e r e n c e between the appearance of chromo-somes f o l l o w i n g treatment in. KC1 and N a C i t r a t e suggests t h a t 70 N a C i t r a t e i n t e r a c t s more v i g o r o u s l y w i t h the chromatin. In g e n e r a l the chromosomes appeared l e s s c o n t r a c t e d , and the i n t e g r i t y o f the chromatin appeared more r e l a x e d . With KC1, the p e r i p h e r i e s of the chromosomes appeared more d i s t i n c t , which seems to i n d i c a t e t h a t KC1 i n t e r a c t s , l e s s d i r e c t l y w i t h the chromatin m a t e r i a l than N a C i t r a t e . S t u d i e s t o t e s t the e f f e c t s o f o t h e r c h e l a t i n g agents, such as EDTA or EGTA, d u r i n g c e l l c u l t u r e or chromosome h a r v e s t , might be u s e f u l i n a c c e n t u a t i n g the frequency of e x p r e s s i o n of the Xq27-28 f r a g i l e s i t e . .These s t u d i e s may a l s o be u s e f u l f o r g a i n i n g a b e t t e r understanding o f the r o l e o f d i v a l e n t c a t i o n s i n f r a g i l e s i t e e x p r e s s i o n . Both f o l a t e d e p r i v a t i o n and NaCitrate. as< a hypotonic have been shown t o i n f l u e n c e the frequency o f e x p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t c u l t u r e . The f i n d i n g of a h i g h e r frequency of expression.when both o f these c o n d i -t i o n s were imposed on c e l l s suggests, t h a t the two f e a t u r e s of the method may i n t e r a c t w i t h one another. On the b a s i s of proposed modes of a c t i o n f o r f o l a t e d e p r i v a t i o n and N a C i t r a t e hypotonic, i t c o u l d be.hypothesized, t h a t by causing abnormal r e p l i c a t i v e behavior, or other d i s t u r b a n c e s i n the DNA, f o l a t e d e p r i v a t i o n may make the Xq27-28 f r a g i l e s i t e p a r t i c u l a r l y s e n s i t i v e t o agents t h a t i n t e r r u p t the condensation process or e f f e c t i v e l y decondense the chromatin a t the f r a g i l e s i t e and so e l i c i t f r a g i l e s i t e e x p r e s s i o n . 4.3 R e l i a b l e d e t e r m i n a t i o n of the frequency of e x p r e s s i o n of the f r a g i l e X chromosome i n f i b r o b l a s t s . 4.31 V a r i a b l e appearance of the f r a g i l e X chromosome. R e l i a b l e d e t e r m i n a t i o n of a frequency of marker e x p r e s s i o n r e q u i r e d r e c o g n i t i o n of the v a r i a t i o n i n appearance of the expressed f r a g i l e s i t e . For the purposes of t h i s study, s l i d e p r e p a r a t i o n s were s t a i n e d w i t h a c e t o - o r c e i n on a r o u t i n e basis.. Although, u n l i k e G-banding, o r c e i n does not permit an a b s o l u t e d i s t i n c t i o n between the X chromosome and the human C group autosomal chromosomes of comparable s i z e , i t was v i s u a l l y more e f f e c t i v e than t r y p s i n G-banding i n r e v e a l i n g e x p r e s s i o n of the f r a g i l e s i t e . The b e t t e r r e s o l u t i o n of the f r a g i l e s i t e , seen w i t h a c e t o - o r c e i n , might be a t t r i b u t e d to i t s higher a f f i n i t y as a s t a i n f o r the n u c l e i c a c i d component of chromatin r a t h e r than the a s s o c i a t e d p r o t e i n components of the chromosome (Pearse, 1961). Giemsa i s thought to s t a i n p r o t e i n com-ponents a s s o c i a t e d , with p a r t i c u l a r DNA r e g i o n s of the chromo-some (Chuprevich e t . a l . , 1973; Brown e t a l . , 1975; Sumner, 1976) which may make i t l e s s u s e f u l f o r d i s t i n g u i s h i n g d i s -c o n t i n u i t i e s i n chromosome s t r u c t u r e . The v a r i a t i o n i n e x p r e s s i o n of the f r a g i l e X chromosome r e p o r t e d i n the r e s u l t s ( F i g . 3) w i t h a c e t o - o r c e i n s t a i n i n g i s c o n s i s t e n t w i t h the observations, of other i n v e s t i g a t o r s (Lubs, 1969; Giraud e t a l . , 1976; Sutherland, 1979b; T u r l e a u e t a l . , 1979). Most o f t e n , a t e r m i n a l s a t e l l i t e was expressed on both chromatids, g i v i n g a b i p a r t i t e appearance to the 72 s a t e l l i t e chromatin as a whole. O c c a s i o n a l l y the t e r m i n a l s a t e l l i t e appeared as s i n g l e chromatin mass w i t h the i n d i v i d u -a l chromatid s a t e l l i t e s p o s s i b l y fused or o v e r l a p p i n g ( F i g . 3 d, e) , and, more r a r e l y , o n l y one of the chromatids expressed a ( s i n g l e ) s a t e l l i t e ( F i g . 3 f ) . A double s a t e l l i t e d X chromosome ( F i g . 3 g) was a morpho-l o g i c a l v a r i a t i o n observed o n l y three times i n the course of t h i s study. T h i s phenomenon has been r e p o r t e d by other i n -v e s t i g a t o r s a t s i m i l a r l y low f r e q u e n c i e s and has been a t t r i -buted to s e l e c t i v e e n d o r e d u p l i c a t i o n of the s a t e l l i t e chroma-t i n (Lubs, 1969; Giraud et a l . , 1976; Sutherland, 1979b; Tu r l e a u , 1979). Analogous d u p l i c a t i o n s of the long arm segment of the chromosome arm d i s t a l t o the f r a g i l e s i t e have been r e p o r t e d f o r the 2ql2 and 10q2 3 f r a g i l e s i t e s (Lejeune, 1966, 1968; F r a c c a r o e t a l . , 1972; Ferguson-Smith, 1973; Noel e t a l . , 1977). For the 2ql2 and 10q23 f r a g i l e s i t e s , d u p l i -c a t i o n of the long arm segment d i s t a l to the f r a g i l e s i t e has g e n e r a l l y been thought t o be the r e s u l t of m i t o t i c non-dys-j u n c t i o n and, subsequent r e p l i c a t i o n of. the chromatids d i s t a l to the f r a g i l e s i t e r a t h e r than s e l e c t i v e e n d o r e d u p l i c a t i o n of the long arm f r a g m e n t — S e l e c t i v e e n d o r e d u p l i c a t i o n has, how-ever, been.the p r e f e r r e d i n t e r p r e t a t i o n of m u l t i p l e c o p i e s of the chromosome, fragment d i s t a l t o the f r a g i l e s i t e when the p i e c e of chromatin i s s m a l l . Magenis et a l . (197 0) r e p o r t e d the presence of m u l t i p l e c o p i e s (2 - 12) of the s m a l l long arm fragment d i s t a l t o the f r a g i l e s i t e a t 16q22 i n lympho-c y t e p r e p a r a t i o n s from s e v e r a l p a t i e n t s . Well extended chromosome p r e p a r a t i o n s p e r m i t t e d b e t t e r d e f i n i t i o n of the f r a g i l e s i t e . However, on the b a s i s o f G-banding, i t was not p o s s i b l e to p r e c i s e l y a s s i g n the f r a g i l s i t e t o e i t h e r t h e . G - p o s i t i v e band a t Xq27 or the G-negative band a t Xq28.. iMMost o f t e n , the f r a g i l e s i t e appeared a t the j u n c t i o n of the two bands. As p r e v i o u s l y s t a t e d ( s e c t i o n 1.22), p r e c i s e assignment of the f r a g i l e s i t e i s to some ex-t e n t a f u n c t i o n of the banding method employed, and, because of r e p o r t e d d i s c r e p a n c i e s i n the l o c a t i o n of the f r a g i l e s i t e on the X chromosome, i t i s r e p o r t e d as Xq27-28 (Sutherland, 1979b). Well extended chromosome p r e p a r a t i o n s s t a i n e d w i t h aceto o r c e i n f r e q u e n t l y showed f i n e threads t r a v e r s i n g the f r a g i l e s i t e , c o n necting the d i s p l a c e d : s a t e l l i t e with the r e s t of the long arm of the chromosome ( F i g . 3 d , e ) . T h i s suggests t h a t the chromatin or DNA i s continuous a c r o s s the f r a g i l e s i t e , and t h a t the f r a g i l e s i t e i s a r e g i o n of l o c a l i z e d decondensation r a t h e r than an a c t u a l i n t e r r u p t i o n or break of the chromosome. That the f r a g i l e s i t e has a tendency to shear or break o f f , however, i s suggested from the observa-t i o n of detached s a t e l l i t e s i n some chromosome p r e p a r a t i o n s . Since detached s a t e l l i t e s were not i n c l u d e d i n a determina-ti o n , of the frequency of e x p r e s s i o n of the f r a g i l e X, they may be r e s p o n s i b l e f o r the unexpectedly low f r e q u e n c i e s of ex p r e s s i o n of the f r a g i l e X o c c a s i o n a l l y observed i n some p a t i e n t s (e.g., detached s a t e l l i t e s occurred. 1% times more f r e q u e n t l y than d i d i n t a c t . f r a g i l e X chromosomes f o r P a t i e n t 74 E i n f i b r o b l a s t s c u l t u r e d i n MEM-FA, T a b l e I I I ) . 4.32 C o n d e n s a t i o n . The r e s u l t s showed t h a t the f r e -quency o f e x p r e s s i o n o f a f r a g i l e X chromosome i n c u l t u r e d s k i n f i b r o b l a s t s was s i g n i f i c a n t l y h i g h e r i n more extended chromosome p r e p a r a t i o n s . I n o r d e r t o o b t a i n a more r e l i a b l e d e t e r m i n a t i o n o f t h e f r e q u e n c y o f e x p r e s s i o n , a c r i t e r i o n was e s t a b l i s h e d which metaphases had t o meet b e f o r e b e i n g s c o r e d f o r t h e f r a g i l e X chromosome. The i m p o s i t i o n o f t h i s s c o r i n g c r i t e r i o n may have con-t r i b u t e d t o h i g h e r f r e q u e n c i e s o f e x p r e s s i o n o f the f r a g i l e X chromosome r e p o r t e d h e r e . Lymphocyte s t u d i e s u s i n g s i m i l a r c u l t u r i n g c o n d i t i o n s conducted by Jacobs e t a l . (1980) o f a l l f i v e p a t i e n t s from F a m i l y No. 1 demonstrated t h e marker chromo-some o n l y i n P a t i e n t s C ( 5 % ) , D (1 3 . 2 % ) , and E (13.5%). Other lymphocyte s t u d i e s by Dunn e t a l . (1980), o f t h r e e p a t i e n t s from F a m i l y No. 1 (A, B, and E ) , demonstrated t h e p r e s e n c e o f the marker chromosome o n l y i n P a t i e n t E ( 1 9 % ) . [ P a t i e n t s A and B, r e p o r t e d h e r e , e x p r e s s e d the marker i n 10.3% and 5.1% o f c e l l s s c o r e d r e s p e c t i v e l y . F o r P a t i e n t C, t h e f r e q u e n c y o f e x p r e s s i o n was 6.5%, f o r P a t i e n t D, 15.9% and 19.3%, and f o r P a t i e n t E, 32.2% o f t h e c e l l s s c o r e d (Table V).] A f i b r o b l a s t s l i d e p r e p a r a t i o n o f P a t i e n t D, t h e f r e q u e n c y o f marker e x p r e s s i o n f o r which was d e t e r m i n e d here t o be 19.3%, was s e n t t o two o t h e r l a b o r a t o r i e s f o r s t u d y . W h i l e t h e s e e x a m i n a t i o n s found t h e marker t o be present., i t was e x p r e s s e d a t a lower f r e q u e n c y ( S u t h e r l a n d , p e r s o n a l communication; J a c o b s and G l o v e r , p e r s o n a l communication). These i n t e r -75 l a b o r a t o r y d i s c r e p a n c i e s i n determining the frequency of mar-ker e x p r e s s i o n are d i f f i c u l t to assess, but may i n p a r t be due to v a r i a t i o n i n what i s c o n s i d e r e d an a c c e p t a b l e degree of chromosome condensation i n s c o r i n g f o r the marker chromosomei Higher f r e q u e n c i e s of e x p r e s s i o n of the f r a g i l e X chromosome i n l e s s condensed chromosome p r e p a r a t i o n s has a l s o been noted by other i n v e s t i g a t o r s (Fox and Gerrard, p e r s o n a l communica-k t i o n ) . Looking a t more extended chromosomes permits b e t t e r d e f i n i t i o n of the s e p a r a t i o n between the s a t e l l i t e and the r e s t of the long arm of the X chromosome. T h i s reduces the s c o r i n g ambiguity encountered w i t h more condensed chromo-some p r e p a r a t i o n s , where the d i s t i n c t i o n between the t e r m i n a l end of the long arm and the s a t e l l i t e i s obscured. Whether e x p r e s s i o n of the Xq27-28 f r a g i l e s i t e i s a c t u a l l y the r e s u l t of an abnormality i n chromosome conden-s a t i o n , perhaps due to p r e m i t o t i c a l t e r a t i o n s i n the chroma-t i n of the f r a g i l e s i t e , or whether the higher frequency of e x p r e s s i o n i n l e s s condensed chromosome p r e p a r a t i o n s i s simply Ibhe-result of b e t t e r r e s o l u t i o n of the f r a g i l e s i t e , i s not known. The f a c t t h a t the f r a g i l e s i t e was o n l y expressed i n 2 A l l o b s e r v a t i o n s here of the f r a g i l e X were done u s i n g phase c o n t r a s t microscopy. While i t has not been determined to what extent t h i s may be important i n the observed frequency of mar-ker e x p r e s s i o n , other s t u d i e s have employed, on a r e g u l a r b a s i s , b r i g h t f i e l d i l l u m i n a t i o n f o r o b s e r v i n g the f r a g i l e X. T h i s t e c h n i c a l d i f f e r e n c e may be, i n p a r t r e s p o n s i b l e f o r d i s c r e -p a n cies between the. f r e q u e n c i e s of e x p r e s s i o n r e p o r t e d by d i f -f e r e n t l a b o r a t o r i e s . . S l i d e s examined here from a l a b o r a t o r y which uses, b r i g h t f i e l d i l l u m i n a t i o n were s t a i n e d to a g r e a t e r i n t e n s i t y than the s l i d e s examined f o r t h i s r e p o r t . I t i s p o s s i b l e t h a t o v e r s t a i n i n g may mask the e x p r e s s i o n of the fragile s i t e i n some metaphases and thereby c o n t r i b u t e to a lower f r e -quency of e x p r e s s i o n of the f r a g i l e X chromosome. 76 a f r a c t i o n of w e l l extended chromosome p r e p a r a t i o n s (Table VI) suggests t h a t condensation e f f e c t s may be secondary to i r r e g u l a r i t i e s i n the chromatin e s t a b l i s h e d e a r l i e r i n the c e l l c y c l e , i . e . , d u r i n g DNA r e p l i c a t i o n . As mentioned above, decondensation a t the 2ql2 f r a g i l e s i t e has been shown t o p a r a l l e l l a t e or f a u l t y r e p l i c a t i v e behavior of the chromatin a t the f r a g i l e s i t e (Bfihler e t a l . , 1970) . In a u t o r a d i o g r a p h i c s t u d i e s of the 2ql2 f r a g i l e s i t e , F r a c c a r o e t a l . (1972) showed t h a t w h i l e the abnormal chromosome No. 2 was synchronous i n r e p l i c a t i o n w i t h i t s homologue i n both arms, 3 the i n c o r p o r a t i o n of H thymidine l a b e l i n t o the f r a g i l e s i t e was not observed. They i n t e r p r e t e d t h i s f i n d i n g as i n d i c a t i n g t h a t e i t h e r the DNA i n the f r a g i l e s i t e d i d not r e p l i c a t e , or there was not enough DNA i n the d i s c o n t i n u i t y i t s e l f to be r e v e a l e d by. autoradiography. Methods employed to improve the frequency of e x p r e s s i o n by i n h i b i t i n g chromosome condensation j u s t p r i o r to the time of h a r v e s t , t h a t i s , the use of 5-BrdU and actinomycin-D, f a i l e d t o s i g n i f i c a n t l y a l t e r the frequency. While both actinomycin-D and.BrdU produced more extended chromosomes, n e i t h e r had any e f f e c t on the frequency of e x p r e s s i o n . Actinomycin-D, i n com-b i n a t i o n with f o l a t e d e p r i v a t i o n , produced such e x t e n s i v e chromosome breakage t h a t i t was f e l t t h a t any s i g n i f i c a n t improvement i n the frequency of e x p r e s s i o n would have to be i n t e r p r e t e d c a u t i o u s l y . BrdU was e f f e c t i v e i n e l i c i t i n g s p e c i f i c decondensation e f f e c t s i n some r e g i o n s of some chromosome. In i t s c a p a c i t y 77 to a c t as a thymidine analog, BrdU produced s i g n i f i c a n t de-condensation e f f e c t s i n the A T - r i c h paracentromeric r e g i o n s of heterochromatin on chromosomes 1, 9, and 16. The 2° c o n s t r i c t i o n r e g i o n s on the D and G group chromosomes a l s o showed s p e c i f i c decondensation e f f e c t s . . These o b s e r v a t i o n s were c o n s i s t e n t w i t h e a r l i e r r e p o r t s of chromosomal seg-ments p a r t i c u l a r l y s e n s i t i v e t o BrdU i n c o r p o r a t i o n and de-condensation (Palmer, 1970; Zakharov e t a l . , 1974). The e f f e c t s of BrdU on the 2° c o n s t r i c t i o n of the D and G. group chromosomes, and the unresponsiveness of the Xq27-28 f r a g i l e s i t e , to BrdU, may be taken as i n d i r e c t evidence i n support of the b e l i e f t h a t the. Xq27-28 f r a g i l e s i t e i s not t h e . r e s u l t = of a ...translocation of chromatin from^one of these I a c r o c e n t r i c chromosomes. 4.33 Autosomal t e l o m e r i c markers. The e x p r e s s i o n of t e r m i n a l markers on some of the autosomal chromosomes under f o l a t e d e p r i v e d c u l t u r i n g c o n d i t i o n s has l e d some i n v e s t i g a -t o r s t o c a u t i o n a g a i n s t assuming a b s o l u t e d i a g n o s t i c s p e c i f i t y f o r the f r a g i l e X chromosome (Jennings e t a l . , 1980). In t h i s study, autosomal markers were expressed i n both p a t i e n t s and c o n t r o l s a t low f r e q u e n c i e s , whereas the f r a g i l e X chromosome was expressed o n l y i n p a t i e n t s a t r e l a t i v e l y much higher f r e -quencies, suggesting t h a t the e x p r e s s i o n of autosomal markers does not r e p r e s e n t a t h r e a t to the d i a g n o s t i c u s e f u l n e s s of the f r a g i l e X chromosome. E x p r e s s i o n of t e r m i n a l l y marked autosomes: i n chromosome p r e p a r a t i o n s from lymphocytes c u l t u r e d i n M19 9 i s w e l l docu-78 merited by o t h e r i n v e s t i g a t o r s s t u d y i n g the f r a g i l e X chromo-some (Howard-Peebles, p e r s o n a l communication; Dunn e t a l . , 1980; Jacobs e t a l . , 1980; J e n n i n g s e t a l . , 1980; M a r t i n e t a l . , 1980; Soudek e t a l . , 1980). W i t h some c o n s i s t e n c y , t e r -m i n a l markers have been noted on chromosomes No. 1, 3, 6, a s m a l l C group chromosome ( p r o b a b l y 1 2 ) , 13, and 19. The median i n t e r s t i t i a l . f r a g i l e s i t e a t 16q22 has a l s o been r e p o r t e d by s e v e r a l w o r k e r s . Because t h e s e autosomal markers a r e expressed i n b o t h normal c o n t r o l s and p a t i e n t s , t h e y have g e n e r a l l y been a c c e p t e d as a p e c u l i a r i t y o f c e l l s c u l t u r e d i n M199. The f r e q u e n c y o f e x p r e s s i o n o f t h e s e autosomal markers i n p a t i e n t s e x p r e s s i n g t h e f r a g i l e X chromosome has a l s o been r e p o r t e d t o be s i g n i f i c a n t l y lower than the f r e q u e n c y o f ex-p r e s s i o n o f t h e f r a g i l e X chromosome (Jacobs, e t a l . , 1980; J e n n i n g s e t a l . , 1 9 8 0 ) . 3 W h i l e some workers have suggested t h a t components o r d e f i c i e n c i e s i n M199 o t h e r t h a n i t s low f o l a t e c o n t e n t may be r e s p o n s i b l e f o r t h e general chromosome i n s t a b i l i t y r e s u l t i n g f r o m . c u l t u r e i n M199 ( E m e r i t e t a l . , 1974; Keck and E m e r i t , 1979), t h e d e m o n s t r a t i o n here of ana-3 l n f i b r o b l a s t s c u l t u r e d i n MEM-FA from P a t i e n t D (Table V I I ) , t h e f r e q u e n c y o f e x p r e s s i o n o f t e l o m e r i c a l l y marked autosomes i n t o t a l was 4 2 , i n 289 c e l l s s c o r e d o r about 7%. I n t h e same p a t i e n t , the f r e q u e n c y o f e x p r e s s i o n o f the f r a g i l e X was 112 i n 589 c e l l s o r about 19%. A more a c c u r a t e a p p r e c i a t i o n of t h e s i g n i f i c a n c e o f t e l o m e r i c a l l y marked autosomes.can be a c h i e v e d i f t h e number o f t h e s e markers i n any one autosomal group i s c o n s i d e r e d s e p a r a t e l y . F o r example, a l l o f t h e autosomal mar-kers, i n t h e A group chromosomes f o r P a t i e n t D (Table: VII) were a c t u a l l y c o n f i n e d t o chromosomes 1 and 3. As t h e r e a r e two r e p r e s e n t a t i v e s o f each o f t h e s e chromosomes f o r e v e r y X i n t h i s male p a t i e n t , t h e f r e q u e n c y o f A group marked autosomes f o r t h i s p a t i e n t would be 13/589 x 4 o r about 0.5%. logous. t e l o m e r i c changes on chromosomes c u l t u r e d i n MEM-FA suggests t h a t the r e l a t i v e l y low l e v e l o f f o l i c a c i d i n M199 i s r e s p o n s i b l e f o r the d e s t a b i l i z i n g e f f e c t s of t h i s medium. Analogous e f f e c t s on chromosomes i n v i v o have been r e -po r t e d i n p a t i e n t s s u f f e r i n g from v i t a m i n d e f i c i e n c y , f o l a t e d e f i c i e n c y , or both (Heath, 1966). Recently, C6t£ and Papadakou-Lagoyanni (1979) r e p o r t e d t h a t an i n v i v o f o l a t e d e f i c i e n c y a s s o c i a t e d w i t h B-thalassaemia may be i n v o l v e d i n the i n c r e a s e d i n c i d e n c e of spontaneous chromosome breakage seen i n some p a t i e n t s . Autosomal t e l o m e r i c markers appear t o r e p r e s e n t a n o n s p e c i f i c chromosomal response t o e i t h e r i n v i v o or i n v i t r o f o l a t e d e f i c i e n c y . 4.4 Co n c l u s i o n s and f u t u r e p r o s p e c t s . T h i s study has demonstrated t h a t the f r a g i l e X chromosome i s expressed i n c u l t u r e d s k i n f i b r o b l a s t s . C u l t u r i n g c o n d i -t i o n s of f o l a t e d e p r i v a t i o n , which had p r e v i o u s l y been r e p o r t e d to i n f l u e n c e the e x p r e s s i o n of the f r a g i l e X chromosome i n p e r i p h e r a l blood lymphocytes, have a s i m i l a r e f f e c t on the e x p r e s s i o n of the marker chromosome i n c u l t u r e d f i b r o b l a s t s . A ' N a C i t r a t e hypotonic treatment a t chromosome h a r v e s t was Shown to. i n f l u e n c e the frequency of e x p r e s s i o n of the f r a g i l e s i t e i n f i b r o b l a s t t i s s u e , and the degree of chromosome conden s a t i o n was shown to be important i n the de t e r m i n a t i o n of a f r e quency o f e x p r e s s i o n of the f r a g i l e X chromosome. While the use of f i b r o b l a s t s does not a t t h i s time r e p r e -80 s e n t an improvement over lymphocytes f o r d e t e r m i n i n g whether an a f f e c t e d male p o s s e s s e s t h e f r a g i l e X chromosome o r whether o r n o t a female i s a c a r r i e r , a t e c h n i q u e f o r d e m o n s t r a t i n g t h e marker i n f i b r o b l a s t s does make t h e p r o s p e c t o f demonstra-t i n g i t i n amniocytes more: l i k e l y . A n t e n a t a l d i a g n o s i s o f f a m i l i a l . s e x - l i n k e d m e n t a l r e t a r d a t i o n a s s o c i a t e d w i t h t h e f r a g i l e . X chromosome w i l l depend on whether o r n o t the f r a -g i l e X chromosome can be r e l i a b l y d e t e c t e d i n c u l t u r e d amnio-t i c f l u i d c e l l s . . S i n c e i t has been. shown t h a t a m n i o c y t e s behave s i m i l a r l y t o p o p u l a t i o n s o f f i b r o b l a s t s i n terms o f chromosome a n a l y s i s (Bryant e t a l , 1978), t h e development of a method t o e l i c i t t h e e x p r e s s i o n o f t h e f r a g i l e X chromosome i n f i b r o b l a s t s i s an i m p o r t a n t s t e p toward t h a t g o a l . I n o r d e r f o r t h e f i b r o b l a s t t e c h n i q u e t o be c l i n i c a l l y v a l u a b l e , f u r t h e r a t t e m p t s s h o u l d be made to. i n c r e a s e t h e f r e q u e n c y o f e x p r e s s i o n o f t h e marker. A l s o , i n o r d e r t h a t i t be c o n s i d e r e d r e l i a b l e f o r d i a g n o s t i c p u r p o s e s , t h i s t e c h -n i q u e s h o u l d be a p p l i e d t o f u r t h e r - - s u b j e c t s i n ' o t h e r " f a m i l i e s i n w hich t h e . d i s o r d e r i s s e g r e g a t i n g . F i b r o b l a s t s do p r o v i d e a u s e f u l l a b o r a t o r y c e l l p o p u l a -t i o n t o s t u d y c e l l c u l t u r i n g and chromosome h a r v e s t i n g c o n d i -t i o n s t h a t i n f l u e n c e t h e e x p r e s s i o n o f t h e Xq27-28 f r a g i l e s i t e . F u r t h e r a p p l i c a t i o n o f h i s t o c h e m i c a l and immunofluor-:: e s c e n t chromosome banding t e c h n i q u e s may p e r m i t b e t t e r d e f i n i -t i o n o f t h e DNA base c o m p o s i t i o n o f the f r a g i l e r e g i o n and add t o o ur g e n e r a l u n d e r s t a n d i n g o f t h e chromosome s t r u c t u r a l i r -r e g u l a r i t i e s o f a f r a g i l e s i t e . These t e c h n i q u e s may a l s o 81 c o n t r i b u t e t o a c l e a r e r u n d e r s t a n d i n g o f t h e r e l a t i o n s h i p t h a t e x i s t s between t h e f r a g i l e X chromosome, i t s c u l t u r i n g pe-c u l i a r i t i e s , and the m e n t a l r e t a r d a t i o n a s s o c i a t e d w i t h t h i s chromosome anomaly. 82 REFERENCES Bahr, G.F. (1977) Chromosomes and chromatin s t r u c t u r e . Chromosomes i n B i o l o g y and Medicine: M o l e c u l a r S t r u c t u r e  of Human Chromosomes. E d i t . J . Yunis, Academic Pr e s s , New York, pp. 143-203. Bowen, P., Biederman, B. and Swallow, K.A. (1978) The X - l i n k e d syndrome of macroorchidism and mental r e t a r d a t i o n : f u r t h e r o b s e r v a t i o n s . Am. J . Med. Genet. 2: 409-414. Brown, R.L., Pathak, S. and Hsu, T.C. (1975) The p o s s i b l e r o l e of h i s t o n e s i n the mechanism of chromosomal G banding. Science 189: 1090-1091. Bryant, E.M., Crouch, E., B o r n s t e i n , P., M a r t i n , G.M., Johnston, P. and Hoehn, H. (197 8) R e g u l a t i o n of growth and gene a c t i v i t y i n e u p l o i d h y b r i d s between neonatal f i b r o -b l a s t s and e p i t h e l o i d amniotic f l u i d c e l l s . Am. J . Hum. Genet. 30: 392-405. Buhler, E.M., Luchsinger, U., Buhler, U.K., Mehes, K. and S t a d l e r , G.R.. (1970) Non-condensation of one segment of a chromosome No. 2 i n a male wi t h an otherwise normal karyo-type (and severe hypospadias). Humangenetik j): 97-104. Cantu, J.M., S c a g l i a , H.E., Go n z a l e z - D i d d i , M., Hernandez-J d u r e g u i , P., Morato, T., Moreno, M.E., Giner, J . , A l c a n t a r , A., H e r r e r a , D. and P e r e z - P a l a c i o s , G. (1978) I n h e r i t e d c o n g e n i t a l normofunctional t e s t i c u l a r h y p e r p l a s i a and mental d e f i c i e n c y . Hum. Genet. 4_1: 331-339. Chaudhuri, J.P. (1972) On the o r i g i n and nature of achromatic l e s i o n s . Chromosomes Today 3_: 147-151. Chuprevich, T.W., Meisner, L.F. and Inhorn, S.L. (1973) Mechanism of t r y p s i n chromosome banding. Lancet _1: 484. Cote, G.B. and Papadakou-Lagoyanni, S. (1977) B-thalassaemia: i n c r e a s e d chromosomal anomalies i n lymphocyte c u l t u r e s . J . Med. Genet. 16: 52-55. Deroover, J . , Fryns, J.P., P a r l o i r , C. and Vanden Berghe, H. (1977) X - l i n k e d r e c e s s i v e l y i n h e r i t e d n o n - s p e c i f i c mental r e t a r d a t i o n : r e p o r t of a l a r g e f a m i l y . Ann. Genet. 2_0: 263-268. Dunn, H.G.. , Renpenning, H. , Gerrard, J.W. , M i l l e r , J.R., Tabata, T. and F e d e r o f f , S. (1963) Mental r e t a r d a t i o n as a s e x - l i n k e d d e f e c t . Am. J . Mental D e f i c i e n c y 67: 827-848. 83 Dunn, H.G., Kalousek, D.K., D i l l , F . J . and Herbst, D.S. (1980) Study i n p r o g r e s s , p e r s o n a l communication. Emerit, I, Levy, A. and Housset, E. (1974) Breakage f a c t o r i n systemic s c l e r o s i s and p r o t e c t o r e f f e c t s of L - c y s t e i n e . Humangenetik 2_5: 221-226. \ Erbe, R.W. (1975) Inborn e r r o r s of f o l a t e metabolism. N. Eng. J . Med. 293: 753-758 & 807-811. Erbe, R.W. (1979) Genetic aspects of f o l a t e metabolism. Advances i n Human Gene t i c s 9, ed. H a r r i s , H. and Hir s c h h o r n , K., pp. 293-253. Ferguson-Smith, M.A. (1973) I n h e r i t e d c o n s t r i c t i o n f r a g i l i t y of chromosome 2. Ann. Genet. 1_6: 29-34. F r a c c a r o , M., K a j j e r , K. and L i n d s t e h , J . (1960) Somatic chromosome. complement i n c o n t i n u o u s l y c u l t u r e d c e l l s of two i n d i v i d u a l s w i t h gonadal dysgenesis. Ann. Hum. Genet. 24:.. 45-61. F r a c c a r o , M., L i n d s t e n , J . , T i e p o l o , L. and R i c c i , N. (1972) I n s t a b i l i t y of the p a r a c e n t r i c r e g i o n and s e l e c t i v e r e d u p l i -c a t i o n of chromosome 2 i n man. Chromosomes Today 3: 138-146. Giraud, F., Ayme, S., M a t t i e , J.F. and M a t t e i , M.G. (1976) C o n s t i t u t i o n a l chromosome breakage. Hum. Genet. 3_4: 125-136. Golomb, H.M. and Bahr, G.F. (1974) E l e c t r o n microscopy of human i n t e r p h a s e n u c l e i : d e t e r m i n a t i o n of t o t a l dry mass and DNA-packing r a t i o . Chromosoma 4j6: 233-245. Hamerton, J.L. (1971) Human C y t o g e n e t i c s . V o l I, General C y t o g e n e t i c s . Academic P r e s s , New York, pp 297-303. Harvey, J . , Judge, C. and Wiener, S. (1977) F a m i l i a l X - l i n k e d mental r e t a r d a t i o n with an X chromosome abnormality. J . Med. Genet. 14: 46-50. Heath, C.W. (1966) C y t o g e n e t i c o b s e r v a t i o n s i n v i t a m i n B, 2 and f o l a t e d e f i c i e n c y . Blood, 27_: 800-815. Hecht, F. and Kaiser-McCaw, B. (1979) The importance of being a f r a g i l e s i t e . Am. J . Hum. Genet. 3JL: 223-225. Herbst, D.S. (1980) A study of X - l i n k e d mental r e t a r d a t i o n i n B r i t i s h Columbia. PhD T h e s i s , U n i v e r s i t y of B r i t i s h Colum-b i a , Vancouver. Howard-Peebles., P.N. and Howell, W.M. (1979) Mental r e t a r d a t i o n , marker X chromosomes, and s i l v e r s t a i n i n g (NORs). Cytogenet. C e l l Genet. 23: 277-278. 84 Howard-Peebles, P.N. and Pryor, J.C. (197 9) Marker X chromo-somes and t i s s u e - c u l t u r e c o n d i t i o n s . N. Engl.. J . Med. 301: 166. Howard-Peebles, P.N., Stoddard, G.R. andMims, M.G. (1979) F a m i l i a l X - l i n k e d mental r e t a r d a t i o n , v e r b a l d i s a b i l i t y , and marker X chromosomes. /Am. J . Hum. Genet. 31: 214-222. Howard-Peebles., P.N. and Stoddard, G.R. (1979) X - l i n k e d mental r e t a r d a t i o n w i t h macrofirchidism and marker X chromosomes. Hum. Genet. 5_0: 247-251. Howard-Peebles, P.N. and Stoddard, G.R. (1980) F a m i l i a l X-l i n k e d mental r e t a r d a t i o n w i t h a marker X chromosome and i t s r e l a t i o n s h i p to. macrottrchidism. C l i n i c a l Genet. 17: 125-128. Hsu, T.C. and Somers, C.E. (1961) E f f e c t of 5-bromodeoxyuri-dine on mammalian chromosomes. PNAS (USA) £7: 396-403. Jacobs, P.A., Mayer, M.,-Rudak, E., Ge r r a r d , J . , Ives, E . J . , Shokier, M.H., H a l l , J . Jennings, M. and Hoehn, H. (1979) More on marker X chromosomes., mental r e t a r d a t i o n and maerottrchidism. N. E n g l . J . Med. 30 0: 737-738,. Jacobs, P.A., Glover, T. , Mayer, M., Fox, P., Ge r r a r d , J . , Dunn, H. and Herbst, D.S. (198 0) X - l i n k e d mental r e t a r d a -t i o n : a study of seven* f a m i l i e s , submitted, Amer. J . Med. Genet. Jennings, M.T., C a n f i e l d , J.E., Bryant, E.M., H a l l , J.G. and Hoehn, H. (1979) S p e c i f i c i t y o f marker chromosomes i n Renpenning syndrome: q u a n t i t a t i v e r a t h e r than q u a l i t a t i v e ? A b s t r a c t , Amer, J . Hum. Genet. 3_1: 98A. Jennings, M., H a l l , J.G. and Hoehn, H. (1980) S i g n i f i c a n c e of phenotypic and chromosomal a b n o r m a l i t i e s i n X - l i n k e d mental r e t a r d a t i o n ( M a r t i n - B e l l or Renpenning syndrome), submitted, Amer. J . Med. Genet. Rabat, D. (1967) F i b r o u s complexes of d e o x y r i b o n u c l e i c a c i d w i t h c e r t a i n g l o b u l a r p r o t e i n s : r o l e of d i v a l e n t metal ions i n the o r g a n i z a t i o n of the n u c l e o p r o t e i n s t r u c t u r e . B i o c h e m i s t r y 6: 3443-3458. K a j i i , T., Ohama, K., Niikawa, N, F e r r i e r , A. and Avira c h e n , S. (197 3) Banding a n a l y s i s of abnormal karyotypes i n spontane-ous a b o r t i o n . Am. J . Hum. Genet. 2_5: 539-547. Keck, M. and Emerit, I. (1979) The i n f l u e n c e of c u l t u r e medium composition on the i n c i d e n c e of chromosomal breakage. Hum. Genet. 50: 277-283. 85 Kondra, P.M. and Ray, M. (1978) A n a l y s i s of DNA r e p l i c a t i o n p a t t e r n s of human f i b r o b l a s t chromosomes: the r e p l i c a t i o n map. Hum. Genet. 43: 139.-149. Lehrke, R.G. (1974) X-Linked Mental R e t a r d a t i o n and V e r b a l D i s a b i l i t y . B i r t h D e f e c t s : O r i g i n a l A r t i c l e S e r i e s , N a t i o n a l Foundation March of Dimes 10(1), E d i t . D. Bergsma, I n t e r -c o n t i n e n t a l M e d i c a l Book C o r p o r a t i o n , New York. LeJeune, J . , Berger, R. and Rethore, M.-O. (1966) Sur 1'endo-r e d u p l i c a t i o n s e l e c t i v e de c e r t a i n s segments du genome. C R . Acad. S c i . (Paris) 263: 1880-1882. Lejeune, J . , D u t r l l l a u x , B., Lafourcade, J . , Berger, R. Abonyi, D. and Rethore, M.-O. (1968) E n d o r e d u p l i c a t i o n s e l e c t i v e du bras du chromosome 2 chez une femme et sa f i l l e . C R . Acad. S c i . (Paris) 266: 24-26. Lubs, H.A. (1969) Marker X chromosome. Am. J . Hum. Genet. 21: 231-244. Magenis, R.E., Hecht, F. , Lovrien,. E.W. (1970) H e r i t a b l e f r a g i l e s i t e on chromosome 16: probable l o c a l i z a t i o n of h a p t o g l o b i n l o c u s i n man. Science 170: 85-87. M a r t i n , J.P. and B e l l , J . (1943) A pedigree of mental d e f e c t showing s e x - l i n k a g e . J . Neurol. P s y c h i a t r y 154-157. M a r t i n , R.H.,; L i n , C . C , Mathies, B.J. a?nd Lowry, R.B. (1980) X - l i n k e d mental r e t a r d a t i o n w i t h macroorchidism and marker X chromosomes, submitted, Amer. J . Med. Genet. McKusick, V.A. (1978).. Mendelian. I n h e r i t a n c e i n Man. 5th ed. Johns Hopkins P r e s s , B a l t i m o r e . Moorhead, P.S., Nowell, P.C., Mellman., W.J., B a t i p p s , D.M. and Hungerford, D.A. (1960) Chromosome p r e p a r a t i o n s of l e u k o c y t e s c u l t u r e d from human p e r i p h e r a l blood.. Exp. C e l l Res. 2_0: 613-616. Neu, R.L., Ortega, C . C , Barg, G.A., P i n t o , W., Gardner, L . I . , Howell, W.M. and Denton, T.E. (1976) I n c l u s i o n of s a t e l l i t e s i n an 18/21 t r a n s l o c a t i o n chromosome shown by ammoniacal-s i l v e r s t a i n i n g (sat-banding) i n a case of p a r t i a l trisomy 18. J . Med. Genet. 13: 520-522. Noel, B., Quack, B., Mottet, J . , Nantois, Y. and D u t r i l l a u x , B. (1977) S e l e c t i v e e n d o r e d u p l i c a t i o n or branched chromosome? Exp. C e l l Res. 104: 423-426. O l i v e r , N., Francke, U. and T a y l o r , K.M. (1978) S i l v e r s t a i n i n g s t u d i e s of the s h o r t arm v a r i a n t of human chromosome 17. Hum. Genet. 42: 79-82. O p i t z , J.M., Sega l , A.T., Klove, H., Mathews,. C. and Lemke, R (1965) X - l i n k e d mental r e t a r d a t i o n , a study of a l a r g e k i n dred with 20 a f f e c t e d members. J . P e d i a t r i c s . 67_: 713-714. Palmer, C.G. (1970) 5-bromodeoxyuridine induced c o n s t r i c t i o n s i n human chromosomes. Can... J... Genet. C y t o l . 12_: 816-830. P a r i s Conference (1971) S t a n d a r d i z a t i o n i n human c y t o g e n e t i c s B i r t h D e f e c t s : O r i g i n a l A r t i c l e S e r i e s , V I I I (7), 1972. The N a t i o n a l Foundation, New York. Pearse, E. (1961) H i s t o c h e m i s t r y : T h e o r e t i c a l and A p p l i e d . 2nd e d i t . , J.& A. C h u r c h i l l , L t d . London, pp. 165-166, 204-205. Reeves, B.R. and Lawler, S.D. (1970) P r e f e r e n t i a l breakage of s e n s i t i v e r e g i o n s of human chromosomes. Humangenetik 8: 295-301. Renpenning, H., Gerrard, J.W., Z a l e s k i , W.A. and Tabata, T. (1962) F a m i l i a l s e x - l i n k e d mental r e t a r d a t i o n . Canad. Med. Ass. J . 87: 954-956. Ruvalcaba, R.H.A.. , Myhre, S.A., Roosen-Runge, E.C. and Beckwith, J.B. (1977). X - l i n k e d mental d e f i c i e n c y megalo-t e s t e s syndrome. JAMA 238: 1646-1650. Savage, J.R. (1977) Assignment of a b e r r a t i o n b r e a k p o i n t s i n banded chromosomes.. Nature 27 0:513-514. Schmid, W. and V i s c h e r , D. (1969) Spontaneous f r a g i l i t y of an abnormally wide secondary c o n s t r i c t i o n r e g i o n on human chromosome No. 9. Humangenetik 7_: 22-27. Se a b r i g h t , M. (1971) A r a p i d banding technique f o r human chromosomes. Lancet 2_: 971-972. Sokal, R.R. and Rohlf, F . J . (1969) Biometry, the P r i n c i p l e s  and P r a c t i c e of S t a t i s t i c s i n B i o l o g i c a l Research. W.H. Freeman and Co., San F r a n c i s c o , pp. 589-599. Sj^rensen, K. , N i e l s e n , J . , Holm, V. and Haahr, J . (1979) F r a g i l e s i t e long arm chromosome 16. Hum. Genet. £8_: 131-134. Soudek, D., P a r t i n g t o n , M.W. and McGregor, T. (1980) X - l i n k e d r e c e s s i v e mental r e t a r d a t i o n : c y t o g e n e t i c s . Canad. C o l l . Med. Genet. Annual Meeting, Scarborough, O n t a r i o , January. S t e f f e n s o n , D.M. (1961) Chromosome s t r u c t u r e with s p e c i a l r e f e r e n c e t o the r o l e of metal i o n s . I n t . Rev. C y t o l . 12: 163-197. S t u b b l e f i e l d , E. (1964) DNA s y n t h e s i s and chromosomal morpho-logy o f Chinese hamster c e l l s c u l t u r e d i n media c o n t a i n i n g N-^deacetyl-N-methylcolchicine (Colcemid) . C y t o g e n e t i c s of  C e l l s i n C u l t u r e . E d i t . R.J. H a r r i s , Academic Pr e s s , New York, pp. 223-225. Sumner, A.T. (1976) The r o l e of p r o t e i n s and dyes i n chromo-some banding. Chromosomes Today 5_: 201-209. Sutherland, G.R. (1977) F r a g i l e s i t e s on human chromosomes: demonstration, o f t h e i r dependence on the type of t i s s u e c u l t u r e medium. Science 197: 265-266. Sutherland, G.R. (1979a) H e r i t a b l e f r a g i l e s i t e s on human chromosomes. 1. F a c t o r s a f f e c t i n g e x p r e s s i o n i n lympho-c y t e c u l t u r e . Am. J . Hum. Genet. 3_1: 125-135. Sutherland, G.R. (1979b) H e r i t a b l e f r a g i l e s i t e s on human chromosomes. 2. D i s t r i b u t i o n , phenotypic e f f e c t s and c y t o g e n e t i c s . Am. J . Hum. Genet. 3_1: 136-148. Sutherland, G.R. (1979c) H e r i t a b l e f r a g i l e s i t e s on human chromosomes. 3. D e t e c t i o n of fra( X ) (q27) i n males with X - l i n k e d mental r e t a r d a t i o n and i n t h e i r female r e l a t i v e s . Am. J . Hum. Genet. _53: 23-27. Sutherland, G.R. and Leonard, P. (1979) H e r i t a b l e f r a g i l e s i t e s on human chromosomes. 4. S i l v e r s t a i n i n g . Am. J . Hum. Genet. 5_3: 2 9-3 0. Suth e r l a n d , G.R. and A s h f o r t h , P.L..C. (1979) X - l i n k e d mental r e t a r d a t i o n w i t h macroorchidism and the f r a g i l e s i t e a t Xq27 or 28. Hum. Genet. 48: 117-120. Sutherland, G.R., Baker, E., and S e s h a d r i , R.S. (1980) H e r i t -able f r a g i l e s i t e s on human chromosomes. 5. A new c l a s s of f r a g i l e s i t e r e q u i r i n g BrdU f o r e x p r e s s i o n , submitted Am. J . Hum. Genet. T u r l e a u , C., Czernichow, P., Go r i n , R., Royer, P. and de Grouchy, J . (1979) D e b i l i t e mentale l i e e au sexe, v i s a g e p a r t i c u l i e r , m a c r o o rchidie e t zone de f r a g i l i t e de l'X. Ann. G4n4t. 22, A: 205-209. Turner, G. and Turner, B. (1974) X - l i n k e d mental r e t a r d a t i o n . J . Med. Genet. 11: 109-113. Turner, G., T i l l , R. and D a n i e l , A. (1978) Marker X chromo-somes, mental r e t a r d a t i o n and macroorchidism. N. E n g l . J . Med. 299: 1472. 88 Turner, G. , Daniel., A. and F r o s t , M. (1980) X - l i n k e d mental r e t a r d a t i o n , macroorchidism and the Xq27 f r a g i l e s i t e . J . P a e d i a t r i c s , i n p r e s s . Yunis, J . J . (1976) High r e s o l u t i o n of human chromosomes. Science 191: 1268-1270. Yunis, J.J.. and Chandler, M.E. (1977). High r e s o l u t i o n chromo-some a n a l y s i s i n c l i n i c a l medicine. Progress on C l i n i c a l  Pathology, V o l . 7, e d i t . S t e f a n i n i . a n d H o s s a i n i , Grune and S t r a t t o n , New York. Zakharov, A.F.. and E g o l i n a , N.A. (1968) Asynchrony of DNA r e p l i c a t i o n and m i t o t i c s p i r a l i z a t i o n along hetero-chromatic p o r t i o n s of Chinese hamster chromosomes. Chromosoma (Berl.) 23: 365-385. Zakharov, A.F., Barabovskaya, L . I . , Ibraimov, A.I., Benjusch, V.A., Demintseva, V.S. and Oblaspenko, N.G. (1974) D i f f e r -e n t i a l s p i r a l i z a t i o n along mammalian m i t o t i c chromosomes. 2. 5-bromodeoxyuridine and ;5-bromodeoxycytidine— r e v e a l e d d i f f e r e n t i a t i o n i n human chromosomes. Chromosoma 4_4: 343-359. 89 APPENDIX A Consent forms and c e r t i f i c a t e o f a p p r o v a l f o r c l i n i c a l r e s e a r c h and o t h e r s t u d i e s i n v o l v i n g human s u b j e c t s . THE UNIVERSITY OF BRITISH COLUMBIA DEPARTMENT OF MEDICAL GENETICS V A N C O U V E R , B.C. , C A N A D A DONALD PATERSON MEDICAL GENETICS UNITS MATHER BUILDING, U.B.C, VANCOUVER. B.C. V6T1W5 TELEPHONE (6041228-5485.228-5483 Consent Form It has been explained to me that the mental retardation that has occurred in my family is a sez-linked hereditary disorder affecting mostly males, while females may act as carriers of the disorder when they-are outwardly quite healthy. I understand that cultures of blood cell s have, in some recent studies, demonstrated a chromo-some irregularity that could provide information for determining whether male infants are liable to develop such retardation, and perhaps whether females are carriers or not. Accordingly, I give my permission to obtain a blood sample by vene-puncture and a tissue sample obtained by skin biopsy. I understand that the skin biopsy procedure involves l i f t i n g a very small piece of skin (about this "0" size) with a hypodermic needle and snipping the skin away with a pair of sharp eye l i d scissors. I understand that cells from the blood and tissue samples w i l l be grown in tissue culture and the chromosomes(material which carries information about inheritance) w i l l be examined. I understand that I am free to withdraw my consent and to discontinue my participation in this study at any time without prejudice to future care. I understand that my participation w i l l be kept confi-dential at a l l times. Date: . 1 9 — Patient's Signature: . Parent's Signature ( i f applicable): Witness' Signature: c o n t i n u e d . APPENDIX BJ. Formulation of t i s s u e c u l t u r e media. MEM M199 MEM-FA MEM-Ad Component mg/L mg/L mg/L mg/L Inorganic S a l t s C a C l 2 200.00 140.00 200.00 200.00 F e ( N 0 3 ) 3 - 9 H 2 O — 0.7 2 — — KC1 400.00 400.00 400.00 400.00 KH 2P0 4 — 60. 00 — — MgS0 4-H 20 200.00 200.00 200.00 200.00 NaCl 6800.00 8000.00 6800.00 6800.00 NaHC03 2200.00 350.00 2200.00 2200.00 NaH 2P0 4-H 20 140.00 — 140.00 140.00 Na 2HP0 4-7H 20 — 90.00 Other Components Adenine s u l f a t e — 10. 00 — — A d e n o s i n e t r i p h o s -phate (Disodium s a l t ) — 1. 00 . — — A d e n y l i c a c i d — 0.20 — — A z a s e r i n e — — — 0.20 C h o l e s t e r o l •— 0.2 0 — — Deoxyribose — 0.50 . — — Glucose 1000.00 1000.00 1000.00 1000.00 G l u t a t h i o n e — 0. 50 — — Guanine HC1 (Free base) — 0.30 — 0.30 Hypoxanthine — 0.30 — — Phenol red 10. 00 20.00 10.00 10. 00 Ribose — 0. 50 — — Sodium a c e t a t e — 50. 00 . — — Thymine — 0. 3 0 — — Tween 8 0 20. 00 — U r a c i l — 0.30 — — Xanthine — 0.30 — — continued. MEM M199 MEM- FA MEM- Ad Component mg/L mg/L mg/L mg/L Amino A c i d s L-Alanine 8. 90 50. 00 8. 90 8. 90 L-Arginine•HC1 126. 00 70. 00 126. 00 126. 00 L-Asparagine•H o 0 15. 00 . — 15-. 00 15. 00 L - A s p a r t i c a c i d 13. 30 60. 00 13. 30 13. 30 L-Cys t e i n e HC1-H 20 0. 11 — — L - C y s t i n e 24. 00 20. 00 24. 00 24. 00 L-Glutamic a c i d -H 20 14. 70 150. 00 14. 70 14. 70 L-Glutamine 292. 00 100. 00 292. 00 292. 00 G l y c i n e 7. 50 50. 00 7. 50 7. 50 L - H i s t i d i n e HC1-H 20 42. 00 21. 88 42. 00 42. 00 L-Hydroxyproline 10. 00 L - I s o l e u c i n e 52. 00 40. 00 52. 00 52. 00 L-Leucine 52. 00 120. 00 52. 00 52. 00 L- L y s i n e HC1 72. 50 70. 00 72. 50 72. 50 L-Methionine 15. 00 30. 00 15. 00 15. 00 L - P h e n y l a l i n e 32. 00 50. 00 32. 00 32. 00 L - P r o l i n e 11. 50 40. 00 11. 50 11. 50 L- S e r i n e 10. 50 50. 00 10. 50 10. 50 L-Threnine 48. 00 60. 00 48. 00 48. 00 L-Tryptophane 10. 00 20. 00 10. 00 10. 00 L- T y r o s i n e 36. , 00 40. 00 36. 00 36. 00 L - V a l i n e 46. . 00 50. 00 46. 00 46. 00 Vitamins A s c o r b i c a c i d 0. 05 Alpha t o c o p h e r o l phosphate ( d i s o -dium s a l t ) 0. 01 d - B i o t i n 1. , 00 0. 01 1. 00 1. 00 C a l c i f e r o l 0. 10 Ca-pantothenate 1. . 00 0. 01 1. 00 1. 00 C h o l i n e c h l o r i d e 1. . 00 0. 50 1. 00 1. 00 F o l i c a c i d 1. . 00 0. 01 1. 00 i - I n o s i t o l 2, . 00 0. 05 2. 00 2. 00 Menadione 0. 01 N i a c i n 0. 025 Niacinamide 1, . 00 0. 025 1. , 00 1. , 00 Para-aminobenzoic a c i d 0. 05 P y r i d o x a l HC1 1, . 00 0. 025 1. ,00 1. , 00 P y r i d o x i n e HC1 0. ,025 R i b o f l a v i n 0 .10 0. 01 0. , 10 0. , 10 Thiamine HC1 1 . 00 0. ,01 1. .00 1. , 00 Vi t a m i n A (acetate) — 0. ,14 APPENDIX C F o l a t e d e t e r m i n a t i o n .on v a r i o u s l a b o r a t o r y l o t s of f e t a l serum and Medium 199 with and without a serum supplement. (Assayed by Dr. George Gray, Department of Hematology, Vancouver General H o s p i t a l , Vancouver). No. Sample Manufacturer Lot No. F o l a t e x l O - 3 mg/L B C D F G H J + * Human cord serum C a l f serum F e t a l c a l f serum (FCS) F e t a l c a l f serum (FCS) F e t a l c a l f serum (FCS) C a l f serum F e t a l c a l f serum (FCS) F e t a l C a l f serum (FCS) Medium 199 w/o FCS Medium 199 w/ FCS Gibco Gibco Wild L i f e Serums Flow Labor-a t o r i e s Gibco Gibco Gibco Gibco Gibco normal human serum v a l u e range 3' heat i n a c t i v a t e d — 7.0+ R191223 9.2 R695618 10.5 802072 14.0 29101111 2.2 R595419 9.1 C184511 5.9 C184511* 5.4 R090814 16.5 A891012 12.0 •16 x 10 3mg/L A l l FCS media supplements i n the study were taken from Flow L a b o r a t o r i e s Lot No. 29101111 (Sample E ) . While i t i s of i n t e r e s t t h a t the f o l a t e l e v e l i n t h i s serum i s p a r t i c u l a r l y low r e l a t i v e t o other samples t e s t e d , i t i s d i f f i c u l t to imagine t h a t a 1:20 d i l u t i o n (5%) of serum i n t o a c u l t u r e medium would c o n t r i b u t e s i g n i f i c a n t l y to the f o l a t e l e v e l . Furthermore, i n comparing Samples I and J of M199 with and without a FCS supplement, there appeared t o be more v a r i a t i o n i n f o l i c a c i d content between d i f f e r e n t l o t s of M199 than would be c o n t r i b u t e d by d i f f e r e n t l o t s of FCS supplemented at 5%. 

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