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Analysis of D/D translocations in man by differential staining Wilson, Robert Douglas 1974

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ANALYSIS OF D/D TRANSLOCATIONS IN MAN BY DIFFERENTIAL STAINING by ROBERT DOUGLAS WILSON B.Sc, University of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Medical Genetics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH DECEMBER, 19 74 COLUMBIA In presenting th is thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion o f th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT With the advent of new cytogenetic techniques, more ques-tions can be answered concerning chromosomal rearrangements. The questions considered in th i s study ask which chromosomes are i n -volved in the t(Dq Dq) t r ans loca t ion , where are the break points located , and what are the poss ible mechanisms of t(Dq Dq) trans-locat ion formation. The segregation pattern of the D/D trans lo-cat ion was considered when the chromosome t rans loca t ion was shown to be f a m i l i a l . Chromosomes were obtained from leukocyte blood cultures and three d i f f e r e n t i a l s t a in ing techniques (Giemsa-banding, f luorescent-banding, C-banding) were used to ident i fy the chromo-somes involved in the D/D t rans loca t ion . These techniques also allowed the break points in each chromosome to be estimated. Four famil ies and a s ingle spontaneous case were examined. The seg-regation pattern of the t(Dq Dq) t rans locat ion was discussed for the f a m i l i a l t rans locat ions . The poss ible mechanisms of forma-tion of these D/D translocat ions was also discussed. The cytogenetic inves t i ga t ion revealed these general re-sults . 1. A homologous t(14q;14q) chromosome, which appeared to be monocentric, was found in a male infant who was ascertained through an inves t iga t ion of h i s short s tature. 2. A homologous t(13q;13q) chromosome, which appeared to be d i c e n t r i c , was found in a family who was ascertained through the b i r t h of a male with numerous congenital malformations. 3. A t(13q;14q) t rans locat ion chromosome, which appeared to be d i c e n t r i c , was found in a family who was ascertained through the b i r t h of a mentally retarded male. 4. A t(13q;14q) t rans locat ion chromosome, which appeared to be monocentric, was found in a family who was ascertained through the b i r t h of a mentally retarded male. 5. A t(13q;14q) t rans locat ion chromosome and a t(13p;14p) t rans locat ion chromosome was found in a family who was ascertained through the b i r t h of a mentally retarded male. TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES ACKNOWLEDGEMENTS Chapter I. INTRODUCTION A. Chromosomal rearrangement which resul t s in the formation of a t(Dq Dq) t rans lo-cat ion B. Incidence of the t(Dq Dq) t rans locat ion in the population C. Chromosomal cons t i tu t ion of the t(Dq Dq) t rans locat ion D. Segregation patterns of t(Dq Dq) trans-locat ions E. F e r t i l i t y in balanced t(Dq Dq) hetero-zygotes F. Other aspects of the t(Dq Dq) trans lo-cat ion II . MATERIALS AND METHODS A. Experimental procedures 1. Fluorescent banding and analys is 2. Trypsin-Giemsa banding and analys i v 3. C-banding and analysis 17 B. C r i t e r i a for e s t ab l i sh ing the pa t i en t ' s karyotype 17 I I I . RESULTS 19 Case 1 19 Case 2 26 Case 3 37 Case 4 48 Case 5 62 IV. DISCUSSION 75 A. Segregation of the t(Dq Dq) translocat ions 75 1. Homologous t(Dq Dq) translocat ions 75 2. Non-homologous t(Dq Dq) transloca-tions 79 B. Possible mechanisms of formation of the t(Dq Dq) t rans locat ion 81 C. C l i n i c a l and chromosome associat ions 86 LITERATURE. CITED 98 APPENDIX 108 v LIST OF TABLES Table Page 1. Analysis of c e l l chromosome number for pedigree 1 22 2. Analysis of c e l l chromosome number for pedigree 2 30 3. Analysis of c e l l chromosome n umb e r for pedigree 3 41 4. Analysis of c e l l chromosome number for pedigree 4 53 5. Analys i s of c e l l chromosome number for pedigree 5 67 6. Comparison of c l i n i c a l signs ; for trisomy-13 93 v i LIST OF FIGURES Figure Page 1. Pedigree - case 1 22 2. Giemsa-banded karyotype - proband case 1 2 3 3. P a r t i a l Giemsa-banded karyotype - proband case 1 23 4. Fluorescent karyotype - proband case 1 24 5. C-banded karyotype - proband'case 1 24 6. Schematic diagram of D/D trans locat ion - case 1 25 7. Pedigree - case 2 30 8. Unhanded karyotype - proband case 2 31 9. Anter ior and f u l l length view - proband case 2 31 10. Hand and foot of proband case 2 with extra d i g i t 32 11. Latera l view of head - proband case 2 32 12. Giemsa-banded karyotype - i n d i v i d u a l pedigree 2 - 1 - 4 33 13. P a r t i a l Giemsa-banded karyotype - ind iv idua l pedigree 2 - 1 - 4 33 14. P a r t i a l Giemsa-banded karyotype - ind iv idua l pedigree 2 - 1 - 4 34 15. P a r t i a l f luorescent karyotype 13/13 trans lo-cat ion chromosome, chromosome 14 p a i r , chromo-some 15 p a i r , s ingle chromosome 3 - ind iv idua l pedigree 2 - 1 - 4 34 16. C-banded karyotype - i n d i v i d u a l pedigree " 2 - 1 - 4 35 17. Schematic diagram of D/D trans locat ion - case 2 36 v i i 18. Pedigree - case 3 41 19. Giemsa-banded karyotype - i n d i v i d u a l pedigree 3 - 1 - 1 42 20. Fluorescent Karyotype - i n d i v i d u a l pedigree 3 - 1 - 1 42 21. Giemsa-banded karyotype - proband case 3 43 22. Giemsa-banded karyotype - ind iv idua l pedigree 3 - 1 - 4 43 23. P a r t i a l Giemsa-banded karyotype - proband case 3 44 24. Fluorescent karyotype - proband case 3 44 25. Fluorescent karyotype - i n d i v i d u a l pedigree 3 - 1 - 4 45 26. P a r t i a l f luorescent karyotype - s ingle chromosome 13, 13./14 t rans locat ion chromosome, s ingle chromo-some 14, chromosome 15 pa i r - proband case 3 45 27. C-banded karyotype - proband case 3 46 28. Schematic diagram of D/D trans locat ion - case 3 47 29. Pedigree - case 4 52 30. Giemsa-banded karyotype - i n d i v i d u a l pedigree 4 - 1 - 15 54, 31. Fluorescent karyotype - i n d i v i d u a l pedigree 4 - 1 - 1 5 54 32. Giemsa-banded karyotype - proband case 4 55 33. Giemsa-banded karyotype - i n d i v i d u a l pedigree 4 - 1 - 2 55 34. Giemsa-banded karyotype - i n d i v i d u a l pedigree 4 - 0 - 1 1 56 35. Giemsa-banded karyotype - ind iv idua l pedigree 4 - 0 - 1 2 56 36. Fluorescent karyotype - proband case 4 57 37. Fluorescent karyotype - i n d i v i d u a l pedigree 4 - 1 - 2 57 v i i i 38. Fluorescent karyotype - i n d i v i d u a l pedigree 4 - 0 - 1 1 58 39. Fluorescent karyotype - i n d i v i d u a l pedigree 4 - 0 - 1 2 58 40. P a r t i a l f luorescent karyotype s ingle chromo-some 13, 13 /14 t rans locat ion chromosome, s ingle chromosome 14, chromosome 15 p a i r - proband case 4 59 41. C-banded karyotype - i n d i v i d u a l pedigree 4 - 1 - 2 59 42. P a r t i a l C-banded karyotype - proband case 4 60 43. Schematic diagram of D/D trans locat ion - case 4 61 44. Pedigree - case 5 67 45. Giemsa-banded karyotype - ind iv idua l pedigree 5 - 1 - 1 " 68 46. Fluorescent karyotype - i n d i v i d u a l pedigree 5 - 1 - 1 68 47. Giemsa-banded karyotype - proband case 5 69 48. Giemsa-banded karyotype - i n d i v i d u a l pedigree 5 - 1 - 2 69 49. Giemsa-banded karyotype - i n d i v i d u a l pedigree 5 - 0 - 1 70 50. Giemsa-banded karyotype - i n d i v i d u a l pedigree 5 - 0 - 4 70 51. P a r t i a l Giemsa-banded karyotype - proband case 5 71 52. Fluorescent karyotype - proband case 5 71 53. Fluorescent karyotype - i n d i v i d u a l pedigree 5 - 1 - 2 72 54. Fluorescent karyotype - i n d i v i d u a l pedigree 5 - 0 - 1 72 55. Fluorescent karyotype - i n d i v i d u a l pedigree 5 - 0 - 4 73 56. Schematic diagram of D/D t rans loca t ion - case 5 74 ix LIST OF APPENDICES Appendix Page A Leucocyte Culture Method 1 0 8 B Fluorescent Banding Technique C Trypsin-Giemsa Banding Technique m D C-Banding Technique E S ta in ing Technology x ACKNOWLEDGEMENTS I wis h t o e x p r e s s my thanks t o t h e members o f my t h e s i s committee a t t h e U n i v e r s i t y o f B r i t i s h Columbia f o r t h e i r s u p p o r t and g u i d a n c e d u r i n g t h e c o u r s e o f t h i s p r o j e c t : Dr. J.R. M i l l e r , Department o f M e d i c a l G e n e t i c s ( C h a i r m a n ) ; Dr. F . J . D i l l , Department o f M e d i c a l G e n e t i c s ( S u p e r v i s o r ) ; Dr. P.J. MacLeod, Department o f M e d i c a l G e n e t i c s ; Dr. D.G. Holm, Department o f Z o o l o g y ; and Dr. C.O. P e r s o n , Department o f Botany. S i n c e r e a p p r e c i a t i o n i s e x p r e s s e d t o Dr. F . J . D i l l and Ms. S. Masui-Smith f o r t h e i r t e a c h i n g and t e c h n i c a l a s s i s t a n c e t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . I thank t h e f o l l o w i n g p e o p l e and p l a c e s f o r t h e i r c o - o p e r a t i o n and a s s i s t a n c e i n c e r t a i n a s p e c t s o f t h i s s t u d y : (1) Ms. D. Gi b s o n and Ms. J . Sa n d e r c o c k f o r t h e i r a s s i s t a n c e i n o b t a i n i n g b l o o d samples n e c e s s a r y f o r t h i s s t u d y . (2) The Woodlands S c h o o l , New W e s t m i n s t e r , B.C. f o r t h e p e r m i s s i o n t o use s e v e r a l p a t i e n t s s t a y i n g a t t h e s c h o o l . (3) The G e n e t i c s L a b o r a t o r y , V.G.H. and Dr. T z e , C h i l d r e n ' s H o s p i t a l , f o r the a s s i s t a n c e i n o b t a i n i n g t h e i n f o r m a t i o n and r e s u l t s f o r c a s e 1. (4) Ms. S. Manning f o r h e r p e d i g r e e i l l u s t r a t i o n s . My s i n c e r e a p p r e c i a t i o n and th a n k s i s g i v e n t o my w i f e , Wendy, whose s u p p o r t and u n d e r s t a n d i n g was a major f o r c e i n a l l o w i n g me t o complete t h i s t h e s i s . x i I acknowledge the support of the Univer s i ty of B r i t i s h Columbia Summer Research Grants during May - August 1973 and 1974. x i i CHAPTER I INTRODUCTION Over the past f i f t een years i t has become poss ible to associate s p e c i f i c a l tera t ions in the human d i p l o i d chromosome number with s p e c i f i c c l i n i c a l s igns . In many syndromes, for example Patau's syndrome (trisomy Dj_) the chromosomal abnormality can be predicted by phenotypic s igns . U n t i l recent ly karyotyping was based on size and centromere p o s i t i o n and hence only numerical and obvious s t ruc tura l changes could be detected. I d e n t i f i c a t i o n of i n d i v i d u a l human chromosomes was possible with autoradiography but i t was a time consuming procedure. Furthermore, small aberra-tions such as invers ions , small equal r ec ip roca l translocat ions could not be i d e n t i f i e d . With the advent of new d i f f e r e n t i a l s ta in ing techniques (Caspersson et a l . ,1969a, 1969b, 1970 ; Seabright, 19 71, 19 73; A r r i g h i and Hsu, 19 71) each human chromosome can be i d e n t i f i e d by i t s c h a r a c t e r i s t i c banding pat tern . As a r e su l t of these new s ta in ing methods, smal ler , more d i screet changes in chromo-some structure can be i d e n t i f i e d and thi s has increased understanding of the possible consequences of chromosome aberrat ions . Also as a r e su l t of these techniques which enable the morphology of the human complement to be examined, the system for the c l a s s i f i c a t i o n of the chromosome was revised (Paris Conference.; 1971). The present study was ca r r i ed out to invest igate D group translocat ions in man, s p e c i f i c a l l y D/D fusions, u t i l i z i n g the new 1 2 s ta in ing techniques. Spec i f i c questions were asked when considering these D/D trans locat ions such as: 1) which chromosome pa i r or pairs are involved in the t rans loca t ion ; 2) where are the poss ible break points on the long or short arms of the chromosomes involved; 3) what i s the segregation pattern of f a m i l i a l t(Dq Dq) t rans loca t ions ; and 4) what i s the poss ible mechanism or mechanisms of formation of the t(Dq Dq) translocat ions? The reason these t(Dq Dq) translocat ions were studied was due to the a v a i l a b i l i t y of famil ies which had pre-v ious ly been shown to have this type of t r ans loca t ion . This type of t rans locat ion was also chosen because of the in te re s t ing pattern of segregation shown by the trans locat ion and the r i s k of producing t rans locat ion trisomy D of f spr ing by male or female D/D trans locat ion c a r r i e r s . This present study was r e s t r i c t e d to D/D trans locat ions there by l i m i t i n g the tr i somic state discuss ion to only the Patau's syndrome ( tr i somy-Di) . This would not have been possible i f trans-locat ions invo lv ing the D group chromosomes and another chromosome group were studied. A. Chromosomal rearrangement which resul t s in the formation of a  t(Dq Dq) t rans locat ion This general type of t rans locat ion was f i r s t described by Robertson (1916) in cer ta in orthopteran populations. It has been termed "Robertsonian t rans loca t ion" or more loosely " c e n t r i c fu s ion" . In the human complement the D group of chromosomes are acrocentr ic in structure and are organized into 3 p a i r s , numbered 13, 14, and 15. 3 The mechanism of formation of the " c e n t r i c fus ion" type t rans locat ion has general ly been regarded as an unequal r e c i p r o c a l t rans locat ion between two acrocentr ic chromosomes with breaks on opposite sides of the centromeres and subsequent re jo in ing of the long arms. This usual ly resul t s in the loss of a minute c e n t r i c fragment, the formation of a new metacentric chromosome and reduc-t ion in the chromosome number in the balanced heterozygote (Hamer-ton, 1972). The minute cen t r i c fragment i s not always l o s t as there are reports of complete r ec ip roca l translocat ions without fragment loss (Emerit et a l . , 1972) and reports where the fragment i s frequently lo s t but i s present in some c e l l s (Palmer et a l . , 1969; de Grouchy et a l . , 1970). Al ternat ive mechanisms have been suggested for the " c e n t r i c fusion" type t rans loca t ion . It has been suggested e i ther that the centromeres simply fuse or that ne i ther of the centromeres are l o s t , but one of them is not v i s i b l e under l i g h t microscopy (Hsu and Mead, 1969; Niebuhr, 1972a). Another p o s s i b i l i t y has been proposed by Niebuhr (1972b). He suggests formation may be due to breaks in the short arms of both acrocentr ics with formation of d i c e n t r i c trans-locat ion chromosomes. He proposes centromeric suppression by yet an unexplained mechanism might explain the monocentric appearance generally not iced in centr i c fusions. 4 B. I n c i d e n c e o f the t ( D q Dq) t r a n s l o c a t i o n i n t h e p o p u l a t i o n S e r g o v i c h e t a l . (1969) s u g g e s t t h a t D/D t r a n s l o c a t i o n h e t e r o z y g o s i s p r o b a b l y r e p r e s e n t s the most f r e q u e n t t y p e o f chromosomal r e a r r a n g e m e n t . From t h e work o f Court-Brown (1967) and newborn s t u d i e s by W a l z e r e t a l . ( 1 9 6 9 ) , S e r g o v i c h e t a l . (1969) , Lubs and Ruddle ( 1 9 7 0 ) , Hamerton e t a l . ( 1 9 7 2 ) , F r i e d r i c h and N e i l s e n ( 1 9 7 3 ) , and J a c o b s e t a l . ( 1 9 7 4 ) , i t has been e s t i m a t e d t h a t t r a n s l o c a t i o n s o f t h e " c e n t r i c f u s i o n " t y p e between two members o f the D group chromosomes o c c u r i n a p p r o x i m a t e l y 1 p e r 1000 t o 2000 i n d i v i d u a l s examined. While t h e t ( D q Dq) t r a n s l o c a t i o n c a n n o t be c o n s i d e r e d common, i t i s not r a r e . F o r c o m p a r i s o n , t h e f r e q u e n c y o f c e n t r i c f u s i o n t y p e t r a n s l o c a t i o n s i n v o l v i n g a c r o -c e n t r i c chromosomes from the D group and t h e G group (2 p a i r s , numbered 21,,22) has been e s t i m a t e d a t 1 p e r 3000 ( G e r a l d and W a l z e r , (1970) . In o t h e r s u r v e y s , where newborn i n f a n t s were n o t u s e d , i n c i d e n c e v a l u e s were s i m i l a r . S a s k i e t a l . ( 1 9 7 1 ) , d o i n g chromosome s t u d i e s i n e a r l y e m b r y o g e n e s i s , f o u n d 1 t ( D q Dq) specimen i n 898 i n d u c e d a b o r t u s e s randomly c o l l e c t e d i n Sapporo. In s t u d i e s done i n p s y c h i a t r i c h o s p i t a l s , t h e f r e q u e n c y o f b a l a n c e d t ( D q Dq) r e a r r a n g e m e n t s was n o t s i g n i f i c a n t l y h i g h e r than t h a t found i n t h e newborn o r t h e g e n e r a l a d u l t p o p u l a t i o n (Newton e t a l . , 1972; N i e l s e n e t a l . , 1973). In a s u r v e y o f males examined f o r m i l i t a r y s e r v i c e , Zeuthen and N i e l s e n (1973) found t h e p r e v a l e n c e o f t ( D q Dq) t r a n s l o c a t i o n among the males s e l e c t e d f o r chromosome i n v e s t i g a t i o n 5 was 2.69 p e r 1000 and the p r e v a l e n c e among t h e t o t a l p o p u l a t i o n sample was 0.78 p e r 1000. The d i f f e r e n c e between t h e s e two v a l u e s i n non-s i g n i f i c a n t . C. Chromosomal c o n s t i t u t i o n o f the t ( D q Dq) t r a n s l o c a t i o n s From an i n v e s t i g a t i o n on c h i l d r e n w i t h D t r i s o m y p l u s a t r a n s l o c a t i o n , Hecht e t a l . (1966) c o n c l u d e d t h a t t h e t r a n s l o c a t i o n s tended t o be o f t h e t ( D q Dq) t y p e r a t h e r than t ( D q Gq) t y p e . They s u g g e s t e d t h a t a l l a c r o c e n t r i c s d i d n o t have an equal chance o f b e i n g f o u n d i n the t r a n s l o c a t i o n w i t h a g i v e n a c r o c e n t r i c chromosome. By the use o f - t h y m i d i n e a u t o r a d i o g r a p h y , the chromosome c o n s t i t u t i o n o f t ( D q Dq) t r a n s l o c a t i o n s c o u l d be i d e n t i f i e d (Schmid, 1963; G i a n n e l l i and H o w l e t t , 1966). Bloom and G e r a l d (1967, c i t e d i n Hecht and K i m b e r l i n g , 1071) p r e s e n t e d a u t o r a d i o g r a p h i c d a t a s u g g e s t i n g the t(Dq Dq) t r a n s l o c a t i o n might be non-random i n c o m p o s i t i o n . T h i s non-randomness has been c o n f i r m e d by numerous a u t h o r s (Rowley and Pergament, 1969; de Grouchy e t a l . , 1970, K r m p o t i c e t a l . , 1970; Hecht and K i m b e r l i n g , 1971; Cohen, 1971). Cohen (1971) r e v i e w e d 64 t ( D q Dq) (58 p r e v i o u s l y r e p o r t e d and 6 new c a s e s ) which had had a u t o -r a d i o g r a p h i c i d e n t i f i c a t i o n , and he showed t h e d i s t r i b u t i o n o f chromosomal c o m b i n a t i o n s was 5 ( 1 3 / 1 3 ) , 49 ( 1 3 / 1 4 ) , 6 ( 1 3 / 1 5 ) , 3 (14/15) and 1 ( 1 5 / 1 5 ) . The homologous 14/14 t r a n s l o c a t i o n was n o t o b s e r v e d . To t h e a u t h o r ' s knowledge, the t ( 1 4 q 14q) t r a n s l o c a t i o n has o n l y been r e p o r t e d once ( H u l t e n and L i n d s t e n , 1970; C a s p e r s s o n e t a l . , 1971). 6 Rowley and Pergament (1969) and Hecht and Kimberling (1971) suggested four possible explanations for the non-random d i s t r i -but ion: 1) that the breaks near the centromere occur at d i f f e r -ent frequencies for each: chromosome; 2) that there i s d i f f e r e n t i a l se lec t ion and s u r v i v a l of t rans locat ions due to cer ta in combinations of de f ic iences ; 3) that meiotic p a i r i n g and crossing over of homologous segments of DNA in non-homologous acrocentr ic chromo-somes may dictate meiotic pa i r ing and cross ing over between cer ta in pairs more frequently than others. The rec iproca l product of this type of cross ing over would be a small metacentric chromosome t(Dp Dp). These have been reported by Palmer et al.(1969) , de Grouchy et a l . (1970) , and Emerit et a l . (1972) ; 4) s p e c i f i c chromo-some breakage points which vary from the long to short arms depend-ing on the chromosome. Certain authors propose that the non-randomness i s not due to d i f f e r e n t i a l breakage frequencies , but that a l l acrocentr ics break with equal frequency and i t i s the s t r i c t d i spo s i t i on of the D group chromosomes around the nucleolus which leads to p r e f e r e n t i a l associat ions (de Grouchy et a l . , 1970 ; Rowley and Pergament, 1969). Hamerton (1968) proposed that prezygotic se lec t ion or meio-t i c drive might explain the s i g n i f i c a n t excess of balanced hetero-zygote t rans locat ion ca r r i e r s over chromosomally normal progeny among the of f spr ing of male t(Dq Dq) c a r r i e r s . He suggested that these translocat ions occur randomly among the D and G chromosomes 7 and that a se lec t ion exists according to the v i a b i l i t y of d i f f e rent combinations. Hamerton (1972) suggested another possible mechanism due to the intermediate r e p l i c a t i n g pos i t ion of chromosome 14. The heterochromatic block in the short arm and the proximal part of the long arm are late r e p l i c a t i n g and e i ther may be more l i a b l e to breakage or once broken take longer to hea l . Krmpotic et al.(1970) proposed that the non-randomness of the D group chromosomes involved in the t(Dq Dq) chromosomes may be due to the time at which cer ta in chromosomal port ions r e p l i c a t e . It i s bel ieved that ear ly r e p l i c a t i n g chromosomes or portions of ear ly r e p l i c a t i n g chromosomes are genet i ca l ly a c t i v e , thus neces-sary for f e t a l development. Late r e p l i c a t i n g chromosomes or por-tions of la te r e p l i c a t i n g chromosomes are general ly considered genet i ca l ly i n a c t i v e ; the i r loss or dup l i ca t ion i s compatible with l i f e i f the de let ion or dupl ica t ion i s not excessive. Chromo-some 14 has a late r e p l i c a t i n g port ion around the centromeric region so i t s loss may be compatible with l i f e while that of chromosome 13 and 15 loss would not. D. Segregation patterns of t(Dq Dq) translocat ions Hamerton (1968) reported that there was a s i g n i f i c a n t excess of heterozygotes by comparison with normal of f spr ing in t(Dq Dq) f ami l i e s . The excess of heterozygotes was accounted for by progeny of heterozygous males and unknown heterozygous parents, but not by the heterozygous female c la s s . It was also reported that the sex r a t i o d id not s i g n i f i c a n t l y depart from equa l i ty . Hamerton found no increase in the frequency of abortion and there 8 was no s i g n i f i c a n t difference in the frequency of abortion for the male or female heterozygote. This data c o n f l i c t e d with re-ports of Court-Brown (1967) which indicated that for male hetero-zygotes, spontaneous abortions occurred in 35.3% of conceptions, while for female heterozygotes, spontaneous abortions occurred in 43.1% of conceptions. These differences were l a t e r thought to ar ise from Court-Brown's smaller sample but complete conceptual h i s t o r i e s and Hamerton's incomplete conceptual h i s t o r i e s . Hamerton (1968) explained that the increase in hetero-zygous progeny from male translocat ions ca r r i e r s and unknown parental t rans locat ion ca r r i e r s was due to the sperm carrying chromosomally balanced translocat ions having a se lec t ive advan-tage over chromosomally normal sperm. Jacobs et al.(1974) found no excess of heterozygotes among the progeny of e i ther male or female ca r r i e r s of a t(Dq Dq) trans-loca t ion . Independent of the sex of the c a r r i e r parent, the segregation r a t i o of balanced t rans locat ion heterozygotes to normals d id not deviate from the expected 1:1 r a t i o . They found no evidence of unbalanced t rans locat ion trisomy among the l iveborn ch i ldren of the balanced c a r r i e r f ami l i e s . In add i t ion , there was no excess of prenatal deaths. Hamerton (1968) stated that balanced t(Dq Dq) heterozygotes have a very low r i s k ( less than 1%). of having affected t rans locat ion trisomy progeny for both male and female c a r r i e r s . 9 Dutr i l l aux and Lejeune (1970) concluded from the progeny of male t(Dq Dq) t rans locat ion ca r r i e r s that : 1) compared to population values, there was no increase in spontaneous abortions (151 - 20%); 2) compared to population values, there was no s ig-n i f i c a n t increase in the frequency of anomalies among the l i v e b i r ths and 3) almost a l l ch i ldren in the progeny (1:1 r a t io trans-loca t ion c a r r i e r to non-carr ier) were phenotypica l ly normal. They concluded from the progeny of female t(Dq Dq) t rans locat ion car-r i e r s that : 1) compared to population values, there was an i n -creased rate of spontaneous abortion (25%); 2); among the l i v e b i r t h s , there was an equal frequency (1%) of trisomy 13 and t r i -somy 21; and 3) among the phenotypical ly normal c h i l d r e n , there was no p r e f e r e n t i a l segregation of the t rans locat ion (1:1 r a t io t rans locat ion c a r r i e r to n o n - c a r r i e r ) . For the female c a r r i e r they suggested an "interchromosomal e f f ec t " was l i k e l y the cause for the 2% frequency of trisomy 13 or trisomy 21, with an o v e r a l l r i s k of anomalies in the order of 5%. This information suggests that t(Dq Dq) female c a r r i e r s have a great ly increased though s t i l l low r i sk of 1 - 2% of having t rans locat ion trisomy D c h i l d r e n . Population estimates of primary trisomy 13 range from 1 per 7602 - 14,500 (Taylor , 1968, 19 71; Conen and Erkman, 1966). E. F e r t i l i t y in balanced t(Dq Dq) heterozygotes I n f e r t i l i t y has been mentioned in associat ion with balanced t(Dq Dq) heterozygotes (Walker and H a r r i s , 1962; Yunis et a l . ,1964 j 10 K jes s le r , 1966; Wilson, 1971). Chandley et al.(1972) suggest that i n f e r t i l i t y or s u b f e r t i l i t y in t rans locat ion heterozygotes can be brought about in two ways: f e r t i l i t y may be reduced as a consequence of the production of gametes carrying a genet ica l ly imbalanced genome, and since aneuploid gametes appear to function at f e r t i l i z a t i o n in man, f e r t i l i t y may be reduced through preim-plantat ion^zygot ic and (or) abortion of chromosomally unbalanced fetuses. A l t e r n a t i v e l y , f e r t i l i t y may be reduced through gameto-genic disturbance which appears confined mainly to male hetero-zygotes and resul t s in o l igo or azospermia. S l i g h t l y lower mean chiasma counts for male t(13q;14q) heterozygotes was observed in two patients by Chandley et a l . (1972). These resul t s were s i m i l a r to a report by Kjes s ler (1966) who found s l i g h t l y lower mean chiasma counts for a s u b f e r t i l e t(Dq Dq) male heterozygote. He suggested that aneuploid gametes may be formed in Dq Dq heterozygotes, but may be s e l e c t i v e l y el iminated during spermiogenesis. A t(Dq Dq) t rans locat ion c a r r i e r showed a homogeneous h i s t o l o g i c a l p ic ture with a normal number of spermatogonia and spermatocytes but an almost t o t a l lack of spermatids (Skakkebach et a l . , 1973). However, one pat ient with a balanced t(14q;l4q) t rans locat ion showed normal spermatogenesis (Hulten and Lindsten, 1970). Chandley et a l . (19 72) suggested that a high proport ion of chromosomally un-balanced zygotes may be l o s t before implantation and thereby escape detection as abortions. 11 Fraccaro et al.(1973) reported 4 cases of t(13q;14q) in 92 men attending a f e r t i l i t y c l i n i c and af ter reviewing three other reports of men attending s i m i l a r c l i n i c s found 8 t(Dq.Dq) male heterozygotes in a sample of 333 men. They concluded that among men attending f e r t i l i t y c l i n i c s , the incidence of D/D translocat ions i s s i g n i f i c a n t l y higher than 1 per 1000 estimated in the general adult populat ion. Analys i s of sperm from t h e i r four patients revealed q u a l i t a t i v e and/or quant i ta t ive semen abnormalities in a l l of them. In another study of male i n f e r t i l -i t y pa t ient s , Koul ischer and Schoysman (1974) found 1 in 202 (0.05%) carr ied a t(Dq Dq) t r ans loca t ion . Palmer et al.(1973) comments on the heterogeneous c l i n i c a l ef fects that the t(Dq Dq) translocat ions produce. The ef fects on f e r t i l i t y are v a r i a b l e , as some famil ies show l i t t l e evidence of reproductive impairment (Hamerton, 1972 ; Palmer et a l . , 1973) while other famil ies are i n f e r t i l e (Wilson, 1971). Palmer et a l . (1973) mention that ear ly studies which did not ident i fy the chromosomes involved when i n f e r t i l i t y was associated, may have been homologous t(Dq Dq) chromosomes. These homologous t(Dq Dq) chromosomes may contribute to the i n f e r t i l i t y by the formation of tr i somic or monosomic zygotes. Palmer (1969) suggested that the v a r i a b i l i t y of f e r t i l i t y may be due to the locat ion of the break points in the t(13q;14q), with some representing true cent r i c fusions and others r ec ip roca l t rans locat ions . Cohen (1971) stated that among the t(Dq Dq) t rans locat ion cases he considered, the f a m i l i a l t rans locat ion was present four 12 times as often as sporadic or non- fami l i a l t rans locat ions . Palmer (1973) suggested this as possible evidence for some se lec t ive ef fect which would cause cer ta in sporadic t(Dq Dq) to become f a m i l i a l t ranslocat ions while others are lo s t due to i n f e r t i l i t y . Cacheiro et a l . (19 74) made observations in the mouse which may suggest some reasons for t(Dq Dq) t rans locat ion i n f e r t i l -i t y . They found that translocat ions which caused s t e r i l i t y , rather than p a r t i a l s t e r i l i t y , in males appeared to be those in which at leas t one of the breaks occurs close to one end of a chromo-some. They proposed that many cases of induced F-^  male s t e r i l i t y may be the re su l t of pos i t ion effects produced when paracentro-meric regions are trans located to euchromatic regions of cer ta in other chromosomes. Where abortions have already been considered in the sect ion on segregating patterns of the t(Dq Dq) t r ans loca t ions , they w i l l b r i e f l y be considered as contr ibut ing to i n f e r t i l i t y . In the family examined by Chandley et a l . (1972) , some male heterozygotes in the family had abortion rates as high as 50% of conceptions while other male heterozygotes had no evidence of miscarriage. They concluded that a high abortion rate i s a s p e c i f i c feature of only a minority of t(Dq Dq) t rans locat ions . In studies on recurrent abortion couples (Kaosaar, 1973; Bhasin et a l . , 1973; Wilson, 1969; Stenchever et a l . , 1968; Pergament et a l . , 1968) only the more recent references (Kaosaar, 1973; Bhasin et a l . , 1973) ident i fy the chromosomes involved. 13 Since translocat ions between homologous pairs can only produce unbalanced zygotes and of th i s type of t rans locat ion only 13/13 translocat ions could poss ib ly come to term, i t would always be a t rans locat ion trisomy -13. Hagen et a l . (1971) mention:- that t(Dq Dq) heterozygotes, with involvement of chromosome 13, have a chance of a healthy c h i l d with normal or balanced karyotype, but in addit ion there i s the chance of repeated abortions of non-viable fetuses or chi ldren with trisomy 13 and severe mal-formations. Heterozygote combinations of chromosome 14 and 15 with unbalanced karyotypes (trisomy 14 or 15) would not survive to term. F. Other aspects of the t(Dq Dq) t rans locat ion Conen and Erkman (1966) found no s i g n i f i c a n t c l i n i c a l difference between primary trisomy 13 and trans locat ion trisomy 13, apart from the greater sever i ty of cardio-vascular les ions in the primary trisomy 13 pa t ient . Magenis et a l . (1968) considered the s u r v i v a l , sex r a t i o and parental age of 221 chromosomally proven trisomy 13 pat ient s . They found that t rans locat ion and mosaic trisomy 13 had a longer surv iva l than primary trisomy 13. There was an i n s i g n i f i c a n t excess of females with primary trisomy 13 and no s i g n i f i c a n t change in the sex r a t i o through three months of age. The bimodal d i s t r i b u t i o n of maternal age for primary trisomy 13 was consistent with age-dependent and age-independent subgroups, while the maternal age of t rans locat ion trisomy 13 infants corresponded to 14 a younger peak. This younger peak is consistent with the idea that translocat ions occur independently of maternal age. Not a l l the cases of trisomy 13 syndrome considered by Magenis et a l . (1968) were of the t(Dq Dq) type. Taylor (1971) reported that approximately 17% of the c l i n i c a l trisomy -13 cases are t(Dq Dq) t rans locat ion c a r r i e r s . This f inding supports the e a r l i e r presented evidence which showed the low r i s k (1 - 2%) of t rans locat ion heterozygotes producing a t rans locat ion trisomy D c h i l d . Other resul t s reported by Taylor (1971) d i f f e red from those of Magenis et a l . (1968). She reported a higher mean maternal age and a bimodal d i s t r i b u t i o n of the maternal ages for the t rans locat ion c a r r i e r mothers. In support of Magenis et a l . (1968), she reported a s l i g h t excess of females (55%) . Taylor et a l . (1970) reached the fol lowing conclus ions : 1) the frequency of translocat ions in trisomy 13 was from 3 to 9 times higher than in trisomy 21; 2) t rans locat ions between two D chromosomes r e s u l t i n g in trisomy 13 was more common than trans lo-cation between two G chromosomes causing trisomy 21. They reported s imi l a r resul t s to Magenis et a l . (1968) concerning the lower maternal age for t rans locat ion trisomy 13 than for primary trisomy 13. CHAPTER II MATERIALS AND METHODS A. Experimental Procedures Chromosome analyses were c a r r i e d out on the .cu l tured human leukocytes, using a modif icat ion of the technique by Moorhead et a l . (1960) , adapted for micro-amounts of whole blood (Arakaki and Sparkes, 1963). A leukocyte culture was i n i t i a t e d by adding approxi-? mately 0.25 cc of whole blood to 5 ml of chromosome medium. For each patient s tudied , two to three cultures were esta-b l i shed . Each culture was incubated for 72 hours at 3 7 ° C . One hour before the c e l l s were harvested, Colcemid was added at a f i n a l concentration of 0.02 ug/ml of cu l ture . At the end of the 72 hour incubation per iod , the c e l l s were treated with a hypotonic so lu t ion (0.075 KC1) and were f ixed in 3:1 absolute methyl a l c o h o l : g l a c i a l acet ic ac id . Flame dr ied s l ides were then prepared from each of the cultures (complete technique in Appendix A) . 1. Fluorescent Banding and Analys i s The f luorescent technique used was a modif icat ion of Caspersson's (1969 a ,b ) . The flame and a i r dr ied s l ides from 15 16 each pat ient were immersed in a so lut ion of quinacrine dihydro-chloride (Atrebin - G.T. Gurr) for a minimum of 20 minutes. The s l ides were then r insed and mounted in d i s t i l l e d water. The covers l ips were sealed with para f f in wax (complete tech-nique in Appendix B) . A l l observations were made with a Zeiss Photomicro-scope using as a source of u l t r a - v i o l e t l i g h t , a mercury lamp with a BG 12 exc i te r f i l t e r and a 500 um b a r r i e r f i l t e r . The photographs were taken using Kodak T r i - X Pan f i lm under o i l immersion (X100) with an exposure time of 4 minutes. 2. Trypsin-Giemsa Banding and Analys i s The t ryps in technique used was a modif icat ion of Sea-br ight ' s (1971). The flame and a i r dr ied s l ides were l e f t at room temperature for 5 - 7 days, which i s the optimum time be-fore t r y p s i n i z a t i o n (Seabright, 1973). The s l ides were immersed in a ser ies of solut ions with the two major steps being the t ryps in digest ion and the Giemsa s ta in s o l u t i o n . Technique modifications made during the study were for the steps between the t ryps in and Giemsa s t a in . The s l ides were placed i n the tryps in so lut ion for per-iods from 5 - 2 0 seconds and were then dipped in various solu-tions to remove or inact ivate the t ryps in . The s l ides were stained in Giemsa so lut ion for 1 - 5 minutes. After the Giemsa s o l u t i o n , the s l ides were b r i e f l y r insed in d i s t i l l e d water, b lo t ted dry and then a i r d r i ed . The s l ides were then dipped in 17 x y l o l and mounted in a sui table mounting medium (complete tech-nique in Appendix C) . A l l observations were made with a Zeiss Photomicroscope under phase contrast . A minimum of t h i r t y c e l l s were observed and 10 - 15 c e l l s were photographed under o i l immersion (X100) using Kodak high contrast copy f i lm 5069. 3. C-Banding and Analys i s The C-banding technique used was a combination of the techniques used by Fore j t (1973) and A r r i g h i and Hsu (1971). The flame and a i r dr ied s l ides were immersed in a NaOH-NaCl so lut ion for 90 seconds, were r insed in d i s t i l l e d water, dip-ped in 70 and 90% methanol and were a i r d r i e d . The dr ied s l ides were then treated in Sorensen buffer pH 6.8 at 6 8 ° C for one hour. The heated s l ides were stained in buffered Giemsa solu-t ion (Fore j t , 1973) for 15 minutes, then r insed in d i s t i l l e d water, b lo t ted dry and a i r d r i ed . S l ides were then dipped in x y l o l and mounted in a suitable mounting medium (complete tech-nique in Appendix D) . Observations and photography were s imi l a r to the t ryps in-Giemsa technique. B. C r i t e r i a for e s t ab l i sh ing the p a t i e n t ' s karyotype For each pa t i ent , 30 - 50 tryps in banded c e l l s were analysed for chromosome number to rule out possible mosaicism. 18 From the banded c e l l s examined, only 10 - 15 having the best reso lut ion were photographed. Chromosome counts were done under X40 magnification to ensure that chromosomes from only one c e l l were being counted. O i l immersion (X100) was used only when c e l l s had morphological cross-overs not separable at X40 magnif icat ion. After the pr in ted photographs were obtained, the karyo-type was completed by fol lowing the s tandardizat ion of the Paris Conference (1971). Karyotypes, e i ther complete or p a r t i a l , were made to e s t ab l i sh the chromosomes involved in the t(Dq Dq) t rans locat ion or to determine normal variants in the p a t i e n t ' s chromosomes. CHAPTER III RESULTS Case 1 This male infant was born to young unmarried parents on November 25, 19 71. No further information on the parents or the pregnancy was avai lable as the c h i l d was adopted at the age of eight months. However, i t is known that the mother was a Canadian Indian (figure 1) . Development was normal except for phys ica l re tardat ion . The avai lable growth date i s summarized as fo l lows: Chronological age (yrs . ) Height (cm) Weight (kg) b i r t h 47 2.38 4/12 - 2.06 6/12 62.8 6.70 1.11/12 71.1 8.60 2.1/12 74.3 8.80 No other past phys ica l measurements are ava i l ab le . Phys ica l examination at two years of age revealed a miniature, w e l l -proportioned male. He appeared quite hyperactive and co-operative, His length was 74.3 cm which is below the t h i r d percent i l e for chronologica l age. The head appeared normal and the sutures 19 20 were a l l c losed. There was marked crowding of the teeth and the jaw appeared smal l . The abdomen and the cardio-vascular system were shown to be i n f a n t i l e by phys ica l examination. The scrotum was underdeveloped. The neurolog ica l examination was normal except for a mild hypotonia. An endocrine i n v e s t i -gation was car r ied out due to a suspected growth hormone de-f i c i e n c y . Tests showed that his growth hormone was normal in i t s response to i n s u l i n . A cytogenetic inves t iga t ion y ie lded the fol lowing ob-servations : a) the proband i s carrying a t(Dq Dq) t rans loca t ion . b) the t(Dq Dq) is a homologous t rans locat ion between two 14 chromosomes (figures 2 and 4). c) based on the G-banding (figures 2 - 3 ) and the C-banding (f igure 5) the t rans locat ion appears to be monocentric. d) based on the G-banding (figures 2 - 3) , the break points were i d e n t i f i e d as being in the short arm of one chromo-some 14 at pl2 and in the long arm of the other chromo-some 14 at q l l . Analysis and chromosome counts were done in this male (Table 1). Fluorescent analysis and karyotype are shown in figure 4. The schematic f igure i s derived from the Paris Conference (1971) to show the i d e a l i z e d form of the translo-cat ion (figure 6) . 21 Summary: Pedigree No. Karyotype 0-1 45, XY, t(14 ; 14) (pl2 ; q l l ) 0-1 C l i n i c a l s i g n s : s m a l l s t a t u r e , m i l d hypotonia, small jaw, underdeveloped scrotum pedigree - case 1 22 a i 2 / M = D/D translocation carrier • O = not tested Figure 1. Pedigree - case 1 TABLE 1 Number on pedigree Number of chromosomes <44 44 45 46 47 . >47 to t a l 0 - 1 2 1 30 - - 33 23 • ; I •: • •» .* • K3 5 - > * * 1 2 3 > - y X V .fit. 13 15 19 * J *~ • 20 6 i2 « a i it 16 1 5 « 3 4 tr a-. 3 . ; . 17 18 4 • • t 21 22 Figure 2. Giemsa-banded karyotype - proband case 1 fc M 1 2 4r Figure 3. P a r t i a l Giemsa-banded karyotype - proband case 1 24 n o » w K \) )) if U (I j; >| I I ! M it i , i t Figure 4. Fluorescent karyotype - proband case 1 il \i It in U 14 6- 12 15 16 17 1 8 19 20 21 22 Figure 5. C-banded karyotype - proband case 1 Figure 6. Schematic diagram of D/D t r a n s l o c a t i o n - case 1 26 Case 2 The proband, a male born on November 10, 1962, was the re su l t of the second pregnancy of an unrelated couple. The pregnancy was uneventful and the proband was f u l l - t e r m . The maternal age was 32 years and the paternal age was 34 years. After four years of marriage, this couple 's f i r s t preg-nancy resul ted in a fu l l - te rm female in fant , born on December 6, 1959. This female died short ly after b i r t h . She had a heart defect and death was sa id to be due to hyaline membrane disease. Chromosome invest igat ions could not be performed at that time. The parents had been invest igated p r i o r to the de-l i v e r y of this f i r s t c h i l d because they had d i f f i c u l t y in con-ce iv ing . This inves t iga t ion was car r ied out in 1958 and sperm analysis had revealed a low count. At b i r t h , the proband was noted to have mult iple congen-i t a l anomalies. The c h i l d died at two months of age and at that time weighed 2400 grams. At autopsy, the infant had Polydactyly with s ix fingers on both hands and s ix toes on the l e f t foot. The ears were markedly hypoplast ic and a c l e f t palate was pre-sent. The omphalocele which had been present at b i r t h had been repaired and a Meckel's d ivert iculum was observed. The v i scera were noted to be hypermobile. The r ight ureter was re trocaval and b i f i d and there were two renal pelves present on the r i g h t . The testes were undescended, ly ing on e i ther side of the i l i o -psoas muscle. A patent ductus arter iosus was present and there 27 was b i l a t e r a l colobomata and microphthalmos. Spotty congestion was noted throughout both lungs. M i c r o s c o p i c a l l y , the lungs were congested and contained a bronchopneumonia process. An i n -d ica t ion of u n i l a t e r a l pye lonephr i t i s was present in the r i g h t kidney, the other organs were normal. Chromosome studies revealed a member missing from the 13 -15 group and an extra body present in the 1 - 3 group. On the basis of the c l i n i c a l pattern of anomalies i t was assumed that the c h i l d was a trisomy D syndrome and the extra body in the group 1 - 3 was a t(Dq Dq) between two chromosomes of the 13 -15 group. The parents were invest igated and i t was found that the mother was 46,XX and that the father carr ied a s i m i l a r trans-locat ion to that observed in the proband but trt was balanced. The father , a school p r i n c i p a l , was phenotypical ly normal. The fa ther ' s mother, s i s t e r , and brother had normal karyotypes. The paternal grandfather was dead (f igure 7). The o r i g i n a l chromo-somal analysis and c l i n i c a l p ictures of the proband are included (figures 8 - 11). A re - inves t i ga t ion of this family i n i t i a t e d during this present study revealed the fo l lowing : a) the t rans locat ion was a homologous t rans locat ion between two 13 chromosomes (f igures 12 - 15). b) on the bas i s of the symmetrical appearance and the suggestion that both the 13 chromosomes centromeres are present as shown 28 by the G-banding (figures 12 - 14) and the C-banding (f igure 16) , the t rans locat ion i s poss ib ly d i c e n t r i c , c) based on G-banding (figures 12 - 14), the break points appear-ed to be in the short arms of both chromosome 13's in the re-gion of p l l - 1 2 . Analys i s and chromosome counts were done only on the father of the proband since he was the only l i v i n g member of the family with the t rans locat ion (Table 2). Due to the nature of the trans-loca t ion ( i . e . invo lv ing both number 13 chromosomes), i t can be assumed that the f i r s t born female was a t rans locat ion trisomy D as w e l l . The f a i lu re to thr ive and the heart defect add strength O O to th i s assumption. Fluorescent resul t s and karyotype are shown in figure IS. The schematic figure i s derived from the Paris Conference (1971) to show the i d e a l i z e d form of the t rans locat ion (figure 17). Summary: Pedigree No. Karyotype 1-1 46, XX 1-4 45 , XY, t d i c (13;13) (pl2;pl2) 0-1 ( t rans locat ion trisomy D 46, XX, t(13q;13q)) 0-2 46, XY, t d i c (13;13) (pl2 ;pl2) 29 C l i n i c a l s i g n s : h e a r t d e f e c t , h y a l i n e membrane di s e a s e , f a i l u r e to t h r i v e C l i n i c a l s i g n s : P o l y d a c t y l y , h y p o p l a s t i c ears, c l e f t p a l a t e , Meckel's d i v e r -t i c u l u m , omphalocele, hyper-mobile abdominal v i s c e r a , b i -f i d r e t r o c a v a l r i g h t u r e t e r , undescended t e s t e s , colobo-mata, microphthalmos, patent ductus a r t e r i o s u s , apneic s p e l l s , f a i l u r e to t h r i v e I pedigree - case 2 30 o a a 2 o ^ 2 3 4 cT3 iTSTi 5 6 7 • # = trisomy-D with D/D translocation • = D/D translocation carrier 0 ® = normal chromosomes 4 miscarriage f dead Figure 7. Pedigree - case 2 TABLE 2 Number on pedigree Number of chromosomes <44 . 44,- 45 '46 47 ' >4'7 total- • 1 - 4 2 2 29 1 - 34 31 ^ U K K U am 1 - 3 + T 4 - 5 6 -12 +X A A AAA 13-15 -D 16-18 19 - 20 A A A 4 ft 2 1 - 2 2 +Y Figure 8. Unbanded karyotype - proband case 2 Figure 9. Anterior and f u l l length view - proband case 2 Figure 11. L a t e r a l view of head - proband case 2 33 S P I 13 B a i 8 « 2 3 IS 4 1 v X 12 6 12 ! | « » * *• M 14 M 15 16 IS 1/ I K • 4 18 Y 19 20 2 1 2 2 Figure 12. Giemsa-banded karyotype - ind iv idua l pedigree 2-1-4 f \ *_ _ l _ _ i 1 2 3 X 6 - 1 2 46 Sfe 1 3 1 4 1 5 Figure 13. P a r t i a l Giemsa-banded karyotype - i n d i v i d u a l pedigree 2-1-4 34 I IV 6 - 1 2 Mi t l 1 4 1 5 Figure 14. P a r t i a l Giemsa-banded karyotype - i n d i v i d u a l pedigree 2-1-4 Figure 15. P a r t i a l f luorescent karyotype 13/13 t rans locat ion chromosome, chromosome 14 p a i r , chromosome 15 p a i r , s ing le chromosome 3 - i n d i v i d u a l pedigree 2-1-4 35 j] 1 U 4* * 111) I ftl II II S* E 1 9 < I. 14 15 6 12 ,3 * X I K 11 II ftl 16 1/ 19 20 IH 21 2? Y Figure 16. C-banded. karyotype - individual pedigree 2-1-4 Figure 17. Schematic diagram of D/D t r a n s l o c a t i o n - case 37 Case 3 This proband, a male born on January 7, 1969, was the re-su l t of the second pregnancy of Jthis unrelated, couple. The ma-ternal age was 23 years and the paternal age was 24 years. The couple was married in 1965 and the i r f i r s t pregnancy eried in spontaneous abortion at 3 - 4 months gestation (figure 18). The second pregnancy had numerous complications throughout. The mother was apparently in contact with rube l l a near the be-ginning of the t h i r d month of gestat ion. For this she was g i -ven Gamma Globul in . At the f i f t h month of gestat ion, she de-veloped f l u - l i k e symptoms and a kidney i n f e c t i o n . The mother had been a d iabet ic for 12 years p r i o r to the b i r t h of the pro-band and during the l a s t 2 - 3 months of the pregnancy she was unable to control her diabetes. As a re su l t she was h o s p i t a l i -zed 7 - 8 weeks p r i o r to the proband's b i r t h and the de l ivery was by Caesarian sec t ion , 30 days p r i o r to term. At this time she was s t e r i l i z e d . The proband was described as act ive and the b i r t h weight was approximately 2500 grams. On two occasions, at 3 and 5 weeks, the proband had an episode of breath holding during which he became s t i f f and tense. During these episodes there were no c l o n i c a c t i v i t i e s and no loss of consciousness. At 4 1/2 months the proband was hosp i t a l i zed and the parents were informed that he appeared developmentally slow. His milestones were delayed from ear ly infancy. He was reported to smile at age 4 months, to blow bubbles at around 7 months, and at 1 year learned to r o l l over 38 from prone to supine p o s i t i o n . He was unable to s i t , crawl , or stand. On phys ica l examination at 4 1/2 months, the proband was s i g n i f i c a n t l y small for hi s chronological age. His weight was -2 standard deviations for his height-age. His head circumfer-ence was -2.5 standard deviations for his chronologica l age, but this was not considered microcephal ic . The head was of normal conf igurat ion and the anter ior fontanelle was s t i l l open. He had spast ic quadriplegia of severe degree and frequently as-sumed a hyperextended p o s i t i o n of his back and head, p a r t i c u l a r l y when handled. There was obvious b i l a t e r a l i n t e r n a l strabismus, with pupi l s equal in size and no evidence of macular or r e t i n a l changes. On examination the palate appeared s l i g h t l y arched. Examination of heart , lungs, abdomen, and g e n i t a l i a were normal, although the testes were undescended. His ref lexes were e i ther absent or exaggerated. Severe retardat ion was evident in both i n t e l l e c t u a l and s o c i a l development. Chromosome invest igat ions on the proband revealed that two chromosomes from the 13 - 15 group were missing and an extra body was present in the 1 - 3 group. It was presumed that a t rans locat ion had occurred between two chromosomes in the 13 -15 group. The parents were examined and i t was found that the father was 46, XY (figures 19 - 20) but the mother was a c a r r i e r of a t rans locat ion s imi l a r to that observed in the proband. 39 A re - inves t i ga t ion of this family i n i t i a t e d during th i s present study revealed the fo l lowing : a) the t rans locat ion was between a chromosome 13 and 14 (figures 21 - 26). b) based on the G-banding (figures 21 - 23) and the C-banding (figure 27), both the centromeres appear to be present. The f luorescent banding (figure 26) shows the br ight centromeric region of chromosome 13 i s present. c) on the basis of the G-banding (figures 21 - 23) and C-banding (f igure 27), the break points were determined to be in the short arms of both chromosome 13 and 14 in the region p l l - 1 2 . The immediate family of the mother refused to co-operate, so the t rans locat ion could not be followed in th i s pedigree. Analys is and chromosome counts were done for each family member studied (Table 3). The schematic f igure i s derived from the Par i s Conference (1971) to show the i d e a l i z e d form of the t rans locat ion (figure 28). Summary: Pedigree No. Karyotype 1-1 46, XY 1-4 45, XX td ic (13;14) (p!2;pl2) Spontaneous abortion poss ible trisomy D 0-1 45, XY, td ic (13;14) (pl2 ;pl2) 40 C l i n i c a l s igns : S i g n i f i c a n t l y small head, s p a s t i c i t y , strabismus, mental retardat ion ( s o c i a l , i n t e l l e c t u a l ) , s i g n i f i c a n t l y small s ta ture , undescended testes pedigree? - case 3 41 0 O 2 O 4 A" D/D translocation carrier normal chromosomes untested miscarriage Figure 18. Pedigree - case 3 TABLE 3 o ® = • o = 9 Number on Number, of, .Chromosomes , Pedigree 1^44 44 45 46 47 >47 TOTAL 1 - 1 1 2 1 30 - 34 1 - 4 3 2 33 - 38 0 - 1 1 5 30 - 36 42 a 3 s 7 6 12 13 i-l A 4 15 a v . . M . 16 17 18 1!) 20 21 22 Y Figure 19. Giemsa-banded karyotype - individual pedigree 3-1-1 \\ Jl II II H |I if (I v« H M II I I I I " » I 4 1 Figure 20. Fluorescent karyotype - individual pedigree 3-1-1 43 5 19 20 g a it A ? s ' V * * * 4 8 A f t t i 15 16 l / 21 18 22 Figure 21. Giemsa-banded karyotype - proband case 3 J < •1 • ) If If 1 118 gill in P 8.4 8 I M 13 19 M IS i.e. 20 11 Mi Ml) 8 III 17 18 21 Figure 22. Giemsa-banded karyotype - ind i v i d u a l pedigree 3-1-4 Figure 23. P a r t i a l Giemsa-banded karyotype - proband case 3 )• iv ir II n i f K . i : ;• i » « • • ' Figure 24. Fluorescent karyotype - proband case 3 45 11 11 i f -.; « n II t ) i i t > • 1 i l . i Figure 25. Fluorescent karyotype - i n d i v i d u a l pedigree 3-1-4 Figure 26. P a r t i a l f luorescent karyotype s ingle chromosome 13, 13/14 t rans locat ion chromosome, s ingle chromosome 14, chromosome 15 p a i r - proband case 3 46 li 17 i A H 13, i a, u ft<t I I I I IB 111 I / in in .'i Figure 27. C-banded karyotype - proband case 3 13 Figure 28. Schematic diagram of D/D t r a n s l o c a t i o n - case 48 Case 4 The proband, a male born on January 24, 1957, was the t h i r d pregnancy of unrelated parents who had been married for eight years. The maternal age was 31 years and the paternal age was 29 years. P r i o r to the proband's b i r t h , there had been two miscarriages . The times of these miscarriages are not knoxvn. The t h i r d pregnancy of th i s mother was abnormal. It was complicated with eclampsia and severe toxemia with convuls ions . As a r e s u l t , a Caesarian section was performed four weeks before f u l l term. The b i r t h weight was 2670 grams. Development appeared normal for the f i r s t year. The pro-band began walking and had spoken his f i r s t words by that time. At 18 months, parental concern about the proband's lack of speech and h i s hyperactive behaviour lead to a more deta i led examination. F u l l inves t iga t ion was ca r r i ed out and the. resul t s showed normal hearing and E . E . G . No phys ica l abnormalities were apparent apart from the b i l a t e r a l epicanthic folds and the appearance of short-ened hands and feet . On tes t ing at three years of age, i t was f e l t he was functioning at a l e v e l of two years o l d . His fea-tures d id not suggest mental dul lness . His height and weight were within the normal range for his age. His head was symmetri-ca l and of normal shape, although the s k u l l circumference was about one standard deviat ion below the mean for his age. A soft s y s t o l i c murmur was noted. The lungs and abdomen were not re-markable. The testes were descended. 49 He showed rapid changes in mood and i n t e r e s t . His speech was i n d i s t i n c t and poorly a r t i c u l a t e d . Limb tone, power, and tendon reflexes were within normal l i m i t s , though the presence of abdominal reflexes were doubtful . From these examinations, the diagnosis made was that the proband suffered from cerebral anoxia in utero during the period of his mother's toxemia and convuls ion. It was thought that th i s may have l e f t him with patchy brain damage character ized by the "organic behaviour syndrome". It was l a t e r c a l l e d i n f a n t i l e autism with poss ible mental re tardat ion . The mother's fourth pregnancy resul ted in a male in f an t , born October 27, 1959, who i s normal except for the presence of a horseshoe kidney. The mother had no toxemia during th i s preg-nancy. The mother's f i f t h pregnancy resul ted in a female i n f a n t , born October 20, 1961, who i s normal. There was a report of neo-nata l achalas ia in th i s female. The mother has been reported as nervous with f i t s of temper. Her E . E . G . , she says, i s " s l i g h t l y abnormal". Chromosome studies on the proband revealed two chromosomes from the 13 - 15 group were missing and an extra body was present in the 1 - 3 group. It was presumed that a t rans locat ion had oc-curred between two chromosomes in the 13 - 15 group. The parents were invest igated and i t was found that the father was 46, XY (figure 30 - 31) and that the mother was a c a r r i e r of a t rans loca t ion 50 s imi la r to that observed in the proband. The male and female s ib l ings were examined and i t was shown that they both car r i ed the t rans locat ion chromosome. Further inves t iga t ion of the maternal family in Ireland traced the t rans locat ion back to the maternal grandmother of the proband. Because of deaths, the t rans locat ion chromosome could not be traced any further . The pedigree i s shown in f igure 29. The maternal grandmother (II-2) had f ive chi ldren (3 males, 2 females) with no reported spontaneous abortions. Four of these f ive of f spr ing car r i ed the t rans locat ion chromosome. These four t rans locat ion ca r r i e r s had 10 o f f spr ing , 5 of whom had the trans-loca t ion . The only spontaneous abortions reported are those of the proband's parents. One t rans locat ion c a r r i e r f a i l e d to pass the t rans locat ion chromosome to e i ther of h i s two o f f spr ing . The three remaining t rans locat ion ca r r i e r s transmitted the transloca-t ion chromosome to at leas t one of t h e i r o f f spr ing . A re - inves t i ga t ion of the proband's immediate family i n i t i a t e d during this present study revealed the fo l lowing : a) the t rans locat ion was between a chromosome 13 and 14 (figures 32 - 40). b) on the basis of the G-banding (figures; 32 - 35) , Q-banding (f igures 37 - 39), and C-banding (f igures 41 - 42), the trans-locat ion appears to be monocentric. The centromere i s that of chromosome 13 as the f luorescent resul t s show the br ight 51 centromeric region i s present and the Giemsa s ta in ing adds further evidence for th i s conclusion, c) on the basis of the G-banding (figures 32 - 35), the break points were determined to be in the short arms of chromosome 13 at pl2 and in the long arms of chromosome 14 at q l l . Analysis and chromosome counts were done for each family member (Table 4). The schematic figure i s derived from the Paris Conference (1971) to show the i d e a l i z e d form of the transloca-t ion (figure 43) . Summary: Pedigree No. Karyotype 1-15 46, XY 1-2 45, XX, t(13;14) ( p l 2 ; q l l ) 0-10 45, XY, t(13;14) ( p l 2 ; q l l ) 0-11 45, XY, t(13;14) ( p l 2 ; q l l ) 0-12 45, XX, t(13;14) ( p l 2 ; q l l ) 0-10 C l i n i c a l s igns : " i n f a n t i l e autism", mental r e ta rda t ion , b i l a t e r a l epicanthic fo lds , shortened hands and feet . 0-11 0-12 C l i n i c a l s igns : horseshoe kidney C l i n i c a l s igns : neonatal achalas ia reaigree - case ^ O 2 n 4-n l6 2 2 3 5 " 4 5 6 7 4 ^ r1\ In] ID] (•) ID] (0) IDI 4> H d (!) 5 6 7 8 9 10 II 12-14 0 (r3KQ) n(Q) J o © ^ i 1 2 3 4 5 6 7 8 9 10 15 16 II 12 13 14 15 16 17 H © = D / D translocation carrier 0(5)= normal chromosomes • O = not tested + - dead Figure 29. Pedigree - case 4 TABLE 4 Number on Number of chromosomes Pedigree -<44 44 45 46 47 >47 TOTAL 1 - 2 4 2 36 1 43 1 - 15 3 2 1 24 30 0 - 10 1 2 35 1 39 0 - 11 2 2 27 - - - 31 0 - 12 5 - 24 1 30 54 I f 1)1 •< 14 3 8 3 J p . R[( A,IU :A> JIMJI 6 12 ^ 0 PM H A 13 l'4 la M l i . i . i/a 16 17 18 19 20 21 22 Figure 30. Giemsa-banded karyotype - individual pedigree 4-1-15 M I i II l» »» II • •1 • • • • Figure 31. Fluorescent karyotype - individual pedigree 4-1-15 55 ^ » 6 ft-' * * ^ m i ? I i 1 i n (i 12 ft 1 M 14 1!> 16 17 4 13 it, ! HI ft! m 19 » i\ r i Figure 32. Giemsa-banded karyotype - proband case 4 l i l y ? l i l t i :' A i Ir * ) l > r i n i i c i f f r* u T w tt u l.l 14 I!) It. 1/ Ii! 19 20 ?1 <j Figure 33. Giemsa-banded karyotype - individual pedigree 4-1 56 Suisse a A «* X 6 - 1 2 A I ft M . M . 13 i4 15 16 17 18 19 20 21 22 Y Figure 34. Giemsa-banded karyotype - individual pedigree 4-0-11 Figure 35. Giemsa-banded karyotype - in d i v i d u a l pedigree 4-0-12 57 Figure 37 . F l u o r e s c e n t karyotype - i n d i v i d u a l pedigree 4-1-2 58 0 i i i ) i t I I H i t * » : « 5 $ i i i i 1 • • • I . Figure 38. Fluorescent karyotype - individual pedigree 4-0-11 M I f I t i t i . i I • 9 ft Figure 39. Fluorescent karyotype - individual pedigree 4-0-12 59 Figure 40. P a r t i a l f luorescent karyotype s ingle chromosome 13, 13 /14 t rans locat ion chromosome, s ing le chromosome 14, chromosome 15 p a i r - proband case 4 iii • i 0 » • s » X 4 l U S ; M ' ! U 13 14 A A 15 6 12 i . A * §_ • | 16 17 18 • i • i i 19 20 21 22 Figure 41. C-banded karyotype - i n d i v i d u a l pedigree 4-1-2 Figure 42. P a r t i a l C-banded karyotype - proband case 4 13 Figure 43. Schematic diagram of D/D t rans loca t ion - case 62 Case 5 The proband, a male born on A p r i l 14, 1969, was the t h i r d pregnancy of th i s unrelated couple. The pregnancy continued un-t i l 38 weeks and was complicated by excessive weight gain, pre-eclampsia without any spec ia l medications and a h i s tory compat-ib le with a general ized convulsion during d e l i v e r y . Maternal age at the b i r t h of the proband was 28 years and the paternal age was 30 years. The b i r t h weight was 1960 grams and in the i n i t i a l new-born per iod , the proband appeared normal. The couple 's f i r s t pregnancy resul ted in a female, born July 25, 1966, who i s normal in development and i n t e l l i g e n c e . The second pregnancy ended in the b i r t h of a 7 1/2'month premature male, who died at 2 days of age with mult iple anomalies inc luding c l e f t palate and Po lydac ty ly . The fourth pregnancy was a female, born June 23, 1972, whose b i r t h weight was 3494 grams (figure 44). The proband was delayed in development through the f i r s t twenty months, he smiled at three months, r o l l e d over at seven months, and could hold himself up at fourteen months. A phys ica l examination was performed before one year of age. He was wel l developed but had a pecu l i a r f ac ie s , with a rather high and bu l -ging forehead. His head circumference was at the mean and his height was one standard deviat ion below the mean for h i s age. His eyes were normal and although his palate was h ighly arched, he had a normal ora l cavity otherwise. His chest was somewhat f l a t t e n e d but there were no a b n o r m a l i t i e s i n the c a r d i o v a s c u l a r , pulmonary, or a l i m e n t a r y systems. There were haemangiomata pre-sent over the p a r i e t o - a c c i p i t a l r e g i o n , lumbar s a c r a l area, and at the t i p of the r i g h t r i n g f i n g e r . He had undescended t e s t e s b i l a t e r a l l y . There were t i n y supernumerary d i g i t s on the u l n a r borders of the proximal phalanges of the small f i n g e r s and the l e f t hand showed a simian crease. He was g e n e r a l l y hypotonic and had h y p e r e x t e n s i b i l i t y of h i s j o i n t s . A l l of h i s p r i m i t i v e r e f l e x e s were absent. During the newborn p e r i o d the parents noted mannerisms in the f o u r t h c h i l d s i m i l a r to those p r e s e n t i n the proband. Developmentally, t h i s female was always slow and at nine months c o u l d s i t with d i f f i c u l t y but r e q u i r e d support. She d i d not crawl, although she had some weight b e a r i n g movements and she had only a few cooing sounds. P h y s i c a l examination at l e s s than one year of age r e v e a l e d that head circumference was above the t h i r d p e r c e n t i l e f o r her age while h e i g h t and weight were w i t h i n normal ranges. Her eyes appeared normal with m i l d strabismus that allowed her to f o l l o w moving o b j e c t s . There was a complete c l e f t of the hard and s o f t p a l a t e , extending through to the uvula. The nasopharynx was q u i t e hyperemic. The c a r d i o v a s c u l a r , r e s p i r -a t o r y , and abdominal systems were normal. Examination of the s k i n showed no a b n o r m a l i t i e s other than a haemangioma on the f o r e -head. The g e n i t a l i a were g r o s s l y normal f o r a female of her age. 64 The neuro log ica l examination showed she had moderate hypotonia with normal reflexes throughout. She had a pecu l i a r posturing of her l e f t hand with the thumb and f i f t h f inger cross ing over the dorsum of the hand to meet in the midl ine . Cytogenetic examination of the proband revealed a chromo-some missing from the 13 - 15 group and an extra body in the 1 -3 group. It was presumed due to the c l i n i c a l pattern of anoma-l i e s that the c h i l d was a trisomy -D syndrome and the extra body in the 1 - 3 group was a t(Dq Dq) between two chromosomes of the 13 - 15 group. The parents were invest igated and i t was found that the father was 46, XY (figures 45 - 46) and the mother was a c a r r i e r of a t rans locat ion s i m i l a r to that observed in the pro-band. She also had a small t(Dp Dp) fragment. The two sibs were examined and i t was found that the f i r s t s ib (0-1) was s imi l a r to her mother as she carr ied both the t(Dq Dq) t rans loca t ion and the t(Dp Dp) fragment. The other sib (0-4) d id not carry the large t(Dq Dq) t rans locat ion but did carry the small t(Dp Dp) fragment. A re - inves t iga t ion of the proband's immediate family i n i t i a t e d during th i s present study revealed the fo l lowing : a) the r ec ip roca l t rans locat ion was between a chromosome 13 and 14 (figures 47 - 55). b) on the basis of the G-banding (figures 47 - 51) and Q-banding (figures 52 - 55), the t(Dp Dp) t rans locat ion involves the 65 centromere and short arms of chromosome 13 and the short arms of chromosome 14. The br ight centromeric region of chromosome 13 was found in the t(Dp Dp) fragment. There i s also c l i n i c a l evidence that the t(Dp Dp) fragment i s mainly composed of chromosome 13. The t(Dq Dq) t rans locat ion involves the centro-mere and long arms of chromosome 14 and the long arms of chromo-some 13. c) on the basis of the G-banding, the break points were determined as in the short arms of chromosome 14 at p l l and in the long arms of chromosome 13 at q l2 . Analys i s and chromosome counts were done for each family member (Table 5). The schematic figure i s derived from the Par i s Conference (1971) to show the i d e a l i z e d form of the t rans locat ion (figure 56) . Summary: Pedigree No. Karyotype 1-1 46, XY 1-2 46, XX, t(13q;14q) ( q l 2 ; p l l ) t(13p;14p) ( q l 2 ; p l l ) 0-1 46, XX, t(13q;14q) ( q l 2 ; p l l ) t(13p;14p) ( q l 2 ; p l l ) 0-2 female, probable t rans locat ion trisomy 13 0-3 46, XY, t(13q;14q) ( q l 2 ; p l l ) 0-4 47, XX, t(13p;14p) ( q l 2 ; p l l ) 66 0-2 C l i n i c a l s igns : 0-3 C l i n i c a l s igns : 0-4 C l i n i c a l s igns : mult ip le anomalies inc luding c l e f t palate and Po lydac ty ly , f a i lu re to thr ive psychomotor re tardat ion , mixed convulsive di sorder , arched pa la te , Po lydac ty ly , simian crease, hypotonia, mult ip le haemangioma, undescended test-i c l e s , profound i n t e l l e c t u a l and s o c i a l re ta rda t ion , hyper-extensible j o in t s psychomotor re tardat ion , c l e f t pa late , strabismus, haemangioma, hypotonia, f inger pos tur ing , moderate i n t e l l e c t u a l and severe s o c i a l re tardat ion , small head pedigree - case 5 67 • # trisomy-D due to Dq Dq translocation B<3 = trisomy-D due to Dp Dp translocation M% - D/D translocation carrier 0 © = normal chromosomes • O = not tested + = dead Figure 44. Pedigree - case 5 TABLE 5 Number on pedigree Number of chromosomes < 44 44 XS 46 47 >47 TOTAL i : - -. i 1 3 1 27 - 32 I. -~i 1 5 3 32 - - 41 (K - ~M 2 - 2 31 - - 35 OL - 3 1 1 2 25 1 - 30 0 - 4 1 - 1 6 23 1 32 68 A * U S ! 1 ^ 3 .Hi 3 4 !. - H I 3 ; l l a | X 6 .2 1.1 JLIM U HI .3 14 15 16 17 18 l l * \ * _ 1!) 20 21 22 C Figure 45. Giemsa-banded karyotype - ind iv idua l pedigree 5-1-1 U If n ir i t ii II II II 1C If 1 1 % A 1 # 1 ft i t i t * # f • 9 t * • Figure 46. Fluorescent karyotype - i n d i v i d u a l pedigree 5-1-1 69 »— *> *• * i M M N * sr •1 H ... *A A * * * 13 *> if. h 14 I I if) * * i t Hi I / in 1!) * % A) ?1 2? Y Figure 47. Giemsa-banded karyotype - proband case 5 » M 13 I* 1 2 3 -I #1 * ft I gift j i g * x 6 12 A i t * * * 13 M 16 i i ; 19 ,'(1 ft • • - a & i n 1 t 21 • Figure 48. Giemsa-banded karyotype - i n d i v i d u a l pedigree 5-1-2 70 ss ** i»' * a 4 t t z: n x 6 1? 13 14 15 |6 1/ 16 - . V * •• • 19 ;o 21 22 Figure 49. Giemsa-banded karyotype - ind iv idua l pedigree 5-0-1 Ul*!) i t If 1 2 3 4 5 11 V I M ! ! Ir If 1.1. I* 6 12 •_t_ U MM. ML 11 13 14 15 16 17 18 J t i J i Mil , M _ 19 20 21 22 Figure 50. Giemsa-banded karyotype - i n d i v i d u a l pedigree 5-0-4 71 I 0 X '. l j _ * 13 14 Figure 51. P a r t i a l Giemsa-banded karyotype - proband case 5 ir i i ii ii ii II II • « II i: I I • i S i « • 1 1 • A » • 1. * • . Figure 52. Fluorescent karyotype - proband case 5 4 ft Figure 53. Fluorescent karyotype - i n d i v i d u a l pedigree 5-1-2 U l) a i . i . t i a i * t ft « # Figure 54. Fluorescent karyotype - i n d i v i d u a l pedigree 5 -0-1 73 Figure 55. F l u o r e s c e n t karyotype - i n d i v i d u a l pedigree 5-0-4 Figure 56. Schematic diagram of D/D t r a n s l o c a t i o n - case CHAPTER IV DISCUSSION In the i n t r o d u c t i o n , cer ta in questions were asked con-cerning the t(Dq Dq) t rans locat ion chromosome in man. These questions were: 1) which chromosome pa i r or pa i r s are involved in the t r ans loca t ion ; 2) where are the poss ible break points involved; 3) what i s the segregation pattern of a f a m i l i a l t(Dq Dq) t r ans loca t ion ; and 4) what i s the possible mechanism or mechanisms of formation of the t(Dq Dq) t rans locat ion? A. Segregation of the t(Dq Dq) translocat ions  1) Homologous t(Dq Dq) translocat ions The segregation of the t(Dq Dq) t rans locat ion d i f f e r s depending on the chromosomes involved in the t rans loca t ion . Re-sults from the present study have shown homologous t(Dq Dq) trans locat ions in two of the cases cy togenet ica l ly examined. The proband in pedigree 1 i s an infant with a homologous t(14q;14q) t rans loca t ion . This is only the second report of th i s type of homologous t(Dq Dq) chromosome, the f i r s t was re-ported by Hulten and Lindsten (19 70) and Caspersson et a l . (1971). The father of the proband in pedigree 2 i s a 45 year old male (1974) with a homologous t(13q;13q) t rans loca t ion . 75 76 This form of homologue has been the most commonly reported amongst the t(Dq Dq) type (de Grouchy et a l . , 1970; Cohen, 1971; Niebuhr, 19 72a; Emberger et a l . , 19 72 ; Hsu et a l . , .19 73; Gardner et a l . , 1974). Lucas (1969) examined a female who had had 13 pregnancies, a l l of which terminated before the 15th week of gestat ion. She was shown to be a c a r r i e r of a homologous 15/15 t rans loca t ion . Caspersson et a l . (1971) ascertained a homologous 14/14 translo-cation in the husband of a female who had recurrent miscarriages . Parslow et a l . (1973) examined a female who had experienced three miscarriages a l l terminating before the 12th week and had no nor-mal pregnancies. They found that she was a c a r r i e r of a homolo-gous 13/13 t rans loca t ion . The reproductive h i s t o r i e s of the homologous t(Dq Dq) t rans locat ion car r i e r s show a high rate of abortion presumably due to the formation of trisomy -D embryos. The trisomy -D cons i t tu t ion would vary depending on the chromosome p a i r involved in the t rans locat ion . K a j i i et a l . (1975) reported that a l l seven of D - t r i somic abortions studied by banding techniques were e i ther trisomy 14 or 15. This i s contrary to the findings in l iveborn in fant s , whose extra D chromosome i s chromosome 13. There has been no instances of trisomy 14 or 15 found in l i v e -born ind iv idua l s although a case of trisomy 14 mosaicism i s known (Gendel et a l . , 1970). There also have been reports of p a r t i a l 77 trisomy -D syndromes in l iveborn ind iv idua l s for chromosome 14 and 15. Cases of p a r t i a l trisomy 14 (Short et a l . , 1972; Muldal et a l . , 1973) and p a r t i a l trisomy 15 (Webb et a l . , 1971; Parker and A l f i , 1972 ; Bucher et a l . , 1973; Crandall et a l . , 1973) are not separable by c l i n i c a l phenotype. The segregation of the homologous t(13q;13q) transloca-t ion in pedigree 2 i s consistent with that shown in previous cases (Sinet et a l . , 1973). The proband in pedigree 2 was shown to be a c a r r i e r of a t(Dq Dq) t rans locat ion which can be presumed to be s i m i l a r to the t(13q;13q) t rans locat ion observed in the pro-band's father. The f i r s t born female of pedigree 2 f a i l e d to survive and was found to have a heart defect and hyal ine mem-brane disease. With the cytogenetic f inding of the t(13q;13q) t rans locat ion in this female's father , i t might be presumed that she was also a t rans locat ion trisomy -13. She could not be studied cytogenet ica l ly at that time of death. Although the l iveborn t rans locat ion trisomy -13 and the spontaneous abortions give evidence of the consistent presence of the homologous t(Dq Dq) chromosome, the nu l l i somic port ion of segregation must be considered. I t i s thought that the mono-somy embryo dies before implantation since monosomy D abortuses are not found. There has been one report of a D group monosomy in a l iveborn i n d i v i d u a l (Pelz , 1971). The 45 XY, -D monosomy karyotype was a consistent observation in a l l studies of the p e r i -pheral blood. There was no evidence of mosaicism or t rans locat ion 78 in 120 c e l l s . The proband, a male, had several phenotypic an-omalies and died at s ix months of age. This report would have been more valuable i f skin biopsies had been used to v e r i f y the monosomy karyotype and i f the monosomy D chromosome had been ident i f i ed . A syndrome for p a r t i a l monosomy 13 (Grosse and Schwanitz, 1973; Ikeuchi et a l . , 1974) is found e i ther as a simple de le t ion or r ing formation (Zink et a l . , 1973; Niebuhr, 1973a, 1973b; Niebuhr and Ottosen, 1973). Another report shows syndrome as-soc ia t ion due to the D - group delet ions (Lehrke et a l . , 1971) of p a r t i c u l a r D chromosomes which resul t s in p a r t i a l D - group monosomy karyotypes. The low sperm count reported in pedigree 2 (1-4) may be due to nu l l i somic sperm not maturing. I f this i s true then the only sperm type in this male would carry the homologous t(13q;13q) t r ans loca t ion . Of in teres t i s the case reported by Parslow et a l . (1973). They reported a female homologous 13/13 t rans locat ion c a r r i e r who never had a pregnancy proceed beyond the 12th week. While in pedigree 2 of the present study, the male homologous t(13q;13q) t rans locat ion c a r r i e r had both his conceptions pro-ceed to f u l l term. The other reports of homologous t(Dq Dq) t rans loca t ion were in new-born pat ients and no reproductive h i s t o r i e s are avai lable (Rowley and Pergament, 1969; de-Grouchy et a l . , 1970 ; 79 Cohen, 1971; M i l l e r et a l . , 1971; Niebuhr, 1972a; Yoshida et a l . , 1972). It may be predic ted that the proband of pedigree 1, a homologous t(14q;14q) t rans locat ion c a r r i e r , w i l l be un-able to father l ive-born i n d i v i d u a l s . A l l conceptions w i l l be e i ther trisomy 14 and w i l l spontaneously abort or i f monosomy for chromosome 14 w i l l probably be lo s t p r i o r to implantat ion. 2) Non-homologous t(Dq Dq) t rans locat ions The segregation patterns of non-homologous t(Dq Dq) trans locat ions d i f f e r from those of homologous t(Dq Dq) trans-loca t ions . The major dif ference i s that non-homologous types may produce normal or balanced c a r r i e r karyotypes while the homologous types can only produce t rans locat ion tr i somic karyo-types . The most commonly reported t(Dq Dq) t rans locat ion in the l i t e r a t u r e involves chromosomes 13 and 14. These are the chromosomes involved in the t(Dq Dq) translocat ions of the fam-i l i e s reported in pedigrees 3 and 4. In pedigree 3, there i s only one l i v e b i r t h which i s a t(13q;14q) t rans locat ion c a r r i e r . This t(13q;14q) t rans locat ion chromosome was found in the mother of the proband as w e l l . There i s one report of a spontaneous abortion but there i s no further information on i t . Owing to the small pedigree, no conclusions can be made on the segrega-tion pattern of the t(13q;14q) t rans locat ion in this family. A much larger family i s ava i lab le in pedigree 4. With the exclusion of the proband and the abortuses from the ana lys i s , 80 the t r a n s l o c a t i o n i s shown t o have been t r a n s m i t t e d t o e i g h t o f f o u r t e e n progeny. T h i s i s a n o n - s i g n i f i c a n t d e v i a t i o n from a 1:1 r a t i o f o r t h e t r a n s l o c a t i o n c a r r i e r t o n o n - c a r r i e r . These r e s u l t s d i f f e r from t h o s e o f Hamerton (1972) and i t c o r r e s p o n d s w i t h t h o s e o f J a c o b s e t a l . ( 1 9 7 4 ) . T h e r e i s no d e v i a t i o n i n t h e sex r a t i o from 1:1 w i t h i n the t r a n s l o c a t i o n h e t e r o z y g o t e s which c o r r e s p o n d s w i t h the r e s u l t s o f Hamerton (1972) and J a c o b s e t a l . ( 1 9 7 4 ) . No chromosomal o r . g e s t a t i o n a l i n f o r m a t i o n i s known ab o u t t h e o n l y two spontaneous a b o r t i o n s i n t h e f a m i l y so no comment can be made about t h e i r s i g n i f i c a n c e t o the s e g r e g a t i o n a n a l y s i s . From the f a m i l y p e d i g r e e s 3 and 4, t h e r e i s no e v i d e n c e o f a d j a c e n t s e g r e g a t i o n i n the male o r f e m a l e t(13q;14q) t r a n s l o c a t i o n c a r r i e r s . A d j a c e n t s e g r e g a t i o n i n the s p e r m a t o c y t e o r o o c y t e o f t h e s e c a r r i e r s would p r o d u c e a d i s o m i c gamete which on f e r t i l i z a t i o n would be t r i s o m i c f o r chromosomes 13 o r 24. P u b l i s h e d r e s u l t s how a d j a c e n t s e g r e g a t i o n t o be o f t h e v e r y low l e v e l o f a p p r o x i m a t e l y 1 - 2 % ( D u t r i l l a u x and L e j e u n e , 1970; Hamerton, 1972; S i n e t e t a l . , 1973; J a c o b s ' e t a l . , 1974). S e g r e g a t i o n i n p e d i g r e e s 3 and 4, as d e t e r m i n e d from the l i v e - b o r n i n d i v i d u a l s , i s a l t e r n a t e , t h e r e b y p r o d u c i n g i n d i v i d u a l s who a r e t(13q;14q) t r a n s l o c a t i o n c a r r i e r s o r have normal k a r y o t y p e s . The chromosomes i n v o l v e d i n the t r a n s l o c a t i o n i n p e d i g r e e 5 a r e chromosomes 13 and 14. The major d i f f e r e n c e between t h i s c a s e and t h o s e i n p e d i g r e e s 3 and 4 i s the p r e s e n c e o f a 81 smaller t(13p;14p) t rans locat ion as wel l as the larger t(13q;14q) t rans loca t ion . The resul t of both alternate and adjacent se-gregation are present in th i s pedigree. The t(13p;14p) trans-locat ion in this family appears to move randomly to e i ther pole at meiosis I. The p o s s i b i l i t y of d i s t r i b u t i v e p a i r i n g (Gre l l and Va lenc ia , 1964; G r e l l , 1967) was considered but since there i s no evidence of non-dis junct ion in chromosomes s imi la r in s ize to the t(13p;14p) t r ans loca t ion , no comment can be made. The alternate segregation of the t rans locat ion complex i s shown by ind iv idua l s in. pedigree 5 (0-1 and 0-4). The f i r s t sib (0-1) i s a c a r r i e r of the t(13q;14q) and t(13p;14p) trans-locations and four normal U chromosomes, while the fourth sib (0-4) has the t(13p;14p) and s ix normal D chromosomes. The adjacent segregation of the t rans locat ion complex is shown by ind iv idua l s in pedigree 5 (0-2 and 0-3). The pro-band (0-3) i s a t rans locat ion trisomy 13 and does not carry the t(13p;14p) t rans loca t ion . Although no autopsy or cytogenetic report was done on the s t i l l b o r n (0-2), mult iple anomalies i n -cluding c l e f t palate and Polydactyly were noted at b i r t h . These c l i n i c a l signs suggest that the infant had the trisomy -13 syndrome. B. Possible mechanism of formation of the t(Dq Dq) t rans locat ion The formation of the homologous t(Dq Dq) trans locat ions on theore t i ca l grounds must have occurred at the f i r s t d i v i s i o n 82 of the f e r t i l i z e d ovum, i f no mosaicism is present. A prezygot ic o r ig in i s u n l i k e l y because the e f fect ive disomic gamete, f e r t i -l i z e d by a normal gamete would produce a tr i somic zygote. If i t was of prezygot ic o r i g i n , i t would be necessary to postulate add i t iona l abnormalities of chromosome d i s t r i b u t i o n in the other gamete to produce the e f f e c t i v e l y balanced chromosome state as shown in pedigrees 1 and 2. Chromosome counts and c e l l analysis on cultured leukocytes showed the modal chromosome number was 45 i n pedigrees 1 and 2. These resul t s suggest there i s no mosaicism present in e i ther case. It appears that the mechanism of formation for the homo-logous t(Dq Dq) t rans locat ion was d i f fe rent in pedigrees l;:and 2. Its formation in pedigree 1 i s due to a rec iproca l translo-cat ion . The chromosome in pedigree 1 (0-1) appears to have one centromere and i s non-symmetrical on e i ther side of the centro-mere, as shown by G and C-banding. This type of r e c i p r o c a l trans-locat ion i s the mechanism o r i g i n a l l y proposed for the formation of the t(Dq Dq) trans locat ions (Hamerton, 1972). By this mech-anism the break points would be in the p region of one chromosome 14 and the q- centromere region of the other chromosome 14. This type of r ec iproca l t rans locat ion may be considered "incom-p le te " because there i s no evidence for the presence of the centromere -short arm fragment due to the break in the long arm-centromere region of one chromosome 14. 83 In pedigree 2, the morphological evidence suggest that the formation of the t rans locat ion was of the cent r i c fusion type. The t rans locat ion chromosome in this pat ient (1-4) ap-pears to have two centromeres and i s completely symmetrical on e i ther side of the centromeric region, as shown by G and C-banding. Hsu et a l . (1973) discussed the centr i c fusion mech-anism of t rans locat ion formation which has the break points through each centromere. Niebuhr (19 72a) assumed that breaks in the short arms of both acrocentr ics involved in the t(Dq Dq) or t(Gq Gq) translocat ions in man are more common than had pre-vious ly been recognized. Niebuhr reported that in one case, he found only 6% of t rans locat ion chromosomes showed two centro-meres. This was interpreted as i n s t a b i l i t y of the d i c e n t r i c centromere region. He explained th i s i n s t a b i l i t y by suggesting that c r i s s -c ros s separations with a breakage - fusion - bridge c y c l e , which tended to diminish the intercentromeric segment could occur, r e su l t ing in fusion of the two centromeres. Hsu et a l . (1973) state that the breaks in the short arms followed by reunion between the chromosomes, cannot be excluded, but is less l i k e l y in view of the s t a b i l i t y of the t rans locat ion chromo-some . The poss ible i n s t a b i l i t y of a centromere region in a f a m i l i a l t(Dq Dq) t rans locat ion was shown in a female who was mosaic for two major c e l l l ine s (Sinha et a l . , 1972). One c e l l l ine had the t(Dq Dq) t rans locat ion and the other c e l l l i n e 84 had 46 chromosomes. In the 46 chromosome c e l l l i n e , however, a normal D-chromosome was replaced by a t e l o c e n t r i c marker chromo-some comparable i n s ize to the long arm of a D chromosome. Sinha and his colleagues suggested that during embryonic l i f e , one c e l l with the t rans locat ion underwent centr i c misd iv i s ion and this was followed by a p e r i c e n t r i c invers ion in one of the te lo-c e n t r i c s . This p e r i c e n t r i c invers ion mechanism resu l ted in the formation of the marker D chromosome with large short arms. The authors suggested that healing occurred at the broken centro-meric regions so that f u l l centromeric a c t i v i t y was restored to each re su l t ing chromosome. Some authors (for example - Emberger et a l . , 1972) have proposed that the homologous t(Dq Dq) translocat ions are due to isochromosome formation. Hsu et a l . (1973) suggested that the presence of two centromeres in the homologous t(Dq Dq)trans-locat ion in the i r case excluded the p o s s i b i l i t y that the i r trans-locat ion was due to isochromosome formation. This would also be the conclusion for the t(13q;13q) t rans locat ion in pedigree 2. Niebuhr (1972 a,b) suggested that in t(Dq Dq) trans lo-cations which appear to be monocentric chromosomes are d i c e n t r i c ; there has been some mechanism of suppressing the a c t i v i t y of one of the centromeres. He suggested that the distance between the centromeres was a determining factor . The breaks would have to occur in the short arms of the involved chromosomes. Other suggestions have been that the dominant centromere suppresses 85 the weaker centromere leaving i t to act only as a connection between the break point and the long arm of the chromosome (Niebuhr, 1972c). It seems more reasonable to favour the hy-pothesis that the breaks may occur i n the short arms of both the involved chromosomes. These breaks may occur close enough to the centromere that no or a minute segment of short arm material i s present. This would allow cent r i c fusion to occur during the formation of the t(Dq Dq) t rans locat ion and may a l -low the d i c e n t r i c structure to be i d e n t i f i e d i n cer ta in cases. Theipe is good evidence of breaks occurring in the short arm regions of the acrocentr ic chromosomes with reports of short arm delet ion chromosomes (Emerit et a l . , 1972; Hoo et a l . , 1974) and complete r ec iproca l t rans locat ions of acrocentr ic chromo-somes (Palmer et a l , , 1969; de Grouchy et a l . , 1970 ; Emerit e_t a l . , 19 72). The mechanism of formation for the t(Dq Dq) transloca-tions are thought to be s imi l a r regardless of whether homologous or non-homologous chromosome pairs are involved. With respect to the G and C-banding r e s u l t s , the t(13q;14q) t rans locat ion chromosome in pedigree 3 appears poss ibly to be d i c e n t r i c . This d i c e n t r i c appearance was not v i s u a l i z e d i n every c e l l analyzed but was observed i n c e l l s from both the proband and his mother. There was no evidence of i n s t a b i l i t y for this d i c e n t r i c chromo-some. The mechanism of formation may be s imi l a r to that in case 2. In pedigree 4, the t(13q;14q) translocated chromosome 86 appeared monocentric and the mechanism of formation was poss i-bly an incomplete r ec ip roca l t rans loca t ion . This c l a s s i c a l mechanism was discussed prev ious ly . In pedigree 5, the mechanism of formation can be con-cluded to be a complete r e c i p r o c a l t rans locat ion due to the presence of the t(13p;14p) t rans loca t ion . This chromosome i s made up of the short arms and the centromere of chromosome 13 and part of the short arms of chromosome 14. The presence of t(Dp Dp) or t(Dp Gp) translocat ions have been previous ly re-ported (Palmer et a l . , 1969; de Grouchy et a l . , 1970; Emerit et a l . , 19 72). The t(13q;14q) t rans locat ion chromosome i s made up of the long arms and centromere of chromosome 14 and the majority of the long arms of chromosome 13. C. C l i n i c a l and chromosome associat ions The c l i n i c a l phenotype shown by the proband in pedi-gree 1 may be co inc identa l to the presence of the homologous t(14q;14q) t rans loca t ion . This proband (0-1) was ascertained because of short stature which is a common abnormality found in a majority of ind iv idua l s with autosomal aberrat ions . The other features reported werejfmild hypotonia, small jaw, and underdeveloped scrotum. These c l i n i c a l observations are seen in many other chromosomal abnormalities and also i n non-chromosomal disorders . They may, therefore , be co inc identa l . In pedigree 2, aside from the report of a low sperm 87 count, there are no phenotypic abnormalities in the homologous t(13q;13q) t rans locat ion c a r r i e r (1-4). The proband (0-2) was shown cytogenet ica l ly to be a t rans locat ion trisomy -D and based on the phenotypic c l i n i c a l signs was diagnosed trisomy-13. In view of f a i l u r e to t h r i v e , heart defect , and hyaline membrane disease, I suggest that the f i r s t born female (0-1) was also a trisomy-13. In pedigrees 3 and 4, i t i s the proband's mother who i s the c a r r i e r of the t(13q;14q) t r ans loca t ion . As mentioned prev ious ly , some authors report an increase in the r i sk of spontaneous abortion for female t(Dq Dq) c a r r i e r s . On the contrary, other authors report no increase i n the r i s k of spon-taneous abortion for female t(Dq Dq) c a r r i e r s . The three fe-male t(13q;14q) ca r r i e r s (one from pedigree 3; two from pedi-gree 4) had three spontaneous abortions in nine pregnancies. A l l the reported abortions were from the mothers of the two probands in pedigrees 3 and 4. The mother (1-4) in pedigree 3 had one spontaneous abortion in two pregnancies while the mother (1-2) in pedigree 4 had two spontaneous abortions in f ive pregnancies. There i s a report of a female t(13q;14q) c a r r i e r who had had three spontaneous abortions whereas her mother who had a (or the same), s i m i l a r t r ans loca t ion , had no abortions i n eleven pregnancies (Kohno and Makino, 1969). Their report was not mentioned with the assumption that the chromosome i s causal of the spontaneous abort ions , as i t i s b i a s e d due t o the f a c t t h a t the f e m a l e was a s c e r t a i n e d due t o t h r e e spontaneous a b o r t i o n s . As m entioned p r e v i o u s l y , t h e s e t(Dq Dq) t r a n s l o c a t i o n chromosomes may have het e r o g e n e o u s e f -f e c t s w i t h i n a f a m i l y . I t must a l s o be c o n s i d e r e d t h a t t h e s e e f f e c t s m i ght be independeny o f the t ( D q Dq) t r a n s l o c a t i o n and due t o o t h e r c a u s e s . The f a m i l i e s i n c a s e s 3 and 4 were both a s c e r t a i n e d t h r o u g h m e n t a l l y r e t a r d e d p r obands. The probands were shown t o be c a r r i e r s o f t(13q;14q) t r a n s l o c a t i o n s as were o t h e r mem-bers o f e ach f a m i l y who were p h e n o t y p i c a l l y n ormal. Modal chromosome numbers o f 45 were f o u n d i n the probands and t h e r e was no i n d i c a t i o n o f m o s a i c i s m i n the l e u k o c y t e c u l t u r e s . T h e r e a r e s i m i l a r r e p o r t s ( P i t t e t a l . , 1964; Glogowska e t a l . , 1971; C r a n d a l l e t a l . , 1972; R a a i j m a k e r s - E n g e l s e n , 1973) o f m e n t a l l y r e t a r d e d t ( D q Dq) t r a n s l o c a t i o n c a r r i e r s whose chromosome number and k a r y o t y p e were s i m i l a r t o o t h e r 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 i n t h e same f a m i l y . V a r i o u s r e a s o n s have been sug-g e s t e d by t h e s e a u t h o r s f o r the p o s s i b l e cause o f t h e mental r e t a r d a t i o n . Most a u t h o r s s u g g e s t t h a t the c l i n i c a l f i n d i n g s i n t h e proband may n o t be c a u s a l l y r e l a t e d t o the chromosome a b n o r m a l i t y , i . e . the a s s o c i a t i o n i s m e r e l y f o r t u i t o u s . In t h e r e p o r t by Glogowska e t a l . ( 1 9 7 1 ) , t h i s i s p r o b a b l y t h e c a s e as t h e probands were a s c e r t a i n e d i n a m e n t a l l y r e t a r d e d p o p u l a -t i o n . A second common s u g g e s t i o n , which would be hard t o a c c o u n t 89 for , i s that an addi t iona l small chromosomal anomaly i s present in the proband and not in the remaining family members. A t h i r d suggestion was given by Crandal l et a l . (1972), who re-ported two s i s te r s with t(Dq Dq) t rans locat ion chromosomes. Both s i s t e r s seemed to have s imi l a r c l i n i c a l syndromes while other family members inc lud ing those with the t(Dq Dq) trans-locat ion were phenotypica l ly normal. The authors suggested that the two s i s te r s maykhave been hemizygous for a recessive mutant gene inher i t ed from the i r father because of the loss of the homo-logous a l l e l e on the t rans locat ion inher i t ed from t h e i r mother. Raaijmakers-Engelsen (1973) made a s i m i l a r proposal for the mental retardat ion shown in two probands with d i f ferent t(Dq Dq) t rans locat ions . Chromosome 14 was involved in each as one was a t(13q;14q) and the other a t(14q;15q). He f e l t the retarda-t ion may be due to hemizygosity for a cer ta in part of chromo-some 14. Jacobs (1974) presented data which suggested that the possession of a de novo euploid s t ruc tura l rearrangement of the autosomes i s , i n some cases, associated with and presumably causal to , severe mental re tardat ion . She suggested three mech-anisms for such an e f f ec t : a) the rearrangement is in r e a l i t y unbalanced and carr ies a small de le t ion below the l e v e l of cyto-l o g i c a l d e t e c t a b i l i t y ; b) chromosome breakage and exchange re-sults in a gene mutation at one or both the break po int s ; and c) cer ta in rearrangements are associated with a p o s i t i o n ef fect 90 detrimental to t h e i r c a r r i e r s . Although her data i s for de novo t rans loca t ions , her suggestions are presented for completeness. In pedigree 4, the proband's retardat ion was thought to be due to cerebral anoxia in utero during his mother's tox-emia and convulsive per iod . Other supportive evidence for this suggestion is the large number of phenotypica l ly normal t(13q;14q) t rans locat ion c a r r i e r s in his family. No add i t iona l s t r u c t u r a l rearrangement could be detected from the proband's karyotype. In pedigree 3, i t i s in te re s t ing that the proband's mother had an abnormal pregnancy owing to her diabetes , exposure to r u b e l l a , f l u - l i k e symptoms and a kidney i n f e c t i o n . Although these compli-cations were not s i m i l a r to those described in the pregnancy of the proband's mother in pedigree 4, they may have had some bear-ing on the r e s u l t i n g re tardat ion . No addi t iona l s t ruc tura l re-arrangement could be detected in the proband's (pedigree 3 - 0-1) karyotype. It must also be considered that the retardat ion in the probands of pedigrees 3 and 4 may be unrelated to the karyo-typic aberrat ion. The c l i n i c a l phenotype, aside from the mental retarda-t i o n , shown by the proband i n pedigree 3 may be co inc identa l to the presence of the t(13q;14q) t rans loca t ion . As mentioned prev ious ly , short stature i s commonly associated with autosomal aberrat ions. Small head, s p a s t i c i t y , strabismus, and mental retardat ion are seen i n many other chromosomal aberrat ions . 91 The strabismus and s p a s t i c i t y may be secondary owing to another factor which has caused retarded brain development as shown by the small head. The apneic- l ike spe l l s in ear ly infancy in the pedigree 3 proband may be co inc identa l or re la ted to the retarded development of the centra l nervous system. In pedigree 4, a l l the t(13q;14q) t rans locat ion ca r r i e r s except the proband (0-10) have normal development. Of in teres t is the presence of a horseshoe kidney in the 0-11 i n d i v i d u a l who is a t(13q;14q) c a r r i e r . Renal anomalies are found in less than 50% of the observed cases of trisomy 13 syndrome but , when pre-sent, a horseshoe kidney is occas ional ly found. This is poss ib ly co inc identa l in this i n d i v i d u a l (0-11). In considering pedigree 5, ascertained because of the chromosome inves t iga t ion of the mentally retarded proband, much of the discussion w i l l be concerned with the d i f fe rent expression of the two types of t rans loca t ions , the large t(13q;14q) and the small t(13p;14p). It i s the t(13q;14q) t rans loca t ion which makes the proband (0-3) tr i somic for most of the long arm of chromosome 13. It i s the t(13p;14p) t rans locat ion vrhich makes i n d i v i d u a l (0-4) tr i somic for a small port ion of the long arm proximal to the centromere, the centromere, and short arms of chromosome 13 and a port ion of the short arm of chromosome 14. Thus these two ind iv idua l s in pedigree 5 are tr i somic for d i f -ferent parts of chromosomes owing to the formation of the two 92 trans locat ion chromosomes by complete r ec iproca l exchange. The other two ind iv idua l s in pedigree 5, 1-2 and 0-1, who are pheno-t y p i c a l l y normal, are completely balanced for these transloca-tions as they have the t(13p;14p) and the t(13q;14q) t rans loca-tions with the remaining four normal D chromosomes (one 13, one 14, and two 15 ' s ) . A summary of the associated c l i n i c a l signs usual ly found in trisomy -13, as adapted from Smith (1970), i s shown by Table 6. Trisomy-13 syndrome summaries also considered were Taylor (1968) and Thomas and Scott (1973). As shown by Table 6, the c l i n i c a l signs which are common to both 0-3 and 0-4 ind iv idua l s are de-velopmental/mental re ta rda t ion , hypotonia and c a p i l l a r y haeman-gioma. The proband (0-3), who i s tr i somic for the majority of the long arm of chromosome 13, has in addit ion a mixed convulsive d i sorder , Po lydac ty ly , a simian crease, undescended t e s t i c l e s , hyperextensible j o i n t s , and a highly arched pa la te . This i n d i v i -dual does not have the c h a r a c t e r i s t i c facies of the majority of patients with trisomy-13. There were no abnormalities in the cardiovascular , pulmonary, or alimentary systems. The female i n d i v i d u a l (0-4) who i s trisomy for the proximal part of the long arm of chromosome 13, the centromere and short arms of chromosome 13 and a port ion of the short arm of chromosome 14, has, in add i t ion , a c l e f t pa late , strabismus, f inger f lex ion and overlapping and a small head (3rd p e r c e n t i l e ) . These c l i n i c a l signs are s i m i l a r to some of the c l i n i c a l signs present 93 TABLE 6 COMPARISON OF CLINICAL SIGNS FOR TRISOMY 13 P5-0-3 P5-0-4 >8CH congenital heart developmental/mental retardat ion + + se izures , j i t t e r i n e s s , apnea moderate microcephaly with s loping + forehead + microphthalmia i r i s colobomata c l e f t palate + c l e f t l i p malformed ears >50l g l a b e l l e r c a p i l l a r y haemangiomata + + cut i s aplas ia ( sca lp , neck) f i n g e r n a i l s , long, hyperconvex f inger s , f lexed, overlapping simian creases Polydactyly d i s t a l t r i r a d i i ta l ipes equinovarus gen i ta l anomalies u m b i l i c a l / i n g u i n a l hernias hypo or hypertelorism thumbs r e t r o f l e x i b l e + + + + C50% f i b u l a r S-shaped h a l l a c a l pattern hypo or hyper tonia strabismus abdominal anomalies renal malformation + + + + Smith (1970) 94 in a p a r t i a l trisomy 13, r e s u l t i n g from a minute extra chromo-some shown to be the centromere and short arms of chromosome 13 (Krmpotic et a l . , 1971). Neither the proband (0-3) nor the female sib (0-4) f i t into the c l a s s i c trisomy -13 syndrome as shown by Table 6. Neither pat ient has the d i s t i n c t i v e facies of trisomy 13 but they both look s i m i l a r to each other. There are no eye defects and no congenital heart defects in e i ther pat ient . The l a t t e r may be the reason why these ch i ldren have l i v e d to 5 years (0-3) and 2 years (0-4). Less than 18% of t r i -somy -13 patients l i ve u n t i l the i r f i r s t b ir thday. There i s very l i t t l e overlap in c l i n i c a l signs between the two patients except in the major area of developmental/mental retardat ion and c a p i l l a r y haemangiomata. However, while the majority of other signs are d i f ferent they appear in greater than 50% of the trisomy -13 ind iv idua l s as shown in Table 6. It may be proposed these differences are due to the difference in the chromosome materia l which i s t r i somic in each pa t ient . For comparison, there are reports of p a r t i a l trisomy syndromes in l i v e b i r t h s for chromosome 14 (Short et a l . , 1972; Muldal et a l . , 19 73) and 15 (Webb et a l . , 19 71; Parker and A l f i , 1972; Magenis et a l . , 1972; Bucher et a l . , 1973; Crandall et a l . , 1973). These two forms of p a r t i a l trisomy cannot be d i s t inguished c l i n i c a l l y . The p a r t i a l trisomy 14 cases have abnormalities common to a majority of autosomal aberrat ions . The p a r t i a l t r i -somy -14 case described by Short et al . , (1972) had phenotypic 95 anomalies such as microphthalmia with hypertelor ism, nasal beaking, hypoplast ic mandible, short neck, b a r r e l chest , and kyphotic spine in addit ion to the abnormalities common to a majority of autosomal aberrat ions . Muldal et al.(1973) found a p a r t i a l trisomy 14 karyotype in a mentally retarded adole-scent with short s tature . The p a r t i a l trisomy 15 cases tend to be moderately retarded and hyperact ive , they do not seem to have any growth retardat ion and have minimal phys i ca l ab-normal i t i e s . The b i r t h weights of autosomal trisomy infants are s i g n i f i c a n t l y lower than those of normal newborns (Chen et a l . , 1972). The order of decreasing b i r t h weights i s trisomy 21 > trisomy 13>tri-somy 18. The proband's (0-3) b i r t h weight was 1960 grams, which is lower than the 2480 gram mean mentioned by Smith (1970). It should be r e c a l l e d that th i s weight was at 38 weeks gestat ion. The b i r t h weight of 0-4 was 3494 grams which was above the mean b i r t h weight. Although these are i so-la ted cases, these b i r t h weights may r e f l e c t the amount of gene-t i c material which i s t r i somic , as 0-3 has the lower b i r t h weight and the larger chromosome port ion in the tr i somic s tate . The defects of mid-face, eye and forebra in , which occur in var iable degree as a feature of the trisomy -13 (Smith, 1970) , appear to be the consequence of a s ingle defect in the ear ly ( t h i r d week) development of the prechordal mesoderm. This meso-derm is not only necessary for the morphogenesis of the mid-face, but exerts an inductive role on the subsequent development of the prosencephalon, the forepart of the b r a i n . This type of 96 defect has been referred to as arhinencephaly and varies in sever i ty from cyc lop ia (Taysi and Tinaztepe, 1972; Fujimoto et a l . , 1973) to cebocephaly and less severe forms. The c l i n i c a l expression of the t(13p;14p) t rans locat ion tends to confuse previous assumptions concerning the short arms of the acrocentr ic chromosomes. Independent of the mechanism of formation of the t(Dq Dq) t rans loca t ions , whether by rec ipro-cal t rans locat ion re su l t ing in a monocentric t rans locat ion chromo-some or by cent r i c fusion re su l t ing in a d i c e n t r i c t r ans loca t ion , there i s always a loss of some short arm mater ia l . The excep-t ion to th i s assumption i s the rare f inding of a complete r e c i -proca l t rans loca t ion with the r e s u l t i n g formation of the t(Dp Dp) fragment. Deletion of the short arm of the D-group chromosomes have been reported for chromosomes 13 (Bias and MigQon, 1967; Lieber et a l . , 1967; Emerit et a l . , 1968; Brogger, 1969;), chromo-some 14 (Emerit et a l . , 1972), and chromosome 15 (Hoo et a l . , 1974). The general conclusion was that the short arm of the D-group chromosomes does not contain important genetic material and the loss has no genetic consequence. S imi la r conclusions were reached when i t was noted that most t(Dq Dq) t rans locat ion car r i e r s showed no phenotypic ef fect due to the loss of short arm materia l from the two chromosomes involved in the t rans loca t ion . The short arms of the acrocentr ic chromosomes were shown to contain the nucleolar-organize.r regions , f i r s t in mi to t i c c e l l s (Ferguson-Smith and Handmaker, 1961) and l a t e r in human 97 meiotic chromosomes (Ferguson&Smith, 1964). By d i f f e r e n t i a l s ta in ing of nucleolus organizers (N banding) of human meta-phase chromosomes (Matsui and Sask i , 1973), the N bands were shown to be r e s t r i c t e d to the s a t e l l i t e regions of a l l acro-centr i c chromosomes. No s ta in was observed in any other chromo-some or chromosomal regions , inc lud ing the secondary cons t r i c -t ion of chromosomes 1, 9, and 16. When a quinacr ine-f luorescent method was used p r i o r to the N s t a i n i n g , i t showed unequivocally that N bands were located exc lus ive ly on tiie s a t e l l i t e s with no banding on the centromeres, short arms, or s ta lk of the s a t e l l i t e . From the reports previously mentioned on the short arm delet ion evidence and the exact pos i t ions of the nucleolus or-ganizers for the D group chromosomes, cer ta in proposals can be made concerning the t(13p;14p) t rans locat ion in the 0-4 female of pedigree 5: a) i t may be that the loss of these short arm-nucleolar organi-zer regions has no e f fect phenotypical ly on the pat ient but i f these regions are in excess there may be some type of cause and ef fect re la t ionship expressed; b) there may be an important gene or gene sequence located close to the centromere in the long arm of chromosome 13 and thi s gene or gene sequence may be included in the t(13p;14p) trans l o c a t i o n . 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Humangenetik 15, 66-70 (1972). Yunis , J . J . , M. A l t e r , E .B . Hook, M. Mayer. F a m i l i a l D/D Trans-l o c a t i o n . New. Eng l . J . Med. 271, 1133-1137 (1964). Yunis , J . J . , 0. Sanchez. G-Banding and Chromosome Structure . Chromosoma 44, 15-23 (1973). Zeuthen, E . , J . N ie l sen . D/D Translocat ion in Males Examined for M i l i t a r y Service . J . med. Genet. 10, 356-361 (1973). Zink, V . , R. Rix, K .P . Grosse, G. Schwanitz. Ring Chromosome D13. Kasui s t ik und Ubers icht . Klm. Padiatr . 185, 192-197 (1973). 108 APPENDIX A: LEUKOCYTE CULTURE METHOD 1) Cultures were grown in p l a s t i c , s t e r i l e , d i spos ib le tubes with screw tops containing 5 cc of Gibco chromosome medium IA. To each tube was added 5-4 drops of venous blood (approximately 0.25 cc) and the cultures were then incubated at 5 7 ° C for 72 hours. 2) Colcemid in a f i n a l concentration of 0.02 ug/ml was added to the cultures approximately one hour p r i o r to the harvest of the c e l l s . The c e l l s were then gently resuspended in the sealed tube. 5) After the incubation period was completed the contents of the tube were emptied into a centr i fuge , covered with Paraf i lm M]_ and centrifuged at 800 rpm for 8 minutes. The supernatant was removed and discarded and the c e l l s were suspended in 5 cc of hypotonic so lut ion (0. 075 M. KC1) at room temperature for 2-5 minutes. 4) The c e l l s were then centrifuged at 800 rpm for 8 minutes and the supernatant was removed and discarded. A small amount of supernatant (approximately 2-5 drops) was l e f t , in which the c e l l s were suspended, in order to reduce clumping on the addi-t ion of the f ixa t ive (5:1 absolute methyl a l coho l : g l a c i a l acet ic ac id ) . The f i r s t amount of f i xa t ive was added slowly (2-5 drops) and the c e l l s were immediately gently agitated with a PSsteur p ipe t te . More f i xa t ive was then added in l a r -ger volumes u n t i l approximately 5 cc of f i xa t ive had been added and then the tubes were allowed to stand at room temperature 109 for approximately 20 minutes. 5) The c e l l s were then centrifuged and the supernatant was re-moved and discarded. Fresh f i xa t ive was used to wash the c e l l s which were suspended in 5 cc of f i xa t ive and then centri fuged. This washing procedure was usual ly done 2-3 times after the i n i t i a l f i x a t i o n . 6) After the f i n a l wash, the supernatant was removed and dis-carded and the c e l l s were suspended in 5-6 drops of fresh f i x a t i v e . 7) The suspended c e l l s were then placed on precleaned, c o l d , wet s l i d e s . The s l ides were then flamed, a i r - d r i e d and stored at room temperature u n t i l required. 110 APPENDIX B: FLUORESCENT BANDING TECHNIQUE 1) The a i r dr ied s l ides were placed in a 0.5 grams/100 ml so lut ion of Atrebin (quinacrine dihydrochloride) (G.T. Gurr) for a per iod of time greater than 20 minutes. 2) The s l ides were removed, r insed in d i s t i l l e d water and a clean covers l ip was then appl ied. 3) The s l ides were then pressed between pieces of bibulous paper and then the covers l ip was sealed along i t s edges by paraf f in wax. I l l A P P E H D I A C: T R Y P S I N - G Iii M S A ii A D D I N G T E C H N I Q U E 1) The a i r d r i e d s l i d e s were taken tnrough a s e r i e s of s o l u t i o n s , the f i r s t being t r y p s i n s o l u t i o n (1 cc of a 5% t r y p s i n (1:250 Difco) s o l u t i o n made up i,i Hank's Ca and f'g free s a l i n e d i l u t e d to 40 cc i n 0.9% s a l i n e ) . The s l i d e s were l e f t in trypsin, for periods from 5-20 seconds depending on the chro:nosores. 2) I n i t i a l l y , the s l i d e s uere tiicn put through two washes of U.91 s a l i n e and one of phosphate b u f f e r (pli 0.5). This was l a t e r modified. The m o d i f i c a t i o n c o n s i s t e d of one wash i n 1% CnCl2 and two washes of d i s t i l l e d water. 3) The s l i d e s were men s t a i n e d in Giemsa s t a i n (1 cc of Giemsa s t a i n (Gurr L1100), 20 cc phosphate h u f f e r (pil b.5), 30 cc d i s t i l l e d water). The s l i d e s were l e f t in s t a i n f o r a v a r i a o l e l e ngth of time, with the l o n g e s t be inf. 5 minutes. 4) A f t e r tne Giemsa s t a i n , tne s l i d e s were r i n s e d i n d i s t i l l e d water, b l o t t e d dry between bib u l o u s paper and allowed to a i r dry. 3) When the banding and s t a i n i n g was s a t i s f a c t o r y , the s l i d e s were dipped i n x y l o l and mounted i n a Pro-texx(Can Lab) mounting me di um. 112 APPENDIX D: C-BANDING TECHNIQUE 1) The a i r dr ied s l ides were taken through a series of so lu t ions , the f i r s t being 0. 03-0. 07 N NaOil in 0.13 M NaCl. The s l ides were l e f t in this so lut ion for 30-90 seconds at room tempera-ture . 2) The s l ides were then r insed in d i s t i l l e d water and then taken through two a lcohol washes (701 and 991 methanol) and allowed to a i r dry. 3) The a i r dr ied s l ides were treated in Sorensen buffer ( p l f 6.8) (KH 2 P0 4 , 6.63 g, Na 2HP0A 7H 20, 2.56g; d i s t i l l e d H 2 0 to 1000 ml) at 6 8 ° C for one hour. 4) The heated s l ides were then stained in buffered Giemsa solu-t ion (50 ml d i s t i l l e d water, 1.5 ml 0.1M c i t r i c a c i d , adjust pl l with 0.2M Na2HP04 to 6 .8-7.2, 1.5 ml pure methanol, 5 ml stock Giemsa (G.T. Gurr) for 15 minutes). 5) Tne s l ides were then r insed in d i s t i l l e d water, b lot ted dry between bibulous paper and a i r dr ied . 6) Tiie a i r dr ied s l ides were then dipped in x y l o l and mounted in a Pro>texx(Can Lab) mounting medium. 113 APPENDIX E : STAINING TECHNOLOGY The d i f f e r e n t i a l s t a in ing techniques now used in cytogenetics have brought a new order of accuracy to chromosome i d e n t i f i c a t i o n . The techniques and the s p e c i f i c i t y of s t a in ing has been reviewed by a number of authors (Pearson, 19 72; Hirschhorn, 19 73; Hsu, 1973) . Certain optimum conditions are necessary to get the maximum from any of the three s ta in ing techniques used in this study. The f i r s t condit ion which i s important to obtain good banding resul t s i s to control the state of hydration of the chromosome preparat ion. This i s p a r t i c u l a r l y necessary for adequate G-bands (Yunis and Sanchez, 1973) and may be achieved by s tor ing s l ides in a dessicant or as in the present study by f i xa t ion with meth-anol. The second condit ion which is c r i t i c a l i s the technique used in making the chromosome preparat ions . The metaphase c e l l s should be cr i sp and wel l spread with a minimum of cytoplasmic.con-tamination. Adequate banding i s achieved by f i xa t ion with fresh acetic-methanol (Crossen, 1973; Yunis and Sanchez, 1973), washing 3 - 4 times with this f i xa t ive and flame-air drying the s l i d e s . Too much heat makes c lear v i s u a l i z a t i o n of the G bands d i f f i c u l t while too l i t t l e heat makes the v i s u a l i z a t i o n of the G bands im-possible as the chromosomes have a shiny unstainable appearance. The t h i r d condit ion which i s important for more d i s t i n c t separa-t ion of the band and inter-band regions is the use of elongated "114 chromosomes. These are obtained from mitoses arrested in ear ly metaphase. The use of lo\v concentrations of arres t ing agents such as colcemid and the use of short in terva l s of one hour a s s i s t in obtaining these elongated chromosomes. F i n a l l y , the fourth condit ion which i s important, i s the type of hypotonic so lut ion and the period of swel l ing of the n u c l e i . Short periods of 2 minutes in KCl y ie lds the best banding r e s u l t s . The quinacrine dihydrochloride technique (Caspersson et a l . , 1969a,b) resul t s in banding patterns which are unique for each chromosome. Every chromosome in the human complement can be re-cognized through the combination of the p o s i t i o n , the width, and the brightness of the quinacrine bands. It was shown by E l l i s o n and Barr (1972) that quinacrine dihydrochloride had a f f i n i t y at the A-T regions of the chromosome instead of the G-C regions as o r i g i n a l l y proposed by Caspersson et a l . (1969a,b). There are some drawbacks to the f luorescent technique, namely: a) the fluorescence i s not permanent; b) the more recent Giemsa-banding procedures reveal more de ta i l ed banding; c) the brightness of the fluorescence var i e s ; and d) there i s fading of the fluorescence on exposure to the u l t r a - v i o l e t l i g h t . For this reason, c e l l s which possessed morphological cross-overs or were spread too large for a s ingle or double frame picture were not analyzed. Although the f luorescent technique has drawbacks, i t i s extremely useful for the br ight fluorescence of the heterochromatin such as in the human Y. It i s also useful for the i d e n t i f i c a t i o n of the polymerphic 115 centromeric f luorescent marker or chromosome 3 and the s a t e l l i t e regions of the human D and G group chromosomes. The Giemsa-banding resul t s have been derived from so many di f ferent techniques (Seabright, 1971; Pearson, 1972; Hsu, 1973; Hirschhorn, 19 73) that no one knows exactly how the G bands are induced. There i s some evidence that the banding involves both the DNA and the prote in which i s associated with i t (Hsu, 1973). Evidence for th i s inc ludes : a) that G bands are comparable to the Q bands; b) that a var ie ty of compounds capable of inducing G bands have chelat ing proper t ie s ; c) urea i s known to cleave proteins from DNA; d) that actinomycin D which does not react with nuclear proteins but does bind to DNA at the guanine moiety can induce G bands without any pos t - f i xa t ion treatment; e) b io-chemical invest igat ions which suggest a r g i n i n e - r i c h histones p r e f e r e n t i a l l y bind G-C r i c h DNA. The induction of G bands by actinomycin D pre f ixa t ion treat-ment support the notion that dark G bands (or br ight Q bands) represent A-T r i c h regions. The Giemsa-banding technique provides more information on the chromosome structure than the fluorescent banding technique in most cases. These cases are s i m i l a r to those mentioned pre-v ious ly as being useful examples for the f luorescent technique. The optimum trypsin-banding is dependent on the concentration and time of exposure of the chromosomes to the t ryps in so lu t ion and the Giemsa s t a i n . The pH of the s ta in seems to af fect the amount 116 of s ta in ing with the optimum range pH 6.5 - 6.8. Other reports which review factors a f fect ing the Giemsa banding and the banding s p e c i f i c i t y are numerous (Wang et a l . , 1972; Yunis and Sanchez, 1973; Crossen, 1973; Meisner et a l . , 1973a,b). The C-banding technique was f i r s t described by A r r i g h i and Hsu (1971). Their method resul ted in intense s ta in ing with Giemsa at the centromeric regions of the chromosomes and the d i s t a l 2/3 of the long arm of the Y. The basis for this technique was the ob-servation of Pardue and Ga l l (1970) that DNA cons i s t ing of h ighly r e p e t i t i v e copies of short segments renatures more rap id ly than other types of DNA after a lka l ine denaturation. The Giemsa s ta in l oca l i ze s in the renatured port ions of the chromosome. In the pre-sent study the C-banding was used to try to ident i fy the centro-meric regions in the t(Dq Dq) translocat ions and determine whether the translocat ions were monocentric or d i c e n t r i c . The assumption was made that the f a m i l i a l chromosome was i d e n t i c a l in structure regardless of the member of the family carrying the t r ans loca t ion . For this reason C-banding was only done on one or two t rans locat ion car r i e r s in each pedigree. C-bands have been produced by methods other than denaturation-renaturation as progressive t ryps in treat-ment shows the decrease of G-banding and the appearance of C-bands (Merrick et a l . , 1973). There has been evidence to show that chromo-some prote in may have a role in C-banding as wel l as in Q and G banding (Chuprevich et a l . , 1973). 

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