Medicine, Faculty of
Medical Genetics, Department of
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UBCV
Stavropoulos, Dimitri James
Master of Science - MSc
University of British Columbia
Confined placental mosaicism (CPM) is detected in approximately 1-2% of viable pregnancies and has been defined as a dichotomy between the chromosomal constitution of the placental and embryonic/fetal tissues. An investigation for the presence of trisomy in the germ cells was initiated in cases diagnosed with CPM since we suspect that the stroma of the placenta and the primordial germ cells (PGCs) have a common developmental origin. Placental and fetal tissues from one conceptus diagnosed with CPM for trisomy 7 and two conceptuses with CPM for trisomy 16, were analyzed using fluorescence in situ hybridization (FISH). Post-termination conventional cytogenetic analysis of amnion and chorionic stroma from case 1 showed only diploid cells in the amnion and mosaicism for trisomy 7 in the stroma. FISH analysis of formalin fixed - paraffin embedded tissues showed significant levels of trisomy in the placenta while all fetal tissues analyzed including testis were diploid. For Case 2, high levels of trisomy 16 were detected using FISH in both chorionic stroma and cytotrophoblast of the placenta while fetal somatic tissues where diploid. FISH analysis of meiotic oocytes showed significant levels of trisomy 16. In case 3, prenatal metaphase analysis of cytotrophoblast and stroma from chorionic villus sampling showed only 47,XX,+16. Culture of placental vill i after termination showed only trisomy 16 while cultures of multiple fetal tissues showed no evidence of trisomy. FISH analysis of formalin fixed - paraffin embedded lung and meiotic oocytes also failed to show evidence of trisomy 16. The results from Case 2 suggest that conceptuses diagnosed with mosaicism in the chorionic stroma are at risk of exhibiting chromosomal mosaicism in their germ cells. Future studies in similar cases are needed to understand the effects this may have on human fertility in the reproductive period.
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An Investigation of Conceptuses with Placental Chromosomal Mosaicism for the Presence of Germline Mosaicism by Dimi t r i James Stavropoulos B.Sc. McMaster University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES GENETICS GRADUATE PROGRAM We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1997 ^c^ Dimitri James Stavropoulos, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Confined placental mosaicism (CPM) i s detected i n approximately 1-2% of viable pregnancies and has been defined as a dichotomy between the chromosomal con s t i t u t i o n of the placental and embryonic/fetal tissues. An investigation for the presence of trisomy i n the germ c e l l s was i n i t i a t e d i n cases diagnosed with CPM since we suspect that the stroma of the placenta and the primordial germ c e l l s (PGCs) have a common developmental o r i g i n . Placental and f e t a l tissues from one conceptus diagnosed with CPM for trisomy 7 and two conceptuses with CPM for trisomy 16, were analyzed using fluorescence in situ hybridization (FISH). Post-termination conventional cytogenetic analysis of amnion and chorionic stroma from case 1 showed only d i p l o i d c e l l s i n the amnion and mosaicism for trisomy 7 i n the stroma. FISH analysis of formalin fixed - p a r a f f i n embedded tissues showed s i g n i f i c a n t levels of trisomy i n the placenta while a l l f e t a l tissues analyzed including t e s t i s were d i p l o i d . For Case 2, high levels of trisomy 16 were detected using FISH i n both chorionic stroma and cytotrophoblast of the placenta while f e t a l somatic tissues where d i p l o i d . FISH analysis of meiotic oocytes showed s i g n i f i c a n t l e v e l s of trisomy 16. In case 3, prenatal metaphase analysis of cytotrophoblast and stroma from chorionic v i l l u s sampling showed only 47,XX,+16. Culture of placental v i l l i a f t e r termination showed only trisomy 16 while cultures of multiple f e t a l tissues showed no evidence II of trisomy. FISH analysis of formalin f i x e d - p a r a f f i n embedded lung and meiotic oocytes also f a i l e d to show evidence of trisomy 16. The res u l t s from Case 2 suggest that conceptuses diagnosed with mosaicism i n the chorionic stroma are at r i s k of exhibiting chromosomal mosaicism i n t h e i r germ c e l l s . Future studies i n sim i l a r cases are needed to understand the effects t h i s may have on human f e r t i l i t y i n the reproductive period. Ill Contents T i t l e Page I Abstract II L i s t of Tables VIII L i s t of Figures X Acknowledgments X I I Introduction 1.1 Chromosomal Mosaicism 1 1.2 Generalized Mosaicism 1 1.3 Confined Mosaicism 2 1.4 C l i n i c a l Consequences of CPM 5 1.5 Confined Placental Mosaicism and Uniparental Disomy ...7 1.6 C l i n i c a l Implications of UPD 9 1.7 Human Embryology 12 1.7.a. Cleavage and Blastocyst Formation 12 1.7.b. Formation of the Embryonic C e l l Lineages 13 1.7 .C Formation of the Yolk Sac 14 1.7.d. Placental Development and Hematopoiesis 15 1.7. e. Primordial Germ C e l l s 18 1.8 Gonadal Mosaicism 22 1.8. a. Genetic Consequences of Gonadal Mosaicism 22 1.8. b. Germline Chromosomal Mosaicism 23 1.9. Aims of the Study 24 2. Materials and Methods IV 2.1 Source of Specimens 2 6 2.2 Sampled Tissues 27 2.2.a Tissues Sampled for Cytogenetic Analysis 27 2.2. b. Tissues Sampled for M i c r o s a t e l l i t e Analysis 28 2.3 Fresh Analysis of Fresh Tissues 2.3. a. Preparation of Tissues for FISH i . Enzymatically Digested Placental Tissues 28 i i . Touch Preparations 3 0 i i i . Squash Preparations of Oocytes 31 2.3.b. Single Colour FISH 32 2.3 . C . Two - Colour FISH 34 2.3.d. S t a t i s t i c a l Analysis 35 2.4 FISH Analysis of Formalin Fixed - Pa r a f f i n Embedded Tissues 3 6 2.5 Conventional Cytogenetic Analysis 3 9 2.6 M i c r o s a t e l l i t e Analysis 40 Results 3.1. Case 1 41 3.1.a. Patient History and Conventional Cytogenetic Analysis 41 3.1. b. FISH Analysis of Case 1 42 3.2 Case 2 3.2. a Patient History and Conventional Cytogenetic Analysis of Case 2 45 3.2.b. FISH Analysis of Tissues from Case 2 46 3.2.c. M i c r o s a t e l l i t e Analysis of Case 2 53 V 3.3. Case 3 56 3.3.a. Patient History and Conventional Cytogenetic Analysis 56 3.3.b. Molecular Cytogenetic Analysis of Case 3 57 3.3 .C . Parental Origin of Chromosome 16 64 4. Discussion 4.1 Embryonic Origin of Tissues and Chromosomal Mosaicism 65 4.1.a. General Comments 65 4.1.b Embryo/Fetus Proper 67 4. I.e. Placental Tissues . . ." 68 4.1.d. Formation of the D e f i n i t i v e Yolk Sac 75 4.I.e. Primordial Germ C e l l s 76 4.1.f. Hematopoiesis 7 8 4.1. g. Amnion 79 4.2. Cytogenetic Analysis of Mosaicism 81 4.3. Cytogenetic Analysis of Mosaic Gestations 83 4. 3. a. Case 1 83 4.3.b. Case 2 87 4.3 .c. Case 3 92 4.4 C l i n i c a l Implications of this Study 96 4.5. Concluding Remarks 98 5. References 102 6. Appendix VI 6.1. Raw Data for Mosaic Conceptuses : I l l 6.2. Raw Data of FISH Analysis Performed on Control Samples for Formalin Fixed - Par a f f i n Embedded Tissues 114 6.3. Raw Data for FISH Analysis Performed on Fresh Control Samples Prepared by the Squash Preparation 120 6.4 Raw Data for FISH Analysis of Touch Preparations of Disomic Fresh Control Tissues 121 6.5 FISH Analysis of Enzymatically Digested Disomic Placenta 126 VII L i s t of Tables 2.1 Suggested pretreatments for formalin fixed - p a r a f f i n embedded tissues 38 3.1 Conventional cytogenetic analysis of Case 1 42 3.2 FISH analysis of tissues from Case 1 43 3.3 Conventional cytogenetics of Case 2. 46 3.4 FISH analysis of tissues from Case 2 53 3.5 Conventional cytogenetic analysis of Case 3 ..57 3.6 Molecular cytogenetic analysis of Case 3 64 3.7 Molecular analysis of the parental o r i g i n of chromosome 16 64 6.1 FISH analysis of tissues from Case 1 using a centromeric DNA probe s p e c i f i c for chromosome 7 I l l 6.2 FISH analysis of tissues from Case 2 using a centromeric DNA probe s p e c i f i c for chromosome 16 112 6.3 FISH analysis of tissues from Case 3 using a centromeric DNA probe s p e c i f i c for chromosome 16. 113 6.4 FISH analysis of disomic kidney control samples using a centromeric DNA probe 114 6.5 FISH analysis of disomic lung control samples using a centromeric DNA probe 115 6.6 FISH analysis of disomic controls for placental samples using a centromeric DNA probe 116 VIII 6.7 FISH analysis of formalin fixed - p a r a f f i n embedded disomic testes using a centromeric DNA probe s p e c i f i c for chromosome 7 117 6.8 FISH analysis of formalin fixed - p a r a f f i n embedded disomic oocytes using a centromeric DNA probe s p e c i f i c for chromosome 16 118 6.9 FISH analysis of formalin fixed - p a r a f f i n embedded oocytes from a trisomic 18 conceptus using a centromeric DNA probe s p e c i f i c for chromosome 18 119 6.10 Raw data for FISH analysis performed on fresh control samples prepared by the squash preparation 12 0 6.11 FISH analysis of a squash preparation of an oocyte from a trisomy 18 syndrome fetus 121 6.12 FISH analysis of disomic fresh kidney using a chromosome s p e c i f i c DNA centromeric probe 121 6.13 FISH analysis of disomic lung touch preparations using a chromosome s p e c i f i c DNA centromeric probe 122 6.14 FISH analysis of disomic fresh brain using a centromeric chromosome s p e c i f i c DNA probe 123 6.15 FISH analysis of disomic cord blood using a chromosome s p e c i f i c centromeric DNA probe 124 6.16 FISH analysis using a centromeric DNA probe s p e c i f i c for chromosome 16, of disomic trophoblast i s o l a t e d from chorionic v i l l i 125 6.17 FISH analysis using a chromosome s p e c i f i c centromeric DNA probe, of fresh stromal c e l l s i s o l a t e d from chorionic v i l l i 126 IX L i s t of Figures 1.1 I l l u s t r a t i o n of a trisomic zygote rescue 8 1.2 Rupture of the primary yolk sac to form the secondary yolk sac at 14 days of development 16 1.3 I l l u s t r a t i o n of the human embryo at four weeks of development 19 3.1 FISH analysis of formalin fixed - p a r a f f i n embedded t e s t i s from Case 1 with a chromosome 7 s p e c i f i c centromeric probe 44 3.2 FISH analysis of an oocyte prepared by the squash preparation obtained from a d i p l o i d fetus at 19m weeks gestational age. : 49 3.3 Squash preparation and FISH analysis of a d i p l o i d oocyte 50 3.4 Squash preparation and FISH analysis of an oocyte obtained from Case 2 51 3.5 FISH analysis of a squash preparation of a trisomic oocyte from Case 2 52 3.6 M i c r o s a t e l l i t e analysis of DNA from parental blood and f e t a l tissues from Case 2 55 3.7 FISH analysis with a chromosome 16 s p e c i f i c centromeric DNA probe of a formalin fixed - p a r a f f i n embedded oocyte obtained from a d i p l o i d gestation 60 X 3.8 FISH analysis with a chromosome 16 s p e c i f i c probe of a fromalin fixed - p a r a f f i n embedded oocyte obtained from a d i p l o i d conceptus 61 3.9 FISH analysis of a formalin fixed - p a r a f f i n embedded oocyte form Case 3 62 3.10 FISH analysis of a formalin fixed - p a r a f f i n embedded oocyte from Case 3 63 XI Acknowledgments With appreciation I thank Dr. D. K. Kalousek for her encouragement and guidance. I thank my committee members Dr. F.J. D i l l , Dr. J.M. Friedman and Dr. D.E. Horsman for th e i r time and suggestions. I would l i k e to express my sincere gratitude to Irene Barrett for her advice, friendship and support which I have received during my graduate degree. I would also l i k e to thank Adele Telenius, Brenda Lomax, V a l i a Lestou and Kamaljeet Opel for th e i r assistance i n the laboratory as well as t h e i r support and friendship. I thank Kim Myers, Barbara Paradice, Richard Mah, Robin Thayne, Debbie Norman and Sylvia Lee from the departments of the B.C. Children's Embryofetopathology and Pathology for assistance i n the c o l l e c t i o n of samples. I also greatly appreciate the technical assistance provided by Dr. Wendy Robinson, Fabiana Bernasconi and Brian Kuchinka for the DNA m i c r o s a t e l l i t e analysis. XII 1 Introduction 1.1 Chromosomal Mosaicism Constitutional chromosomal mosaicism i s defined as the presence of two or more c e l l l i n e s with d i f f e r e n t chromosomal complements i n an i n d i v i d u a l . In most instances the mosaic i n d i v i d u a l presents with two c e l l l i n e s - a normal d i p l o i d c e l l l i n e and an aneuploid c e l l l i n e . This usually arises from a post-zygotic mutational event i n early embryonic development. Mosaicism can occur i n both chromosomally normal and abnormal conceptions as a r e s u l t of a mitotic error such as nondisjunction, anaphase lag or a s t r u c t u r a l rearrangement. Different types of c o n s t i t u t i o n a l mosaicism can aris e depending on the exact timing of the mutational event, c e l l lineage involved and v i a b i l i t y of the mutant c e l l l i n e . The mosaicism can be generalized a f f e c t i n g both the fetus and the placenta, or confined to the placenta or the embryo/fetus proper (Kalousek and D i l l , 1983). 1.2 Generalized Mosaicism In cases of generalized mosaicism, multiple tissues of both embryonic and extraembryonic o r i g i n are affected. This occurs when the viable mutant c e l l l i n e arises at the f i r s t postzygotic d i v i s i o n or shortly thereafter (Kalousek and Vekemens, 1996) . An early mutational event results in.a 1 greater proportion of mutant c e l l s i n the developing morula, thus increasing the p o s s i b i l i t y that tissues of both embryonic and extraembryonic o r i g i n are affected (Kalousek, 1993) . Data from amniocentesis performed i n the second trimester of pregnancy i n women 3 5 years of age have been used to estimate the prevalence of generalized mosaicism. This procedure makes available c e l l s derived from the amniotic membrane, skin and e p i t h e l i a l c e l l s of the g a s t r o i n t e s t i n a l , urinary and respiratory tracts of the fetus for cytogenetic analysis. Several studies have shown that approximately 0.1 to 0.3 per cent of viable pregnancies are generalized mosaics (Hsu et a l . , 1984; Worton and Stern, 1984; Bui et a l . , 1984). Approximately 70% of these p o s i t i v e findings have been confirmed i n f e t a l tissues (Hsu et a l . , 1984). F a i l u r e to confirm prenatally diagnosed mosaicism i n the fetus following pregnancy termination has been explained by contamination of amniotic f l u i d by chorionic c e l l s during the amniocentesis i n gestations which exhibit chromosomal mosaicism confined to the placenta (Kalousek et a l . , 1987). 1.3 Confined Mosaicism Before the studies of Kalousek and D i l l (1983), mosaicism observed i n placental tissue was interpreted to represent the entire conceptus. However, since the timing 2 of the mutational event and the c e l l lineage involved determines the d i s t r i b u t i o n of mutant c e l l s i n the conceptus, Kalousek and D i l l postulated that a postzygotic nondisjunctional event can produce mosaicism confined to either the placenta or the fetus but not necessarily both. They showed that some infants diagnosed with unexplained intrauterine growth r e s t r i c t i o n (IUGR) exhibited aneuploidy i n tissues of extraembryonic o r i g i n while f e t a l tissues were d i p l o i d . For example, analysis of the term placenta from a gestation with unexplained IUGR revealed trisomy 22 i n cultured chorion with no evidence of aneuploidy i n amnion, cord blood or skin. Confined placental mosaicism (CPM) has been defined as a dichotomy between the chromosomal constit u t i o n of the placental tissues and embryonic/fetal tissues. CPM i s most often prenatally diagnosed i n chorionic v i l l u s sampling (CVS) at 9 to 12 weeks of gestation. CVS involves obtaining a small piece of chorionic v i l l u s for cytogenetic analysis which could either be processed by short term incubation (direct preparation) or the culture method. The d i r e c t preparation involves cytogenetic analysis of r a p i d l y d i v i d i n g c e l l s derivet from the trophoblast lineage; the long term culture allows the analysis of v i l l u s stroma (Sadler, 1995). CPM has been reported i n approximately 1 to 2% of viable pregnancies analyzed by CVS followed by a f a i l u r e to confirm mosaicism i n f e t a l tissues (Ledbetter et a l . , 1991; Wang et a l . , 1993). Also, several studies have 3 shown that mosaicism i n the placenta p e r s i s t s to term i n the majority of cases analyzed (Schwinger et a l . 1989; Kalousek et a l . 1991; Henderson et a l . , 1996 ). A number of hypotheses have been proposed to explain the more frequent occurrence of chromosomal mosaicism i n the placenta when compared to the fetus. Kalousek and D i l l (1983) proposed that the incidence of mosaic placentas should be higher than the incidence of mosaicism i n fetuses because the embryo i s derived from three or four c e l l s of the mammalian blastocyst while the remaining c e l l s give r i s e to the extra-embryonic membranes (Markert and Petters, 1978). A second hypothesis i s that trisomic c e l l s have a growth disadvantage i n f e t a l c e l l lineages and s e l e c t i o n of normal c e l l s during embryologic development would ensue, while the abnormal c e l l s are tolerated i n the placenta (Gosden, 1993). CPM has been described to exist i n three d i f f e r e n t forms depending on the placental tissues involved (Kalousek 1990). Mosaicism confined to the trophoblast c e l l lineage of the placenta i s designated as Type I CPM. Certain autosomal trisomies such as those for chromosomes 3, 7, 11, 13, 16 and 18 are most common i n Type I CPM. Rare trisomies include 5, 10, 14, 17 and 19, while no cases of chromosomes 1, 4 and 6 have been described. For sex chromosome aneuploidies, monosomy for chromosome X i n a complete or mosaic form i n the placenta i s most common. (Godsen et a l , 1995) . 4 Aneuploid- fetuses may also exhibit type I CPM. Kalousek et a l . (1989) have reported non-mosaic trisomy 13 or 18 i n f e t a l tissues, amnion and placental stroma while a d i p l o i d c e l l l i n e was present i n the cytotrophoblast. Type II CPM represents chromosomal mosaicism confined to the chorionic stroma. The frequency of Type II CPM i s si m i l a r to that reported for Type I, although the frequency of s p e c i f i c chromosomal trisomies involved d i f f e r (Godsen et a l . 1995). The most commonly observed trisomies include chromosomes 2, 7, 8, 9, 12, 16, 18 and 21. Trisomies 3, 5, 10, 11, 13, 14, 15, 17, 2 0 and 22 are rare while 1, 4, 6 and 19 have not been reported. Sex chromosome aneuploidy mainly involves monosomy of the X chromosome ( i b i d . ) . Mosaicism involving both the trophoblast and stroma of the placenta has been designated as Type III CPM. This i s the least common form of CPM and usually involves trisomies for chromosomes 7, 15,16 and 22. Aneuploidy for chromosomes 2, 9, 12, 18, 2 0 and 21 are occasionally observed and trisomies for the remaining chromosomes have not been reported (Godsen et a l . , 1995). 1.4 C l i n i c a l Consequences of CPM The p o s s i b i l i t y of poor pregnancy outcome as a r e s u l t of chromosomal mosaicism confined to the placenta was f i r s t suggested by Kalousek and D i l l (1983) when they found CPM i n two out of nine pregnancies with unexplained IUGR. These 5 findings have been further supported by Kalousek et a l . (1991) when they reported 6 of 34 term pregnancies diagnosed with CPM at 9 to 12 weeks gestation, with fetuses small for gestational age. A number of reports have followed i n which a p o s i t i v e c o r r e l a t i o n of CPM and f e t a l manifestations including intrauterine death of chromosomally normal fetuses or IUGR have been found (Wolstenholme et a l . , 1994). Johnson et a l . (1990) evaluated 4319 pregnancies by both d i r e c t and long-term culture CVS techniques and found that 16.7% of pregnancies diagnosed with CPM ended i n f e t a l loss versus 2.7% of the non-CPM group. These findings have been further supported by Breed et a l . (1991) who found a s i m i l a r rate of 16.6% of pregnancy loss i n the CPM group. Additional evidence suggesting that CPM may a f f e c t p e r i n a t a l outcome was reported by Kalousek et a l . (19 89). They found intrauterine s u r v i v a l of chromosomally abnormal fetuses with trisomy 18 or 13 may be enhanced by the presence of a normal c e l l l i n e confined to the trophoblast of the placenta. However, other studies suggest that the e f f e c t of CPM i s minimal or non-existent (Schwinger et a l . , 1989). Many of these studies attempt to correlate the pregnancy outcome with the prenatal CVS r e s u l t . However, since the anueploid c e l l l i n e i n the placenta does not p e r s i s t to term i n a l l pregnancies diagnosed with CPM prenatally, the outcome of the pregnancy should be compared to the extent of chromosomal mosaicism i n the term placenta (Henderson et a l . , 1996). 6 The e f f e c t of CPM may depend on the type of chromosomal abnormality, the placental tissues affected and the percentage of aneuploid c e l l s i n the placenta. For example, CPM for trisomy 16 i s most often found as type III and i s more frequently associated with pregnancy complications than trisomies involving other chromosomes (Kalousek and Barrett, 1994). CPM type I seems to be associated with IUGR and spontaneous abortion (Johnson et a l . , 1991) while the e f f e c t of CPM II has not been c l e a r l y defined (Kalousek et a l . , 1987; Robinson et a l . , 1997). Further, Robinson et a l . (1997) have found that the l e v e l of trisomy i n the term trophoblast was higher when the o r i g i n of the extra chromosome was meiotic as compared to cases i n which the trisomy was a r e s u l t of a mitotic error. Also, a meiotic o r i g i n of trisomy was correlated with a high frequency of IUGR. 1.5 Confined Placental Mosaicism and Uniparental Disomy The occurrence of a d i p l o i d fetus/newborn with a high l e v e l or non-mosaic complete trisomy i n both placental lineages most l i k e l y r esults from a trisomic zygote rescue (Kalousek and Vekemans, 1996). In such cases i t i s thought that a meiotic non-disjunction event gives r i s e to a trisomic zygote. Loss of one chromosome from a postzygotic mitotic error results i n the formation of a d i p l o i d c e l l l i n e (Figure 1.1). 7 Figure 1.1: I l l u s t r a t i o n of a trisomic zygote rescue. CPM type III usually r e s u l t s from meiotic nondisjunction followed by a post-zygotic loss the extra chromosome i n an embryonic progenitor c e l l . Depending on which chromosome i s l o s t , the d i p l o i d fetus may be affected with uniparental disomy. (From Kalousek, 1994) BIPARENTAL CKSOMY BIPARENTAL CKSOMY UNIPARENTAL DISOMY present h one third 8 When the trisomic chromosome i s l o s t i n the trophoblast, Type I CPM with a non-mosaic aneuploid fetus results (Kalousek et a l . , 1989). When the trisomic chromosome i s lo s t i n the embryonic progenitor c e l l , a d i p l o i d fetus with Type III CPM develops. Theoretically, i n one t h i r d of these cases, the consequence of the mitotic error w i l l be uniparental disomy (UPD) i n which both chromosomes of a pai r originate from the same parent (Hall, 1990). 1.6 C l i n i c a l implications of UPD When considering the phenotypic consequences of UPD i n the fetus and the placenta several d i f f e r e n t factors must be taken into account: (1) the ef f e c t due to imprinted gene(s); (2) metabolic effects due to a mutated a l l e l e being present i n homozygous form; and i n the case of f e t a l UPD (3) the e f f e c t of placental trisomy on the placental function during intrauterine development. Genomic imprinting refers to the d i f f e r e n t i a l genetic marking of genes which i s dependent on parental o r i g i n . A number of l o c i i n the mammalian genome are only expressed from one chromosome and are thus said to maintain a "parental imprint". The role of imprinted genes i n development was demonstrated by Surani et a l . (1984) and Barton et a l . (1984) by the production of androgenic and 1 gynogenic mice. Androgenic mice were constructed by removing the female pronucleus from a zygote and replacing 9 i t with a male pronucleus. The embryo contained a d i p l o i d set of paternally derived chromosomes and gave r i s e to predominantly extra-embryonic placental tissues with severely stunted embryos. For gynogenic gestations which only contained maternally derived chromosomes, development of the embryo proper was normal to early somite stages and died in utero with unusually small extraembryonic placental tissue. These studies suggest that cer t a i n genes required for placental development are expressed from the paternally derived chromosomes while those on the maternal homologues are inactive. Conversely, early development of the embryo proper i s dependent on genes p r e f e r e n t i a l l y expressed from the maternal homologues. Two extensively studied examples of human disorders caused by uniparental disomy of imprinted genes are Prader-W i l l i syndrome (PWS) and Angelman syndrome (AS). Individuals affected with PWS have mental retardation, hypophagia, obesity and are hypoactive while AS i s characterized by mental retardation, hyperactivity, seizures and a happy puppet-like appearance with bouts of inappropriate laughter (Nicholls, 1993). The role of genomic imprinting i n the pathogenesis of these syndromes was suggested when a deletion i n the 15qll-13 region of the paternal a l l e l e was described i n patients with PWS while a deletion on the maternal chromosome i n the same region was observed i n patients affected with AS (Knoll et a l . , 1989; Williams et al.., 1990). Further evidence for a role of 10 genomic imprinting comes from the observation that UPD for chromosome 15, i n which both homologues are of maternal o r i g i n causes PWS while paternal UPD for chromosome 15 leads to AS (Nicholls et a l . , 1989; Malcolm et a l . , 1991). Also, PWS has been reported i n cases of CPM 15 with maternal UPD for t h i s chromosome pair, suggesting that t h i s arose due to a trisomic zygote rescue (Purvis-Smith et a l . , 1992; Morichon-Delvallez et a l . , 1993). Depending.on the mutational event involved i n the formation of UPD, an i n d i v i d u a l may have heterodisomy i n which the uniparental p a i r i s made up of two homologous chromosomes, or isodisomy i n which the p a i r contains duplicates of the same chromosome. The l a t t e r case provides a genetic mechanism i n which an autosomal recessive disease cqn be inherited from a single c a r r i e r parent. For example, UPD 7 has been associated with c y s t i c f i b r o s i s (Voss et a l . , 1989) and Bloom syndrome has been associated with UPD 15 (Woodage et a l . , 1994). Presumably, the affected c h i l d has acquired two copies of a single chromosome from one parent who i s a c a r r i e r of the mutant gene. UPD of chromosomes other than 15 has been described i n which trisomy mosaicism was present i n the placenta(Langlois et a l . , 1995; Kalousek et a l . , 1993)'. In such cases, the phenotypic effects of UPD may be due to imprinting effects or the functional i n s u f f i c i e n c y of the trisomic placenta or both. The best studied example of t h i s i s CPM 16 which most often arises by a nondisjunction event i n meiosis followed 11 by trisomic zygote rescue. Kalousek et a l . (1993) and Bennett et a l (1995) have reported a high incidence of IUGR and intra-uterine death i n CPM 16. It has been found that a high l e v e l of trisomy 16 i n the placenta rather than UPD 16 i s associated with IUGR (Kalousek and Vekemans, 1996). IUGR i s found i n cases with biparental inheritance and high leve l s of trisomy 16 i n the placenta, while normal b i r t h weight has been recorded i n a patient with UPD 16 and low levels of trisomy 16 i n the placenta (Kalousek and Barrett, 1994). 1.7 Human Embryology 1.7.a. Cleavage and Blastocyst Formation To understand the concept of confined mosaicism, the d i f f e r e n t i a t i o n of embryonic and extraembryonic tissues i n early human embryology must be considered. Human development begins with the union of the spermatozoon and the oocyte to give r i s e to the zygote. The act of f e r t i l i z a t i o n releases the ovum from a depressed metabolic state and the zygote begins a series of mitotic d i v i s i o n s . The r e s u l t i n g c e l l s are known as blastomeres and become smaller with each c e l l d i v i s i o n . At the eight c e l l stage, the loosely arranged clump of c e l l s undergoes a process of compaction i n which they maximize t h e i r contact with each other and form tight junctions. At approximately three days 12 a f t e r f e r t i l i z a t i o n the embryo undergoes the fourth cleavage to form the 16 c e l l morula. The blastomeres at t h i s stage acquire t h e i r i d e n t i t y as a r e s u l t of t h e i r p o s i t i o n i n the morula. The outer surrounding c e l l s w i l l develop into the outer c e l l mass while the inner c e l l s of the morula w i l l constitute the inner c e l l mass. A stage of c a v i t a t i o n follows i n which the blastocyst forms a f l u i d f i l l e d c avity referred to as the blastocele. The inner c e l l mass i s located at one pole and w i l l eventually give r i s e to the embryo/fetus proper as well as the extraembryonic structures including the amnion, yolk sac and stroma of the chorion. The outer c e l l mass fla t t e n s to form the e p i t h e l i a l wall of the blastocyst known as the trophoblast, which w i l l eventually form the cytotrophoblast and syncytiotrophoblast of the placenta (Sadler, 1995; Carlson, 1994). 1.7.b. Formation of the Embryonic C e l l Lineages At eight days development the c e l l s of the inner c e l l mass d i f f e r e n t i a t e into two layers: 1) the epiblast, a layer of high columnar c e l l s ; and 2) the hypoblast, a layer of small cuboidal c e l l s adjacent to the blastocele. Together these layers form the bilaminar disk (Carlson, 1994). The epiblast contributes c e l l s to the formation of embryonic as well as extraembryonic tissues. The f i r s t tissue observed to derive from the epiblast at eight days i s the amnion, which ultimately encloses the embryo proper i n a 13 f l u i d f i l l e d sac c a l l e d the amniotic cavity. The extraembryonic mesoderm has been described to ar i s e from the caudal region of the epiblast (Luckett, 1978). However the o r i g i n of the extraembryonic mesoderm i n the human i s controversial since Enders and King (1987) described development of the extraembryonic mesoderm from the hypblast. It now seems that both the epiblast and hypoblast contribute to the formation of the extraembryonic mesoderm (Carlson, 1994). The embryonic ectoderm i s derived d i r e c t l y from the epiblast c e l l s while the embryonic mesoderm and endoderm are formed through a process of c e l l migration c a l l e d gastrulation. Gastrulation begins i n the t h i r d week of development when the primitive streak appears as a short l i n e a r depression on the dorsal surface of the epiblast. C e l l s of the epiblast migrate i n the d i r e c t i o n of the primitive streak and s l i p beneath i t through a process of invagination to form the embryonic mesoderm and endoderm ( i b i d . ) . 1.7.c. Formation of the Yolk Sac It i s generally accepted that flattened c e l l s o r i g i n a t i n g from the hypoblast and l i n i n g the inner surface of the cytotrophoblast form the primary yolk sac during the ninth day of development (Luckett, 197 8). In the thirteen day old embryo, the multipotent extraembryonic mesoderm 14 if. surrounds the primary yolk sac and amnion. This i s followed by the rupture of the primary yolk sac at 14 days; repair then occurs to form a smaller secondary yolk sac (Luckett, 1978) (Figure 1.2). 1.7.d. Placental Development and Hematopoiesis The placenta consists of chorion, amnion and the umbilical cord. The embryologic o r i g i n of the chorion can be traced to two c e l l lineages: 1) the trophoblast and 2) the extraembryonic mesoderm. The cytotrophoblast and syncytiotrophoblast which begin to invade the uterine wall at eight days p o s t f e r t i l i z a t i o n are derived from the trophoblast c e l l lineage (Carlson, 1994). The extraembryonic mesoderm contributes to the formation of the v i l l u s stroma, chorionic plate, the mesoderm of the umbilical cord, secondary yolk sac and amnion. During the umbilical cord, the amnion becomes constricted around the body stalk to form a cord with two veins and one artery, which connect the embryonic c i r c u l a t i o n with the placenta (Drews, 1995). 15 Figure 1 . 2 : Rupture of the primary yolk sac to form the secondary yolk sac at 14 days of development. Notice the multipotential extraembryonic mesoderm surrounding the ruptured yolk sac. Note: embryonic ectoderm = epiblast; embryonic endoderm = hypoblast; trophoblast i s blue surrounding the extraembryonic mesoderm (Modified from Drews, 1995) 16 At the t h i r d week of development the mesoderm of the v i l l i and the secondary yolk sac begin to form blood vessels which w i l l become continuous with the intraembryonic c i r c u l a t o r y system. At the same stage of development blood islands begin to form i n the mesoderm of the yolk sac which i s the sight of embryonic hematopoiesis up to the s i x t h week of development (Drews, 1995). Hematopoiesis is' then taken over by the l i v e r followed by the spleen and bone marrow. It has been generally accepted that the primitive blood c e l l s from the secondary yolk sac serve as progenitors for a l l hematopoiesis i n l a t e r l i f e (Schwartz et a l . , 1991). However, recent evidence from studies i n the mouse suggest that hematopoietic contribution from the yolk sac progenitor c e l l s i s transient and d e f i n i t i v e hematopoiesis i s autonomously i n i t i a t e d i n the.para-aortic splanchnopleura, also known as the aorta-gonad-mesonephrous (AGM) region. Dickson et a l . (1995) reported that TGFbl mutant mouse embryos, which die as a r e s u l t a developmental deficiency of endothelial d i f f e r e n t i a t i o n and hemopoiesis i n the yolk sac, appeared to have erythrocytes i n the para-a o r t i c regions within the embryos. Medvinsky and Dzierzak (1996) demonstrated that the AGM region exclusively i n i t i a t e s long-term repopulating hematopoietic stem c e l l s (LTR-HSC) independently from the yolk sac. CulturedOtissues from the yolk sac, l i v e r and AGM region at 10 days gestation were transplanted into l e t h a l l y i r r a d i a t e d mouse recipients, and only the AGM was able to contribute to the rec i p i e n t 17 d e f i n i t i v e c i r c u l a t i o n . Also, Muller et a l . (1994) have i d e n t i f i e d hematopoietic stem c e l l s i n the AGM before they appear i n the f e t a l l i v e r , suggesting that the AGM i s the major contributor of progenitor c e l l s to the l i v e r for f e t a l hematopoiesys i n the mouse. It i s important to r e a l i z e that although the mouse has been used extensively as a model for early human hematopoiesis and other aspects of embryological development, these re s u l t s must be taken with caution. The embryological events leading to the establishment of the d e f i n i t i v e hematopoietic system i n the human are not well understood. However, Huyhn et a l . (1995) have shown that progenitor blood c e l l s are present i n extrahepatic embryonic tissue at 3 0 to 3 5 days of development, before hematopoiesis predominates i n the human f e t a l l i v e r . These observations are i n agreement with those from the mouse which suggest that progenitor blood c e l l s found i n the l i v e r originate from extrahepatic embryonic tissues such as the AGM region. 1.7.e. Primordial Germ C e l l s The f i r s t , i d e n t i f i a b l e primordial germ c e l l s (PGCs) are observed i n the secondary yolk sac endoderm at four weeks of development i n the human (Figure 1.3) (Fujimoto et a l . , 1977) . 18 Figure 1.3: I l l u s t r a t i o n of the human embryo at four weeks development. The primordial germ c e l l s are f i r s t i d e n t i f i i n the endoderm of the secondary yolk sac. (Modified from Drews, 1995) The PGCs then separate from the endoderm and migrate by amoeboid movements into the developing embryo. They a r r i v e at the pri m i t i v e gonads at the f i f t h week and begin to invade the genital ridges at the si x t h week of development. The ge n i t a l ridges consist of a population of c e l l s derived from the coelomic epithelium and c e l l s from the mesonephric ridge (Carlson, 1994) . Early i n the s i x t h week the primitive sex cords grow into the genital ridge from the mesonephrous, and approximately 1000 to 2000 PGCs migrate into sex cords. There are no distinguishable male or female c h a r a c t e r i s t i c s of the gonad at th i s stage, hence i t i s referred to as the i n d i f f e r e n t gonad. In the absence of t e s t i s d i f f e r e n t i a t i o n signals, the gonad undergoes ovarian development. In the ovary PGCs are known as oogonia. They undergo a series of mitotic d i v i s i o n s while the surface epithelium of the ovary gives r i s e to another population of c e l l s c a l l e d f o l l i c u l a r c e l l s . The number of germ c e l l s i n each ovary reaches approximately 7 000 000 at eighteen weeks of development. Most of these c e l l s die by apoptosis, and by the seventh month only 400 000 viable oocytes remain (Sadler, 1995). The f i r s t meiotic figures i n the female gonad have been reported as early as eight weeks of development (Baker, 1963). Once meiosis i s i n i t i a t e d the oocytes enter into prophase I, which i s characterized by f i v e stages. The f i r s t stage i s known as leptotene, i n which the chromatin begins to condense and chromosomes begin to take form. This i s 20 followed by the i n i t i a t i o n of p a i r i n g between homologous chromosomes i n the zygotene stage and the completion of p a i r i n g and formation of synaptonemal complexes (SC) during pachytene. In diplotene, the l a t e r a l elements of the SC's begin to separate and the oocytes then arrest i n dictyotene when the l a t e r a l elements disperse forming a fine background of chromatin threads (Speed, 1985). The oocytes remain i n distyotene u n t i l ovulation, when meiosis I completed and meiosis II occurs, r e s u l t i n g i n the formation of haploid gametes. In the male embryo, the germ c e l l s are surrounded by the developing sex chords which eventually give r i s e to the seminiferous tubules of the t e s t i s . Within the tubules the PGCs p r o l i f e r a t e to form the spermatogonia which are accompanied by S e r t o l i c e l l s derived from the surface epithelium of the gonad. The Leydig c e l l s derived from the o r i g i n a l mesenchyme of the gonadal ridge are f i r s t recognizable i n the i n t e r s t i t i u m surrounding the tubules at 6 weeks development (Trainer, 1987). Unlike oogonia, the spermatogonia due not enter into meiosis u n t i l puberty. 21 1.8 Gonadal Mosaicism 1.8.a. Genetic Consequences of Gonadal Mosaicism Germline mosaicism has been suggested i n families where phenotypically normal parents have more than one c h i l d affected with a dominantly inherited disorder (Hall, 1988). These disorders include osteogenesis imperfecta (Byers et a l . , 1988), Duchenne muscular dystrophy (Edwards, 1986), neurofibromatosis type I (Ricardi and Lewis, 1988)and inc o n t i n e t i a pigmenti (Kirchman et a l . , 1995). Molecular analysis for the presence of mutations i n the affected children and phenotypically normal parents suggests that the mutation may be confined to the germline or i t may be found i n somatic as well as germ c e l l s . Raghunath et a l . (1995) described a family i n which two children severely affected with osteogenesis imperfecta c a r r i e d the mutation for which the mildly affected father was mosaic i n blood, s a l i v a , hair root bulb c e l l s and f i b r o b l a s t s . Conversely, Prior et a l . (1992) detected a deletion i n the DMD gene i n two out of three s i b l i n g s but not i n the leukocytesof the mother who t h e o r e t i c a l l y should be the c a r r i e r . It has been suggested that the apparent confined germline mosaicism reported i n several families i s a r e s u l t of an early mutation i n the progenitor c e l l s of the gametes 22 but not somatic c e l l s (Hall, 1988). Another scenario i s that the mutation occurs before the a l l o c a t i o n of somatic and germ c e l l s , accounting for cases i n which the parent i s mosaic i n both c e l l lineages. Further, apparent confined germline mosaicism may be due to the i n a b i l i t y of somatic c e l l s to tolerate the mutant c e l l s . Also, since most studies only examine blood and/or skin of mosaics, i t i s possible that the mutation i s present i n other somatic tissues which have not been sampled ( i b i d . ) . 1.8.b. Germline Chromosomal Mosaicism The occurrence of individuals with germline chromosomal mosaicism has been scarcely reported i n the l i t e r a t u r e . There have been reports of recurrence of trisomy 21 i n families with phenotypically normal parents. Given that the expected frequency of trisomy 21 i s 1 i n 600 to 800 l i v e b i r t h s (McKusik, 1994) there has been speculation that families with multiple affected pregnancies either have genetic and/or- environmental predisposing factors to nondisjunction «r have germline chromosomal mosaicism (Pangalos et a l . , 1992). Cytogenetic analysis of skin f i b r o b l a s t s and/or blood leukocytes has shown parental mosaicism i n some cases (Harris et a l . , 1982; Uchida and Freeman (1985).;. Neilson et a l . , 1988; Pangalos et a l . , 1992). A comprehensive analysis of multiple somatic tissues 23 and gametes however i s lacking i n the l i t e r a t u r e due to the d i f f i c u l t y of obtaining multi-lineage tissue samples. 1.9 Aims of the Study The purpose of th i s study i s to explore the p o s s i b i l i t y that gestations diagnosed with type II and type III confined placental mosaicism w i l l also exhibit gonadal mosaicism. The embryologic o r i g i n of the PGCs i n the human i s not f u l l y understood. They are f i r s t observed i n an extraembryonic location i n the endoderm of the secondary yolk sac (Fujimoto et a l . , 1977); however i t i s not known whether they are d i r e c t descendants of the primary yolk sac endoderm ( i . e . hypoblast) or another c e l l lineage. Clonal analysis of epiblast c e l l s i n the mouse have shown that the same clones which give r i s e to the primordial germ c e l l s also contribute to the extraembryonic mesoderm (Lawson and Hage, 1994). This suggests that the extraembryonic mesoderm and the PGCs have a common embryologic o r i g i n i n mammals. In the developing human, the primary yolk sac ruptures at 14 days of development and at the same time i s surrounded by multipotent extraembryonic mesoderm. We propose that the extraembryonic mesoderm i s involved i n the repair of the primary yolk sac to form the secondary yolk sac and i n doing so, contributes to the formation of the PGCs. Thus we suggest that conceptuses with an aneuploid c e l l l i n e i n the 24 stroma of the placenta may also exhibit aneuploidy i n the oogonia/spermatogonia. A comprehensive analysis using fluorescence in situ hybridization was undertaken i n spontaneously or therapeutically terminated pregnancies diagnosed with CPM, to test for chromosomal mosaicism i n placental and f e t a l tissues, including the germ c e l l s . .When possible, the analysis was performed on the trophoblast and stromal lineages of the placenta separately, as well as the brain, lung, kidney, cord blood and germ c e l l s . This study may lead to a greater understanding of the developmental aspects of CPM and the embryological o r i g i n ,of the PGCs. It may also provide insight into human f e r t i l i t y i n the reproductive period i n individuals whose placentas exhibited chromosomal mosaicism. 25 2. Materials and Methods 2.1 Source of Specimens The three mosaic gestations included i n this study were co l l e c t e d for analysis on the basis of diagnosed CPM types II or III a f t e r conventional cytogenetic analysis of placental and/or f e t a l tissues. The mosaic gestations for cases 1 and 3 were formalin fixed and p a r a f f i n embedded aft e r pregnancy termination i n 1993 and 1991 respectively. Tissue sections of cases 1 and 3, as well as a l l formalin fixed - p a r a f f i n embedded control samples were obtained from the B.C. Children's Surgical Pathology and Embryofetopathology Laboratories. Fresh tissues for Case 2 were obtained from Dr. David Bick at the Genetics and IVF Institute, Fairfax, VA. A l l fresh tissues used for control samples were obtained from the B.C. Children's Hospital Embryofetopathology Laboratory. The gestational age of mosaic conceptuses and control samples i s defined as the period of time elapsed since the date of the l a s t menstrual period and i s two weeks greater than the developmental age ( i . e . i f developmental age i s 9 weeks than gestational age i s 11 weeks). When possible, the gestational age was determined from information provided by the mother. If information of gestational age was not provided by the mother, i t was calculated as two weeks greater than the developmental age. The developmental age 26 was established by measurements of f e t a l hand and foot lengths. 2.2 Sampled Tissues 2.2.a. Tissues Sampled for Cytogenetic Analysis For formalin fixed - p a r a f f i n embedded specimens, tissue sections from the chorionic v i l l i , kidney, lung and gonad were sampled for FISH analysis. The brain, kidney, lung, chorionic v i l l i , cord blood and gonad were sampled for fresh tissues analyzed by FISH analysis. Disomic controls were used to e s t a b l i s h the minimum proportion of nuclei with three signals required for a diagnosis of trisomy. Cut-off values were calculated separately from disomic controls for each formalin fixed - p a r a f f i n embedded and fresh tissue analyzed. Conventional cytogenetic analysis of metaphases using trypsin G-banding was performed on the amnion and chorionic plate for case 1; amnion, chorionic plate and v i l l u s stroma for case 2; and the amniotic f l u i d , amnion, skin, blood, kidney, spleen, cytotrophoblast, and v i l l u s stroma for case 3 . 27 2.2. b. Tissues Sampled for M i c r o s a t e l l i t e Analysis Molecular analysis of amnion, v i l l u s stroma, cytotrophoblast, lung, adrenal gland and parental blood of case 2 was performed i n the Medical Genetics Laboratory of Dr. Wendy Robinson i n the B.C. Research I n s t i t u t e for C h i l d and Family Health. Molecular analysis of f e t a l and parental blood was performed on case 3 and published by Kalousek et a l . (1993). 2.3 FISH analysis of Fresh Tissues 2.3. a. Preparation of Tissues for FISH i . Enzymatically Digested Placental Tissues Fresh chorionic v i l l i were separated into trophoblast and stromal tissues v i a enzymatic digestion. Samples of 3 0 to 50 mg of chorionic v i l l i were washed i n transport medium which consisted of minimum essential medium (Gibco BRL, Grand Island, NY) and 4% (v/v) of antibiotic-antimyocotic solution (Gibco BRL, Grand Island, NY) to remove contaminating blood. Maternal decidua was i d e n t i f i e d using a Nikon (Japan) dissecting microscope with 3OX magnification and removed from the v i l l i with forceps. The v i l l i were then digested with 1 ml of collagenase type 1A (lmg/ml; Sigma Chemical Company, St. Louis, MO) for 10 to 2 0 min. 28 The solution was vigorously agitated and allowed to s i t for 2 min, and the c e l l suspension of trophoblast was placed i n a 15 ml polystyrene conical centrifuge tube (Falcon, F a i r Lawn, NJ, USA). The c e l l suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The p e l l e t was resuspended i n 1% (w/v) sodium c i t r a t e solution (Fisher S c i e n t i f i c , F a i r Lawn, NJ) prewarmed to 37°C and allowed to s i t for 2 0 min at room temperature. Then the c e l l suspension was centrifuged at 1000 rpm for 5 min, and the p e l l e t was fixed i n 3:1 (v/v) methanol (Baker, Ph i l l i p s b u r g , N J ) / g l a c i a l acetic acid (BDH, Toronto, ON). The fixed c e l l s were washed i n 3:1 methanol/ g l a c i a l acetic acid twice and a c e l l suspension was made for s l i d e preparation. The trophoblast c e l l suspension was then dropped on twin frosted microscope s l i d e s (FGR Steinmetz Inc., Surrey, BC) cleaned with methanol. Slide preparations were a i r dried and then dehydrated i n ethanol series (70%, 80%, 90% and 100% for two min each). Enzyme pretreatments were c a r r i e d out with 0.000075% (w/v) trypsin (DIFCO BACTO, Detroit, MI) for 2 to 4 sec, washed i n phosphate buffered saline solution (PBS) (GIBCO BRL, Grand Island, NY) twice for 5 min and treated with 10% formalin (Fisher S c i e n t i f i c , F a i r Lawn, NJ) for 10 min. They were then washed twice i n PBS for 5 min and placed i n 50% formamide (Fisher S c i e n t i f i c , F a i r Lawn, NJ) i n 2X SSC for 15 min at room temperature. The 2X SSC consists of 0.15 M sodi um chloride and 0.015 M sodium 29 c i t r a t e solution with a pH of 7 (Fisher S c i e n t i f i c , F a i r Lawn, NJ). The remaining v i l l u s stromal material from the collagenase digested v i l l i was washed twice i n PBS and placed i n -70°C freezer for at least 2 h. Then i t was minced with two scalpel blades, placed i n a 15 ml centrifuge tube and washed with 0.9% sodium chloride (w/v), pH 1.5. The digestion with 0.125% (w/v) pepsin (Sigma Chemical Company, St. Louis, MO) i n 0.9% sodium chloride solution, pH 1.5 at 37°C for 10 to 15 min followed. The solution was then agitated vigorously, allowed to stand for 2 min and the supernatant was placed i n a new 15 ml centrifuge tube. This was then washed twice with PBS and the c e l l suspension was dropped on s i l a n i z e d precleaned s l i d e s (Fisherbrand Superfrost/Plus, Fisher S c i e n t i f i c , F a i r Lawn, NJ) and allowed to a i r dry overnight. Microscope s l i d e s were then placed i n 10% formalin for 2.5 h, washed i n PBS twice for 5 min and dehydrated i n ethanol series. i i . Touch Preparations Fetal lung and kidney were washed i n transport medium and s l i c e d i n h a l f . The freshly s l i c e d tissue was touched onto precleaned s i l a n i z e d microscope s l i d e s and allowed to a i r dry. Blood dropped out of the umbilical cord onto the end of a precleaned s i l a n i z e d microscope s l i d e was smeared across the s l i d e with the end of a second s l i d e and a i r 30 dried. Fetal brain tissue was smeared across precleaned s i l a n i z e d microscope s l i d e s and allowed to a i r dry. A l l touch preparations were then placed i n 100% methanol for 5 min and allowed to a i r dry. They were then placed i n 3:1 methanol/glacial acetic acid for 5 min, a i r dried and dehydrated i n ethanol series. Slide preparations were treated with 0.00005% trypsin (w/v) for 2 to 4 sec, washed i n PBS twice for 5 min and placed i n 10% formalin for 10 min. This was followed by two PBS washes for 5 min and incubation i n 5 0% formamide i n 2X SSC for 15 min at room temperature p r i o r to DNA denaturation for FISH. i i i . Squash Preparation of Oocytes The f e t a l ovary was cut i n 4 to 6 pieces and each piece was placed i n a drop of PBS on precleaned s i l a n i z e d microscope s l i d e s . A second s l i d e was placed on top of the tissue which was then squashed. The second s l i d e was c a r e f u l l y removed and the oocytes were allowed to a i r dry on the bottom s l i d e . The s l i d e preparations were placed i n 100% methanol for 5 min, a i r dried, followed by 3:1 methanol/glacial acetic acid for 5 min and a i r dried. The sl i d e s were then dehydrated i n ethanol series. Enzyme pretreatments were c a r r i e d out with 0.00075% trypsin (w/v) for 4 to 8 sec, followed by washes i n PBS twice for 5 min and treatment with 10% formalin for 10 min. They were then washed twice i n PBS for 5 min and placed i n 50% formamide i n 31 2X SSC for 15 min at room temperature p r i o r to the DNA denaturation for FISH. 2.3.b. Single Colour FISH One colour FISH was performed on a l l fresh tissues except oocytes. The s l i d e preparations were denatured i n 7 0% formamide i n 2X SSC at 7 0°C for 5 min. The s l i d e s were then dehydrated i n -2 0°C p r e c h i l l e d ethanol series and allowed to a i r dry. A hybridization mixture of 15 (ll Hybrizol VI (Oncor, Gathersburg, MD) + 0.75 | l l digoxigenin labeled centromeric DNA probe mixture (Oncor, Gaithersburg, MD) was denatured at 7 0°C for 5 min and then placed on i c e . The hybridization mixture was applied to each s l i d e , covered with a 22 X 22 glass coverslip, sealed with rubber cement and incubated overnight at 37°C. On the next day, the rubber cement was removed, and microscope s l i d e s were placed i n 2X SSC u n t i l the coverslips could be e a s i l y removed. Post-hybridization washes consisted of 65% formamide i n 2X SSC at 43°C for 15 min followed by two washes i n 2X SSC at 37°C for 8 min each. The s l i d e s were then washed three times for 2 min i n phosphate buffered detergent (PBD) which consisted of 0.1 M sodium phosphate dibasic anhydrous (Fisher S c i e n t i f i c , F a i r Lawn, NJ), 0.06 M sodium phosphate monobasic (Fisher S c i e n t i f i c , F a i r Lawn, NJ) and 0.1% Tr i t o n X-100 (v/v) .(Sigma, St. Louis, MO). 32 Immunodetection of hybridized probe was c a r r i e d out with a digoxigenin antibody system. A l l antibodies were dissolved i n PMN solution which consists of PBD with 0.048% instant skim milk powder (w/v) (Nestle, Don M i l l s , ON) and 0.02% sodium azide (Sigma, St. Louis, MO). 60 (0.1 of 0.4 |lg/ml anti-digoxigenin (sheep) IgG i n (Boeringer Mannheim, Laval, QE) was placed on the microscope s l i d e s , covered with p l a s t i c coverslips and incubated for 15 min at 37°C. The sl i d e s were then washed i n PBD three times for 2 min and incubated with 60 \il of 2 |ig/ml rabbit anti-sheep IgG (Pierce, Rockford, IL) for 15 min at 37°C. This was followed by three PBD washes and incubation with 60 | l l of 20 jig/ml a n t i - r a b b i t IgG fluorescine-isothiocyanate (FITC) . The s l i d e s were then washed three times i n PBD, and the nuclei were counterstained with 2 (lg/ml propidium iodide (Sigma Chemical Company, St. Louis, MO) i n vectashield mounting medium (Vector Laboratories, Burlingame, CA). If needed, the fluorescent signals were amplified by repeating the l a s t two incubations of the antibody treatments. The nuclei were analyzed under a Nikon (Japan) fluorescence microscope at 600X magnification. For each sample approximately 500 nuclei were scored, and the proportion of nuclei displaying one, two, three and four or more signals were recorded. Interphase nuclei with disrupted or overlapping nuclear membranes were not scored. 33 2.3 . C Two-Colour FISH Two colour FISH was u t i l i z e d i n order to v i s u a l i z e two d i f f e r e n t chromosome s p e c i f i c centromeric DNA probes per nucleus. One probe was s p e c i f i c for the chromosome of interest, and the second probe was used as an in t e r n a l control. In order to select oocytes with unpaired chromosomes, only oocytes with two hybridization signals for the i n t e r n a l control were scored for the test probe. The s l i d e preparations were denatured as described i n section 2.3.b. The hybridization solution contained 1.5 | l l of digoxigenin labeled centromeric DNA probe mixture + 1.5 (ll of b i o t i n labeled centromeric DNA probe mixture (Oncor, Gathersburg, MD) and 27 [il of Hybrizol VI. The hybridization solution was denatured at 70°C, applied to the s l i d e preparation, covered with a 22 X 60 mm coverslip, sealed with rubber cement and incubated overnight at 37°C. The post-hybridization wash was c a r r i e d out as described i n section 2.4.b., and s l i d e s were washed three times i n PBD for 2 min. A l l antibodies applied to the s l i d e preparation were dissolved i n PMN. A mixture of 3 0 | l l of 5 |lg/ml FITC-avidin (Vector Laboratories, Burlingame, CA) and 3 0 | l l of 1 |ig/ml mouse anti-digoxigenin (Sigma Laboratory, St. Louis, MO) per s l i d e was c h i l l e d on ice for 5 min. This was applied to- each s l i d e and incubated at 3 7°C for 15 min. The s l i d e s were washed three times i n PBD and incubated with 60 JU.1 of 2 jig/ml anti-mouse Ig-digoxigenin, F(ab) 2 fragment 34 (Boehringer Mannheim, Laval, QE), per s l i d e for 15 min followed by 3 more PBD washes. The next antibody applied to each s l i d e was 60 | l l of anti-dig-rhodamine, Fab fragment (Boehringer Mannheim, Laval, QE) which was incubated for 15 min at 37°C followed by three PBD washes. Then 60 | l l per s l i d e of 5 |ig/ml b i o t i n y l a t e d a n t i - a v i d i n D (Vector Laboratories, Berlingame, CA) was applied and incubated for 15 min. The s l i d e s were washed i n PBD and incubated with 6 0 |i l of 5 |jg/ml FITC-avidin (Vector Laboratories, Berlingame, CA) for 15 min and washed with PBD as before. The nuclei where then counterstained with 0.04 (lg/ml 4, 6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) (Sigma Chemical Companies, St. Louis, MO) i n 2X SSC for 4 min. The s l i d e s were washed three times i n PBD for 5 min, and 22 X 60 mm coverslips were mounted with vectashield mounting medium. Approximately 10 0 to 500 oocytes were analyzed per sample with a Zeiss (Germany) fluorescent microscope. The proportion of oocytes with one, two, three or four or more hybridization signals for the test probe were recorded. 2.3.d. S t a t i s t i c a l Analysis FISH analysis was performed on disomic controls for each tissue tested, and cut-off values for detection of trisomy were calculated as described by Lomax et a l . (1994) . To determine the minimum proportion of c e l l s with three i 35 hybridization signals representing a trisomic c e l l l i n e , an upper reference l i m i t (L) was calculated as follows: L = x + t ( { X / n _ 1 ) S 1 + 1/n) , where x was the mean proportion of nuclei with three hybridization signals i n the disomic control samples, s was the standard deviation and n was the number of specimens studied. The 95% confidence i n t e r v a l (CI) of th i s upper reference l i m i t was calculated using the following equation: CI = L 1.96 J [ l + ( t 2 ( a n _ 1 ) (1 + 1/n)/2]s 2/n The upper reference 95% confidence l i m i t was designated as the cut - off value which represents the minimum proportion of nuclei required to diagnose trisomy i n a s p e c i f i c tissue. 2.4 FISH analysis of Formalin Fixed - Pa r a f f i n Embedded Tissues. 50 |lm sections from tissue blocks were cut into narrow s l i v e r s and placed into 15 ml polypropylene conical centrifuge tubes (Fisher S c i e n t i f i c , Pittsburgh, PA). The tissues were dewaxed with 10 ml xylene (Fisher S c i e n t i f i c , F a i r Lawn, NJ) for 10 min at room temperature. The xylene was removed, and fresh xylene was added a second time for 10 min. The xylene was removed, and the tissues were treated i n 10 ml of 100% ethanol four times for 10 min each. the 100% ethanol was removed, and the tissues were rehydrated with 10 ml of 90%, 70% and 50% ethanol twice for 10 min each. The tissues were then washed i n d i s t i l l e d water twice 36 and incubated i n 10 ml Hanks balanced s a l t solution (HBSS) with Ca/Mg (GIBCO, Grand Island, NY) overnight at 4°C. The HBSS was removed, and the tissues were washed with fresh HBSS twice. A nuclear suspension was then prepared by enzymatic digestion of the tissues. F i r s t , the tissues were incubated i n 2 ml of collagenase type XI (1.25 mg/ml; Sigma Chemical Company, St. Louis, MO) for 1 h with vigorous ag i t a t i o n every 10 min. The solution was centrifuged at 15 0 0 rpm for 5 min, arid the supernatant was discarded and replaced with HBSS without Ca/Mg (Gibco BRL, Grand Island, NY). The tissues were washed i n HBSS without Ca/Mg twice, and resuspended i n 4 ml trypsin/EDTA (Gibco BRL, Grand Island, NY). This was incubated for 1 h at 37°C and examined under the Nikon (Japan) disse c t i o n microscope to determine the state of digestion. If the digestion was not complete, then the tissues were incubated i n trypsin/EDTA for another 1 to 2 h. When large numbers of single c e l l s could be seen i n solution, the tubes were agitated vigorously, allowed to s i t on the bench top for 3 min and the supernatant was decanted into another conical centrifuge tube. The supernatant was centrifuged, and the c e l l s were resuspended i n 4 to 10 drops of HBSS without Ca/Mg to make s l i d e s . The c e l l suspension was dropped onto precleaned s i l a n i z e d s l i d e s and baked overnight on a Labline Instruments (Melrose Park, IL) s l i d e warmer at 37°C. The next day the sl i d e s were dehydrated i n the ethanol series and allowed to a i r dry. They were then treated with 3 0% 37 sodium b i s u l f i t e (w/v)p(Aldrich Chemical Company, Milwaukee, WI) at 43°C, washed three times i n 2X SSC for 1 min, and dehydrated i n ethanol series. This was followed by incubation with proteinase K (250 (ig/ml to 1 mg/ml; Sigma Chemical Company, St. Louis MI) at 37°C for 15 min to 1 h. The enzyme was washed off with 2X SSC three times for 1 min and the s l i d e s were then dehydrated i n ethanol ser i e s . The most successful pretreatments for each tissue are summarized i n Table I. Table 2.1: Suggested pretreatments for formalin fixed -p a r a f f i n embedded tissues. Tissue 30% Sodium B i s u l f i t e at 43°C Proteinase K at 37°C (concentration) kidney, lung, placenta 15 to 30 min 15 to 30 min (250 |lg/ml) oocytes 3 0 min 15 min to 1 h (250 Jig/ml to lmg/ml) The s l i d e s were then hybridized with a digoxigenin labeled centromeric DNA probe. The hybridization mixture consisted of 10 (ll Hybrysol VIII (Oncor, Gaithersburg, MD) + 2 | l l probe mixture and were applied to the s l i d e . This was covered with a 22 X 22 mm glass coverslip, sealed with rubber cement and baked at 45°C u n t i l the cement was 38 completely dry. The s l i d e preparation was placed i n a 90°C Blue M oven (Blue Island, IL) for 12 min to denature the probe and genomic DNA. The s l i d e s were then hybridized overnight at 37°C. The next day the coverslips were removed as described above i n section 2.3.b. Post-hybridization washes consisted of a 50% formamide i n 2X SSC wash at 37°C for 5 to 7 min, followed by a 0.1 X SSC wash at 37°C for 5 to 7 min. The immunodetection steps and evaluation of hybridization signals were performed as described i n section 2.3.b. The placenta, lung and kidney s l i d e preparations were counterstained with 2 |ig/ml propidium iodide i n vectashield mounting solution and analyzed using a Nikon fluorescent microscope at 600X magnification. The oocytes were counterstained with DAPI as described i n section 2.3.c. and analyzed under a Zeiss fluorescent microscope at 1000X magnification. Approximately 100 to 500 nuclei were analyzed per sample, and the proportion of nuclei with one, two, three or four or more hybridization signals were recorded. S t a t i s t i c a l analysis for detection of trisomy was performed as described i n section 2.3.d. 2.5 Conventional Cytogenetic Analysis Cytogenetic analysis of metaphases using trypsin G-banding was performed on f e t a l and placental tissues by standard cytogenetic methods (Rooney and Czepulkowski, 39 1992). Analysis of cultured amnion and chorion of case 1 was performed by the B.C. Children's Hospital Cytogenetic Laboratory. Conventional cytogenetic analysis of cultured amnion, v i l l u s stroma and chorionic plate for case 2 was performed by Irene Barrett i n the Cytogenetic Laboratory of the B.C. Research I n s t i t u t e for C h i l d and Family Health. Results from cytogenetic analysis of prenatal amniotic f l u i d culture, cytotrophoblast, v i l l u s stroma, amnion, skin, f e t a l blood, kidney, lung and spleen for case 3 were obtained from Kalousek et a l . (1993). 2.6 M i c r o s a t e l l i t e Analysis Molecular analysis of parental o r i g i n of chromosome 16 for case 2 was performed on parental blood and: f e t a l tissues by Fabiana Bernasconi i n the B.C. Research I n s t i t u t e for Chi l d and Family Health. The highly polymorphic m i c r o s a t e l l i t e markers, D16S398 and D16S423 of chromosome 16, were amplified using polymerase chain reaction and analyzed by gel electrophoresis as previously described by Robinson et a l . (1995). For case 3, the polymorphic marker D16S83 of chromosome 16, was analyzed by r e s t r i c t i o n enzyme digestion and Southern blot analysis by standard techniques as described by Kalousek et a l . (1993) . 40 3 Results 3.1 Case 1 3.1.a. Patient History and Conventional Cytogenetic Analysis This pregnancy was terminated at 21 weeks of gestation a f t e r a prenatal diagnosis of f e t a l anomalies. The mother was a 29 year old, Gravida 2, Para 1 and Aborta 1 woman with a healthy c h i l d from a previous pregnancy. Ultrasound showed a hyperechogenic bowel along with ventriculomegaly. Gross examination of damaged f e t a l parts a f t e r termination f a i l e d to reveal any developmental abnormalities. A normal male karyotype was found by cytogenetic analysis of cultured amnion subsequent to termination (Table 3.1). Two metaphases of cultured chorionic plate showed a male karyotype with trisomy of chromosome 7, while two metaphases were normal. 41 Table 3.1: Conventional Cytogenetic Analysis of Case 1. Tissue Number of Trisomic C e l l s / T o t a l Number of C e l l s Analyzed Chorionic Plate 2/4 Amnion 0/5 Note: Cytogenetic analysis was performed by the Cytogenetic Laboratory of the B.C. Children's Hospital. 3.1.b. FISH Analysis of Case 1 Results from the molecular cytogenetic analysis of formalin fixed - p a r a f f i n embedded f e t a l tissues are summarized i n Table 3.2. The proportion of nuclei with three hybridization signals for the chromosome 7 s p e c i f i c probe i n the lung and kidney i s 7.2 and 2.2%, respectively. These are less than the cut-off values, 10.91 and 11.78%, established by the disomic control samples. FISH analysis of three s i t e s from the placenta, which includes c e l l s derived from both stroma and trophoblast, indicates mosaicism for trisomy 7. The proportion of c e l l s with three signals representing chromosome 7 are s i g n i f i c a n t l y greater than the mean of disomic control samples with 95% confidence (cut - off value = 7.89%). The l e v e l of trisomy detected by FISH i n placental s i t e s 1, 2 and 3 are 11.4, 12.2 and 23.5%, respectively. 42 The nuclear preparation of the t e s t i s represents a population of somatic and germ c e l l s . FISH analysis was performed on a l l c e l l types of the t e s t i s since the germ c e l l s could not be morphologically distinguished from somatic c e l l s (Figure 3.1). No evidence of trisomy for chromosome 7 has been detected since the proportion of nuclei with three signals i n the t e s t i s of the CPM gestation i s less than the cut-off values set by the control samples (7.69%). Table 3.2: FISH analysis of tissues from Case 1. Tissue % Nuclei with Three Signals (Number of Nuclei Scored) Cut-Off Values (Number of Controls) Placenta Site 1 11.4 (501) 7.89 (8) Placenta Site 2 12.2 (501) 7.89 (8) Placenta Site 3 23.5 (601) 7.89 (8) Lung 7.2 (501) 9.92(9) Kidney 2.2 (501) 11.78 (10) Testis 2.5 (708) 7.69 (5) 43 Figure 3.1: FISH analysis of formalin f i x e d - p a r a f f i n embedded t e s t i s from Case 1 with a chromosome 7 s p e c i f i c centromeric probe. The germ c e l l s are indistinguishable from the somatic c e l l s i n the t e s t i s as observed with the propidium iodide DNA counterstain. 3.2 Case 2 3.2.a. Patient History and Conventional Cytogenetic Analysis of Case 2. This conceptus was terminated at 12 weeks of gestation and was the t h i r d pregnancy of a 37 year old woman, Gravida 3, Para 1 and Aborta 2. She has a healthy daughter and had a spontaneous abortion i n a previous pregnancy. Ultrasound examination of t h i s pregnancy did not show developmental abnormalities. Prenatal diagnosis for advanced maternal age by culture of chorionic v i l l u s sampling revealed trisomy for chromosome 16 i n a l l 21 metaphases scored (Table 3.3). Post-termination conventional cytogenetic analysis of cultured chorionic plate revealed that a l l f i v e metaphases analyzed were trisomic for chromosome 16; i n cultured v i l l u s stroma eight out of ten c e l l s exhibited trisomy 16. Culture of amnion was discordant from the results obtained from the placenta. No trisomic c e l l s were detected i n f i v e metaphases scored from the amnion. 45 Table 3.3: Conventional Cytogenetics of Case 2. Tissue Number of Trisomic Metaphases/Total Number of Metaphases Analyzed Culture of CVS# 21/21 Chorionic Plate* 5/5 V i l l u s Stroma* 8/10 Amnion* 0/5 # Analysis performed at the Genetics and IVF Centre, Fairfax, VA. * Analysis performed by Irene Barrett, B.C. Research In s t i t u t e for C h i l d and Family Health. 3.2.b. FISH Analysis of Tissues from Case 2. FISH analysis was performed on trophoblast and v i l l u s stroma from two s i t e s of the placenta. The v i l l u s stroma from s i t e s 1 and 2 showed s i g n i f i c a n t proportions of nuclei with three hybridization signals, consistent with 50.4% and 68.2% trisomy for chromosome 16 (Table 3.4). The r e s u l t s obtained from the trophoblast lineage of the placenta also indicated that there were high levels of trisomy for chromosome 16 (site 1 = 72.5% and s i t e 2 = 79.6%). Conversely, the proportion of c e l l s with three hybridization signals from cord blood (5.8%), brain (6.6%), lung (1.2%) and kidney (2.2%) were not greater than the cut-off values 46 established from the disomic controls (5.92, 7.67, 8.82 and 6.17%, resp e c t i v e l y ) . For molecular cytogenetic analysis of the gonad, only oocytes which were morphologically distinguishable from the surrounding somatic c e l l s were scored. As i l l u s t r a t e d i n Figure 3.2, oocytes i n prophase of meiosis I can be i d e n t i f i e d by t h e i r large size and d i f f u s e chromatin. The oocyte shown i n t h i s figure was obtained from a d i p l o i d fetus at 19 weeks gestational age which was used as a disomic control. The two d i s t i n c t hybridization signals representing chromosomes 16 and 18 can be seen by the FITC and rhodamine fluorochromes respectively. The lack of p a i r i n g between homologous chromosomes i n t h i s oocyte suggests that i t i s i n the leptotene stage. The smaller c e l l below the oocyte may either be a somatic c e l l or an oogonium which has not yet entered into prophase of meiosis I. Figure 3.3 i l l u s t r a t e s an oocyte from the same disomic control ovary. This oocyte i s i n the pachytene stage of meiosis I and condensed chromatin which forms thick chromosomes can be observed. The single hybridization signal for chromosome 16 and 18 indicates that homologous chromosomes are paired. Oocytes from both Figures 3.2 and 3.3 where harvested from the same f e t a l gonad at approximately 19 weeks gestational age. The finding of oocytes at various stages of meiosis i n any one ovary i s consistent with the r e s u l t s published by Monotaya and Potter (1963), Speed (1985) and Cheng and Gartler (1994). 47 Progression of oocytes through the stages of prophase I i s not synchronized i . e . a l l oocytes within the gonad are not at the same stage of development. Rather, oocytes c h a r a c t e r i s t i c of leptotene, zygotene and pachytene stages can be i d e n t i f i e d at th i s gestational age. In order to enrich for oocytes with chromosomes that were unpaired, only those oocytes i n which the in t e r n a l control (chromosome 18 i n Figure 3.2) showed two hybridization signals were scored during the cytogenetic analysis. As reported i n Table 3.4, 2 6.0% of oocytes from Case 2 exhibited three hybridization signals for chromosome 16. This number was greater than the cut-off value derived from the disomic controls (7.06%) in d i c a t i n g a s i g n i f i c a n t l e v e l of trisomic oocytes i n this ovary. Figures 3.4 and 3.5 i l l u s t r a t e the appearance of oocytes i d e n t i f i e d i n th i s gestation. No oocytes c h a r a c t e r i s t i c of pachytene stage were i d e n t i f i e d . Previous studies also d i d not i d e n t i f y pachytene stage oocytes i n ovaries from fetuses at 11 to 12 weeks gestational age (Monotaya and Potter, 1963; Speed, 1984) . 48 Figure 3.2: FISH analysis of an oocyte prepared by the squash preparation, obtained from a d i p l o i d fetus at 19 weeks gestational age. Two colour FISH with chromosome 16 (green) and chromosome 18 (red) s p e c i f i c DNA probes labeled with FITC and rhodamine fluorochromes respectively, i s shown. The chromosome 18 s p e c i f i c probe was used as an int e r n a l control to select oocytes for scoring, which have unpaired homologous chromosomes. This chromatin i s counterstained with DAPI. Notice the large s i z e of the oocyte with d i f f u s e chromatin. The smaller c e l l i s either a somatic c e l l or an oogonium which has not entered into meiosis. 49 Figure 3.3: Squash preparation and FISH analysis of a d i p l o i d oocyte exhibiting one signal for the chromosome 16 (green) and chromosome 18 (red) s p e c i f i c probes. Oocytes sim i l a r to th i s which only exhibit one signal for the int e r n a l control were not scored i n the FISH analysis. Notice the thick condensed chromatin and chromosome formation. One signal for the chromosome 16 and 18 s p e c i f i c probes suggest that t h i s oocyte i s i n the pachytene stage of meiosis i n which the homologous chromosomes have paired. 50 Figure 3.4: Squash preparation and FISH analysis of an oocyte obtained from Case 2. Notice the large s i z e of th i s oocyte and di f f u s e appearing chromatin. Two signals for the chromosome 18 (red) s p e c i f i c and chromosome 16 (green) s p e c i f i c probes indicate that t h i s oocyte i s d i p l o i d . 51 Figure 3.5: FISH analysis of a squash preparation of a trisomic oocyte from Case 2. Notice the larger nucleus with dif f u s e chromatin which has been i d e n t i f i e d as an oocyte. The three signals for the chromosome 16 (green) s p e c i f i c probe indicate that t h i s oocyte i s trisomic for chromosome 16. The smaller d i p l o i d nucleus i s either a somatic c e l l or oogonium which has not entered into meiosis. 52 Table 3.4: FISH analysis of tissues from Case 2. Tissue % Nuclei with Three Signals (Number of Nuclei Scored) Cut-Off Values (Number of Controls) Stroma Site 1 50.4 (1148) 9.66 (8) Stroma Site 2 68.2 (500) 9.66 (8) Trophoblast Site 1 72.4 (500) 6.59 (6) Trophoblast Site 2 79.6 (504) 6.59 (6) Cord Blood 5.8 (501) 5.92 (9) Brain 6.6 (502) 7.67 (8) Lung 1.2 (500) 8.82 (5) Kidney 2.2 (500) 6.17 (4) Oocytes 26.0 (227) 7.06 (8) 3.2.c. M i c r o s a t e l l i t e Analysis of Case 2. M i c r o s a t e l l i t e analysis was performed with polymorphic markers s p e c i f i c for chromosome 16 (Figure 3.6). The PCR amplification products for marker D16S423 show that there was maternal contribution to the f e t a l lung and adrenal while neither of the father's a l l e l e s were found i n these 53 f e t a l tissues. Results from marker D16S398 showed three d i f f e r e n t a l l e l e s for the trophoblast, stroma and amnion while the f e t a l lung has two a l l e l e s which are the same size as the maternal a l l e l e s . 54 Figure 3.6: M i c r o s a t e l l i t e analysis of DNA from parental blood and tissues from Case 2. (Abbreviations: M = mother, F = father, A = amnion, T = trophoblast, S = v i l l u s stroma, L = f e t a l lung and Ad = f e t a l adrenal gland) A) PCR amplification of marker D16S423, which i s from the short arm of chromosome 16, shows no paternal contribution to the f e t a l tissues. M L A d F B) PCR amplification of marker D16S398, from the long arm of chromosome 16, shows three d i f f e r e n t a l l e l e s i n the amnion, stroma and trophoblast, while the f e t a l lung has two a l l e l e s . A S T L M Note: Molecular analysis was performed by the laboratory of Dr. Wendy Robinson at the B.C. Research I n s t i t u t e for C h i l d and Family Health. 55 3.3 Case 3 3.3.a. Patient History and Conventional Cytogenetic Analysis This pregnancy was terminated at 2 5 weeks gestation. The mother was a 27 year old Gravida 2, Para 1 and Aborta 1. At 21 weeks gestation the mother developed severe hypertension and proteinuria. An appropriately grown fetus with a thick placenta containing echogenic regions was shown on ultrasound. Prenatal diagnosis by CVS showed trisomy 16 i n a l l metaphases examined by the d i r e c t and culture method while amniocentesis revealed a normal female karyotype (Table 3.5). The pregnancy was terminated at 2 5 weeks gestation due to severe maternal hypertension, persistent vomiting and elevated hCG. Postmortem examination revealed a growth retarded fetus with no congenital defects. Cytogenetic analysis of f e t a l kidney, spleen, skin, amnion and blood showed a normal female karyotype, while cultured chorionic plate and stroma of v i l l i were trisomic for chromosome 16. 56 Table 3.5: Conventional cytogenetic analysis of Case 3. Tissue Number of Trisomic Nuclei/Total Number Scored Amniotic F l u i d 0/36 CVS - Direct Method 9/9 CVS - Culture Method 20/20 Chorionic Plate 3/3 V i l l u s Stroma 24/24 Amnion 0/15 Skin 0/15 Kidney 0/15 Spleen 0/15 Blood 0/63 Note: Previously published re s u l t s from Kalousek et a l . , 1993 . 3.3.b. Molecular Cytogenetic Analysis of Case 3. FISH analysis was attempted on formalin f i x e d - p a r a f f i n embedded placenta, kidney, lung and oocytes. The nuclear preparations of kidney and placental tissues f a i l e d to hybridize with the chromosome 16 s p e c i f i c probe, regardless of several attempts with varying enzyme pretreatments. A successful hybridization was achieved with 57 the lung which showed no s i g n i f i c a n t levels of trisomy (Table 3.6). For analysis of formalin f i x e d - p a r a f f i n embedded oocytes, a nuclear preparation was hybridized with a chromosome 18 s p e c i f i c centromeric probe (internal control) and a chromosome 16 s p e c i f i c centromeric probe. However, attempts to amplify the signals with antibodies for two-colour FISH resulted i n unacceptable levels of background fluorescence. Therefore, only one-colour FISH with a chromosome 16 s p e c i f i c probe was used i n the analysis of formalin f i x e d - p a r a f f i n embedded oocytes, since t h i s resulted i n lower levels of background fluorescence. The distinguishing features of oocytes prepared by th i s method were not as dramatic as i n fresh oocytes prepared by the squash method. As seen i n Figures 3.7 and 3.8, the appearance of di f f u s e chromatin i s not as obvious i n p a r a f f i n embedded-formalin fixed oocytes (Figures 3.2 to 3.5). However, oocytes were i d e n t i f i e d by the appearance of s t r i n g - l i k e chromatin. In t h i s analysis i t was not possible to enrich for oocytes with unpaired chromosomes since an in t e r n a l control was not u t i l i z e d when selecting c e l l s to be scored (Figure 3.7). This resulted i n levels of apparent monosomy as high as 36.6% i n the disomic controls (Table 6.8 i n Appendix). The single hybridization signal of the chromosome 16 s p e c i f i c probe i l l u s t r a t e s t h i s point i n Figure 3.8. It i s also apparent that the p a i r i n g c h a r a c t e r i s t i c s of oocytes 58 resulted i n only 39% trisomy 18 i n a formalin fixed -p a r a f f i n embedded ovary which was obtained from a trisomy 18 syndrome fetus (Table 6.9 i n Appendix). When fresh oocytes from a trisomy 18 syndrome fetus were analyzed with an int e r n a l control, 71% of the oocytes analyzed showed three signals (Table 6.11 i n Appendix). Similar to the disomic control samples, the l e v e l of monosomy i n the patient was found to be 39% as a r e s u l t of paired homologues as i l l u s t r a t e d i n Figure 3.9. Figure 3.10 shows an example of a disomic oocyte from the mosaic conceptus. Two signals for chromosome 16 were observed i n 56% of the oocytes analyzed from the mosaic conceptus (Table 6.3 i n Appendix). Also, 5.0% of the oocytes from t h i s fetus exhibited three hybridization signals while the cut-off value for a diagnosis of trisomy i s 6.87% (Table 3.6). 59 Figure 3.7: FISH analysis with a chromosome 16 s p e c i f i c centromeric DNA probe (green signals) of a formalin fi x e d -p a r a f f i n embedded oocyte obtained from a d i p l o i d gestation. Close examination of the chromatin structure of t h i s nucleus reveals the s t r i n g - l i k e chromatin which f a c i l i t a t e d the i d e n t i f i c a t i o n of th i s nucleus as an oocyte. Two green signals representing the chromosome 16 centromeric probe confirm that t h i s oocyte i s disomic for chromosome 16. 60 Figure 3.8: FISH analysis with a chromosome 16 s p e c i f i c probe of a formalin fixed - p a r a f f i n embedded oocyte obtained from a d i p l o i d conceptus. Notice the d i f f u s e chromatin and s t r i n g - l i k e chromosome structures which are ch a r a c t e r i s t i c of an oocyte i n meiosis. The single green signal represents chromosome 16 and may be a r e s u l t of paired homologous chromosomes. 61 Figure- 3.9: FISH analysis of a formalin fixed - p a r a f f i n embedded oocyte form Case 3. One signal was observed for •the chromosome 16 s p e c i f i c centromeric DNA probe i n 3 9% o ..the oocytes examined as shown i n this figure. This i s presumed to be a re s u l t of paired chromosome 16 homologue i n the meiotic oocytes. Figure 3.10: FISH analysis of a formalin fixed - p a r a f f i n embedded oocyte from Case 3. The two green signals represent the centromeric region of chromosome 16 and indicate that t h i s oocyte i s disomic for th i s chromosome. Table 3.6: Molecular cytogenetic analysis of Case 3. Tissue Proportion of Trisomic Nuclei (Total Number of Nuclei) Cut-Off Values (Number of Samples) Lung 3.2 (500) 9.92 (9) Oocytes 5.0 (100) 6.87 (10) 3.3.c. Parental Origin of Chromosome 16 Molecular analysis of DNA from parental blood, f e t a l and placental tissues was performed to determine parental o r i g i n of chromosome 16. As reported i n Table 3.7, there i s maternal o r i g i n of the extra chromosome i n the trisomic placenta. Further the d i p l o i d fetus has maternal uniparental disomy for chromosome 16. Table 3.7: Molecular analysis of the parental o r i g i n of chromosome 16. Probe Mother Father Placental V i l l i Fetus D16S83 a, c b,b a, b, c a, c Note: These are previously published results from Kalousek et a l . , 1993 64 4 Discussion 4.1 Embryonic Origin of Tissues and Chromosomal Mosaicism 4.1.a. General Comments In order to understand the concept of confined versus generalized mosaicism, several factors a f f e c t i n g the d i s t r i b u t i o n of the mutant c e l l l i n e i n the developing conceptus must be considered. Tracing the c e l l lineage of s p e c i f i c tissues affected with.chromosomal mosaicism helps one understand how the mutant c e l l l i n e contributes to s p e c i f i c tissues of the conceptus during the process of d i f f e r e n t i a t i o n . During early embryogenesis c e l l fate or d i f f e r e n t i a t i o n i s thought to proceed through a number of developmental mechanisms including: c e l l p o l a r i z a t i o n , cleavage, compaction, p o s i t i o n effects, c e l l adhesion and c e l l death (Vogler, 1987). A l l o c a t i o n of a c e l l or group of c e l l s by these processes and the genetic changes within the c e l l s and t h e i r mitotic progeny give r i s e to a s p e c i f i c pattern of development (Carlson, 1994). The d i s t r i b u t i o n of a mutant c e l l l i n e throughout the conceptus i s determined by the effects of a mutational event such as nondisjunction, anaphase lag or chromosomal rearrangement, on these developmental processes. The outcome can be affected by the timing of the mutational 65 event, the v i a b i l i t y of the mutant c e l l l i n e and the c e l l lineage affected. Much of what we know of c e l l lineage i n human embryology i s based on observations of h i s t o l o g i c a l preparations of embryos recovered at successive stages of pregnancy. The r e s u l t i n g fate maps are based on the interpretations of the investigator as to what occurs between each of the stages which have been sampled. As Gardner (1983) states "Clearly, this approach i s an inherently unreliable one, and i t i s hardly surprising to f i n d opinion divided about one or more c e l l lineage i n v i r t u a l l y every species whose early development has been looked at c l o s e l y i n this way. This i s e s p e c i a l l y so i n man, i n whom specimens are lim i t e d both i n number and q u a l i t y and often of uncertain gestational age." A more r e l i a b l e method has been to mark c e l l s or tissues i n early mammalian embryos and follow the c e l l lineage. These types of experiments were performed i n rodent embryos and have contributed a great deal towards the understanding of mammalian embryology. This knowledge has been used to gain insight into human c e l l lineage, however such comparisons must be considered with caution since human development d i f f e r s from murine development. (Gardner, 1983). 66 4.1.b. Embryo/Fetus Proper The precursor c e l l s which give r i s e to the embryo/fetus proper are found within the inner c e l l mass of the early blastocyst. The inner c e l l mass also contributes to the development of extraembryonic tissues such as the yolk sac and extraembryonic mesoderm (Luckett, 1975, 1978). The proportion of c e l l s of the inner c e l l mass which contribute to the embryo/fetus proper i s not known, but experiments i n the mouse suggest that three c e l l s are involved. Markert and Petters (197 8) produced chimeric mice by aggregating three cleavage stage embryos g e n e t i c a l l y d i s t i n c t with respect to t h e i r coat colour. The coat of some mice had contributions from a l l three colours, suggesting that at least three c e l l s of the blastocyst give r i s e to the embryo. Aggregation of four g e n e t i c a l l y d i s t i n c t embryos gave r i s e to mice with no more than three contributions to t h e i r coat colour. These re s u l t s have been interpreted to suggest that three to f i v e c e l l s from the inner c e l l mass i n the developing human blastocyst give r i s e to the embryo/fetus proper (Kalousek and D i l l , 1983; Simoni and Serchia, 1994). Since so few c e l l s give r i s e to the fetus as compared to the extraembryonic tissues, Kalousek and D i l l (1983) predicted that there would be many more instances of mosaicism i n the placenta than the embryo/fetus proper. It has been since reported that the incidence of generalized mosaicism a f f e c t i n g both the fetus and placental tissues i s 67 0.1 to 0.3% of viable pregnancies tested by amniocentesis at 16 to 20 weeks of gestation (Hsu et a l . , 1984; Worton and Stern, 1984; Bui et a l . , 1984). While the incidence of chromosomal mosaicism confined to the placenta i s approximately 1 to 2% i n viable pregnancies tested by CVS at 9 -to 12 weeks of gestation followed by cytogenetic analysis of f e t a l tissues (Ledbetter et a l . , 1991; Wang et a l . , 1994) . Another hypothesis explaining the higher incidence of CPM as compared to generalized mosaicism i s that the embryo has control mechanisms to l i m i t the r e p l i c a t i o n of aneuploid c e l l s , i . e . d i p l o i d c e l l s may have a growth advantage and outgrow the abnormal c e l l s . In the extraembryonic tissues, the abnormal c e l l s may p r o l i f e r a t e c l o n a l l y and are simply eliminated with the placenta at delivery (Simoni and Serchia, 1994) . 4.I.e. Placental Tissues The chorionic v i l l i of the placenta are composed of an outer layer of hormonally active and invasive syncytiotrophoblast, a middle layer of cytotrophoblast, from which the syncytiotrophoblast i s derived, and an inner mesodermal core containing blood c a p i l l a r i e s . The cytotrophoblast and syncytiotrophoblast are derived from the trophoblast c e l l lineage. In the developing human, the trophoblast i s the f i r s t lineage to d i f f e r e n t i a t e from the 68 multipotential c e l l s of the morula. D i f f e r e n t i a t i o n begins as the outer c e l l s of the 16 c e l l morula develop a polar shape and eventually form the trophoblast c e l l lineage i n the blastocyst stage (Vogler, 1987). The remaining inner c e l l mass w i l l constitute the embryo/fetus proper, amnion, yolk sac and the chorionic stroma. Therefore i t i s assumed that Type I CPM arises as a r e s u l t of a mitotic error i n the progenitor c e l l s of the trophoblast soon a f t e r they have been determined to form the trophoblast c e l l lineage (Crane and Cheung, 1988) . The developmental hi s t o r y of the chorionic stroma i s more complex, and thus the mechanism of CPM Type II and III formation i s less understood. Examination of the Carnigie c o l l e c t i o n by Luckett (1978) suggests that the extraembryonic mesoderm f i r s t arises at 12 days of development i n a thickening of the caudal pole of the epiblast. This modification of the epiblast i s interpreted as the caudal margin of the primitive streak. In 13 day human embryos the caudal margin of the primitive streak i s observed to give r i s e to extraembryonic mesoderm which spreads p e r i p h e r a l l y over the amnion and primary yolk sac and eventually gives r i s e to the stroma of the chorionic v i l l i . Luckett's interpretations of the Carnigie sections are i n agreement with fate mapping experiments performed i n the mouse. Parameswaran and Tarn (1995) u t i l i z e d lac Z expressing epiblast c e l l s from transgenic mice grafted to 69 nontransgenic early primitive streak stage mouse embryos to assess the fate of epiblast c e l l s . They found that c e l l s i n the proximal region of the epiblast d i f f e r e n t i a t e d into extraembryonic mesoderm. When considering the c e l l lineage map established by Luckett et a l . (1978), i t i s d i f f i c u l t to explain how a mosaic blastocyst may contribute to the formation of CPM Types II and III . Since the epiblast i s thought to give r i s e to both the v i l l u s stroma and embryonic tissues, the chromosomal constitution of the embyro/fetus i s expected to be concordant <with that of- the v i l l u s stroma. However, the experiments by Markert and Petters (197 8) suggest that three to four c e l l s of the epiblast give r i s e to the embryo/fetus proper. A mutational event occurring i n epiblast c e l l s which are allocated for extraembryonic mesoderm or during the p r o l i f e r a t i o n of the extraembryonic mesoderm i t s e l f can re s u l t i n CPM type II. Type III CPM, i n which both the trophoblast and chorionic stroma are trisomic, probably arises from a trisomic zygote rescue (Kalousek et a l . , 1993). A meiotic non-disjunction produces a trisomic zygote, and post-zygotic loss of one of the three chromosomes res u l t s i n a d i p l o i d c e l l l i n e . When the d i p l o i d c e l l s i n the mosaic gestation contribute to a l l the embryonic/fetal tissues while chorionic stroma and trophoblast have s i g n i f i c a n t l e v e l s of trisomy, type III CPM res u l t s . When considering Luckett's (1978) observations, i t i s expected that the epiblast of the developing gestation 70 would be mosaic i n type III CPM. The c e l l s which give r i s e to the embryo/fetus proper would be d i p l o i d , and the caudal region which d i f f e r e n t i a t e s into extraembryonic mesoderm would contain trisomic c e l l s . Enders and King (1988) suggest that the hypoblast contributes c e l l s to the extraembryonic mesoderm. H i s t o l o g i c a l examination of sectioned 10.5 day rhesus monkey embryos showed branching processes from the primary yolk sac extending across the chorionic cavity toward the trophoblast. U l t r a s t r u c t u r a l studies of these c e l l s revealed expanded endoplasmic reticulum cisternae which are c h a r a c t e r i s t i c of the extraembryonic.mesodermal c e l l s i n s l i g h t l y l a t e r stages. Between day 13 and 16 the network of c e l l s between the yolk sac and the trophoblast seemed to d i f f e r e n t i a t e into mesenchymal c e l l s i n a r e l a t i v e l y uniform fashion. The authors interpreted these re s u l t s to suggest that extraembryonic mesoderm i s derived from the hypoblast before the formation the primitive streak. Bianchi et a l . (1993) incorporated the observations by Enders and King and reviewed the cytogenetic data obtained by the U.S. collaborative study on CVS (Ledbetter et a l . , 1992) and the Munster CVS program (Miny et a l . , 1991) to assess c e l l lineage i n humans. Bianchi et a l . (1993) interpret the discordance observed between the chromosomal constitu t i o n of the chorionic v i l l u s and embryo/fetus proper i n CPM type II as evidence that the o r i g i n of the extraembryonic mesoderm i s not the epiblast but rather the 71 hypoblast. Since the inner c e l l mass i s known to s p l i t into the hypoblast and epiblast during embryologic development, CPM type II would r e s u l t when the mosaic c e l l s of the inner c e l l mass contribute to the formation of the hypoblast but not the epiblast. Similarly, i t i s expected that type III CPM r e s u l t s when a trisomic zygote rescue occurs i n d i p l o i d progenitors of the epiblast. However, the hypothesis presented by Bianchi et a l . (1993) by no means has been established as correct. F i r s t l y , Enders and King (1988) were not able to dismiss the observations by Luckett (1978) i n which the caudal margin of the epiblast seems to be involved i n contributing to the formation of the extraembryonic mesoderm. Further, Luckett (1978) described a d i f f e r e n t fate for the network of c e l l s derived from the hypoblast which are observed between the primary yolk sac endoderm and trophoblast. Luckett suggested that t h i s tissue develops an endodermal meshwork, the inner layer of which eventually constitutes the primary yolk sac endoderm. As the blastocyst grows, the meshlike tissue unfolds u n t i l only a few single strings are recognizable on day 12. Luckett (1978) also pointed out that the primordium of mesodermal v i l l i develop i n i t i a l l y i n a l o c a l i z e d region overlying the precociously d i f f e r e n t i a t e d p r i mitive streak. The close temporal and s p a t i a l r e l a t i o n s h i p between primitive streak d i f f e r e n t i a t i o n and the i n i t i a t i o n of the chorionic v i l l u s formation i n the 12 to 14 day human embryo suggests that the sheet of 72 extraembryonic mesoderm spreading from the p r i m i t i v e streak and the overlying trophoblast have an inductive i n t e r a c t i o n i n the formation of the chorionic v i l l i ( i b i d . ) . Another hypothesis i s that the hypoblast contributes to the formation of the extraembryonic mesoderm i n the chorionic cavity, which i s l a t e r joined by extraembryonic mesoderm derived from the epiblast (Carlson, 1994). In t h i s scenario the extraembryonic mesoderm from the hypoblast i s expected to be trisomic while that from the epiblast i s d i p l o i d r e s u l t i n g i n a mosaic placenta i n gestations with CPM types II and I I I . Kalousek (1993) has suggested that.the complete dichotomy between the aneuploid placenta and d i p l o i d fetus i n conceptuses diagnosed with CPM type III arises from an i n i t i a t i o n of the twinning process. A meiotic nondisjunction event gives r i s e to a trisomic zygote and develops into a nonmosaic trisomic blastocyst. The trisomic zygote rescue occurs i n a f r a c t i o n of the c e l l s of the inner c e l l mass of the blastocyst destined to d i f f e r e n t i a t e into the embryo. The d i p l o i d embryonic progenitors aggregate to form one embryo and the trisomic embryonic progenitors form a second embryo. The trisomic nonviable twin "vanishes" and the d i p l o i d twin develops as a singleton supported by a trisomic placenta. There are two l i n e s of evidence found i n the l i t e r a t u r e which support this hypothesis. F i r s t l y , there have been reports of monozygotic twins who d i f f e r i n t h e i r chromosomal constitution. Nielsen (1967) summarized 73 twin studies which indicate that one twin of monozygotic pairs has trisomy 21 or monosomy for the X chromosome- while the other has a normal karyotype. Secondly, Landy et a l . (1986) performed sonographic analysis of 1000 pregnancies with viable gestations i n the f i r s t trimester and showed that 3.29% were twins and 21.2% of theses gestations demonstrated the "vanishing twin" phenomenon with a good prognosis for the remaining fetus. Regardless of several e f f o r t s to e s t a b l i s h the embryological o r i g i n of the extraembryonic mesoderm, a conclusive r e s u l t has not been reached to t h i s day. C l i n i c a l material obtained from gestations with CPM provides an opportunity to assess the embryologic relatedness between d i f f e r e n t tissues, but the unknown embryologic stage at which the mutational event occurs during development undermines the r e l i a b i l i t y of making conclusive statements from such studies. Further, possible forces of c e l l s e l e c t i o n which l i k e l y take place i n chromosomally mosaic gestations complicate the interpretations of the developmental processes leading to CPM. 74 4.1.d. Formation of the D e f i n i t i v e Yolk Sac Luckett (1978) was the f i r s t to hold the now generally accepted view that the hypoblast i n humans i s derived from the inner c e l l mass. Also, i t i s now clear that the hypoblast gives r i s e to c e l l s which l i n e the trophoblast of the blastocyst cavity to form the endoderm of the primary yolk sac. Formation of the secondary yolk sac i s a developmental process unique to the anthropoid primates including monkeys, apes and humans. In the rhesus monkey, the secondary yolk sac i s formed by a folding and pinching off of the endoderm of the primary yolk sac (Luckett, 1978). The development of the secondary yolk sac i n the human i s poorly understood. Luckett (197 8) describes the collapse of the primary yolk sac i n the human between 12 and 13 days gestation. Analysis of day 12 to 14 human embryonic s e r i a l sections prompted Luckett (197 8) to hypothesize that the intact remnants of the primary yolk sac which come to l i e immediately beneath the embryonic disc are pinched off to form the secondary yolk sac. However, at this stage of human development the ruptured secondary yolk sac i s surrounded by multipotential c e l l s of the extraembryonic mesoderm. It i s d i f f i c u l t to conclude with certainty that c e l l s of the primary endoderm contribute exclusively to the development of the secondary yolk sac. Thus, i t i s also possible that c e l l s of the 75 extraembryonic mesoderm are involved i n the formation of the secondary yolk sac. At 16 days of gestation the endoderm of the secondary yolk sac i s completely covered with extraembryonic mesoderm which forms the d e f i n i t i v e yolk sac splanchnopleure (Luckett, 1978) . The chromosomal constitution of the secondary yolk sac i n gestations with CPM has not been documented. The amnion and v i l l u s stroma are d i p l o i d i n gestations affected with CPM type I. Since the yolk sac endoderm l i k e the amnion and v i l l u s stroma, i s derived from the inner c e l l mass, i t i s also expected to be d i p l o i d . We hypothesize that the extraembryonic mesoderm contributes to the formation of the secondary yolk sac. Therefore we expect that i n conceptuses with CPM types II and III i n which there i s chromosomal mosaicism or complete aneuploidy i n the extraembryonic mesoderm, the endoderm of the secondary yolk sac w i l l be mosaic or completely aneuploid. 4.I.e. Primordial Germ C e l l s It i s well established that human PGCs are f i r s t i d e n t i f i a b l e i n the secondary yolk sac endoderm at four weeks of development (Fujimoto et a l . , 1977). This i s followed by a separation of the PGCs from the endoderm and migration into the embryo by amoeboid movements (Fujimoto et 76 a l . , 1977). The PGCs continue t h e i r migration u n t i l they reach the gonadal ridge where they invade the pri m i t i v e sex cords. In female development, the PGCs undergo a series of mitotic d i v i s i o n s u n t i l the number of oogonia i n each ovary reaches approximately 7 000 000 at 18 weeks of development. The oogonia enter into prophase of meiosis I at eight weeks and arrest i n dictyotene of meiosis at seven months. The majority of oocytes undergo apoptosis so that the number declines to 400 000 by the seventh month of development and only 3 0 000 i n the seven year old c h i l d . In the f e t a l t e s t i s the germ c e l l s p r o l i f e r a t e from week 8 u n t i l week 11, at which time they enter a res t i n g stage. Then at puberty, subpopulations of spermatogonia undergo waves of mitosis, and the progeny of these d i v i s i o n s enter meiosis as synchronous groups to form spermatozoa (Carlson, 1994) . Although the PGCs are f i r s t i d e n t i f i a b l e by t h e i r a l k a l i n e phosphatase a c t i v i t y i n the endoderm of the secondary yolk sac, the c e l l lineage from which they evolve i s not understood i n the human (Fujimoto et a l . , 1977). It i s possible that the hypoblast contributes to t h e i r formation since t h i s c e l l lineage i s involved i n the development of the secondary yolk sac endoderm. However, since the extraembryonic mesoderm may also contribute to the formation of the secondary yolk sac, i t i s possible that the PGCs ar i s e from the d i f f e r e n t i a t i o n of the extraembryonic mesoderm. 77 F i n a l l y , there i s evidence i n mouse embryological studies that the PGCs arise from the same c e l l s of the epiblast that give r i s e to the extraembryonic mesoderm. Epiblast c e l l s i n the proximal region give r i s e to the extraembryonic mesoderm as well as the PGCs (Lawson and Hage, 1994). This suggests that the extraembryonic mesoderm and the PGCs have a common embryological o r i g i n i n mammals. The chromosomal constitution of the gametes has not previously been studied i n cases diagnosed with CPM. Since the PGCs seem to have a common embryological o r i g i n with the extraembryonic mesoderm, we expect that conceptuses diagnosed with type II and III CPM may show chromosomal mosaicism of the germ c e l l s . 4.1.f. Hematopoiesis I It has been generally accepted that the progenitor blood c e l l s which contribute to hematopoiesis throughout the embryonic, f e t a l and adult periods of l i f e a r i s e from the mesoderm of the secondary yolk sac and migrate to the l i v e r , spleen and bone marrow during the embryonic/fetal period (Schwartz et a l . , 1991). The yolk sac mesoderm i s the f i r s t s i t e of hematopoiesis during the t h i r d week of development. Blood c e l l production i s then taken over by the f e t a l l i v e r at s i x weeks, followed by the spleen and bone marrow (Dzierzak and Medvinsky, 1995). If the o r i g i n of hematopoiesis i s the mesoderm of the yolk sac, then i t i s 78 d i f f i c u l t to explain the finding of d i p l o i d blood i n cases diagnosed with confined placental mosaicism types II and III i n which the extraembryonic mesoderm i s mosaic (Kalousek and D i l l , 1983; Ledbetter et a l . , 1992). It has been suggested that t h i s discrepancy i s due to s e l e c t i v e pressures during hematopoietic d i f f e r e n t i a t i o n , during which only d i p l o i d stem c e l l s survive (Bianchi et a l . , 1993) . Another p o s s i b i l i t y i s that yolk sac hematopoiesis i s transient and d e f i n i t i v e stem c e l l production occurs i n the embryonic mesoderm. There i s very persuasive evidence emerging from studies of hematopoiesis i n the mouse which support the hypothesis that d e f i n i t i v e blood c e l l production occurs i n the AGM region. Dickson et a l . (1995) have shown that mutant mouse embryos with a d e f f i c i e n c y of yolk sac hematopoiesis contain erythrocytes i n the AGM region. Medvinsky and Dzierzak (1996) demonstrated that only the AGM region and neither the yolk sac nor the l i v e r of 10 day post coitum mouse embryos was able to contribute to d e f i n i t i v e hematopoiesis of l e t h a l l y i r r a d i a t e d mouse re c i p i e n t s . 4.1.g. Amnion In his investigation, Luckett (1975) describes the process of amniogenesis as c a v i t a t i o n of the dorsal epiblast. This cavity enlarges and the roof becomes thinner u n t i l i t opens up. The edges of the epiblast grow towards each other u n t i l they j o i n to form the ectoderm of the 79 amnion. The thin amniotic ectoderm i s l a t e r joined by a layer of extraembryonic mesoderm which p r o l i f e r a t e s from the caudal margin of the epiblast to form the outer mesoblastic layer of the amnion (Luckett, 1978). In mosaic gestations affected with CPM, amniocentesis i s assumed to represent the karyotype of the fetus and not the placenta. While chromosomal mosaicism i s found i n the placenta i n 1 to 2% (Ledbetter et a l . , 1992; Wang et a l . , 1994) of chorionic v i l l u s samplings, the incidence i n pregnancies tested by amniocentesis i s 0.1 to 0.3% (Hsu et a l . , 1984; Worton and Stern, 1984; Bui et a l . , 1984). Since amniocentesis involves the culture of f i b r o b l a s t s from amniotic ectoderm as well as f e t a l tissues including skin c e l l s and e p i t h e l i a l c e l l s of the g a s t r o i n t e s t i n a l , respiratory and urinary tracts, i t has been established i n the l i t e r a t u r e that the chromosomal constit u t i o n of the amniotic ectoderm i s most often concordant with the f e t a l tissues i n cases diagnosed with CPM. Also, chromosomal mosaicism detected i n amniotic f l u i d has been confirmed i n the fetus i n 70% of the cases (Hsu et a l . , 1984). F a i l u r e to confirm mosaicism i n the fetus has been explained by contamination of amniotic f l u i d by chorionic c e l l s during amniocentesis i n gestations with CPM. It i s also possible that there are low levels of mosaicism i n the fetus which are not confirmed at term because tissues sampled are not representative of a l l f e t a l c e l l lineages, i . e . , only f e t a l skin and/or blood are usually used to confirm mosaicism. 80 Discrepant CVS and amniocentesis may also be due to the presence of an undetected degenerated dizygotic twin (Reddy et a l . , 1991; C h i t i a t et a l . , 1991). 4.2 Cytogenetic Analysis of Mosaicism Fluorescence in s i t u hybridization has allowed the detection of s p e c i f i c numerical chromosome aberrations i n interphase n u c l e i . This was accomplished with a labeled DNA probe s p e c i f i c for the r e p e t i t i v e a l p h a - s a t e l l i t e sequences found i n the centromeric region of the chromosome of i n t e r e s t . Subsequent immunochemical detection with conjugated fluorochromes allowed d i r e c t v i s u a l i z a t i o n of the number of copies of s p e c i f i c chromosomal regions. In t h i s way hundreds of nuclei were scored for numerical abnormalities of a s p e c i f i c chromosome r e l a t i v e l y quickly. At the same time the power of the s t a t i s t i c a l analysis was increased compared to conventional cytogenetic analysis since hundreds of interphase nuclei were analyzed. Only 15 metaphases are routinely analyzed by conventional cytogenetic analysis. Assay s e n s i t i v i t y was enhanced because the p r o b a b i l i t y of detecting low le v e l s of mosaicism was increased by the analysis of hundreds of n u c l e i . Another advantage of FISH was that non-dividing c e l l s were amenable to analysis including archival tissues fixed i n formalin and p a r a f f i n embedded. Further, since conventional cytogenetic analysis most often requires tissue 81 culture to obtain analyzable metaphases the possible growth advantage of one c e l l l i n e over another can r e s u l t i n tissue culture bias (Lomax et a l . , 1994). By using FISH analysis to detect aneuploidy this tissue culture bias was eliminated. Although FISH analysis i s a powerful technique i t i s l i m i t e d by the probe used to target a s p e c i f i c sequence whereas conventional cytogenetic analysis can provide information on numerical and s t r u c t u r a l abnormalities of the entire genome (Eastmond et a l . , 1995). Thus analysis of the mosaic gestations f i r s t required conventional cytogenetic techniques to i d e n t i f y the chromosome involved i n the trisomy before molecular cytogenetics could be employed. For example, culture of v i l l u s stroma followed by conventional cytogenetic analysis of Case 1 revealed the trisomy of chromosome 7. The d i s t r i b u t i o n of trisomy i n f e t a l and placental tissues could then be explored by the u t i l i z a t i o n of FISH analysis with a chromosome 7 s p e c i f i c probe. In addition, technical a r t i f a c t s can p o t e n t i a l l y r e s u l t i n d i p l o i d controls with fa l s e p o s i t i v e findings of trisomy or monosomy. Thus s t a t i s t i c a l analysis of control data to set cut-off values for the detection of trisomy was performed as described i n the Materials and Methods. Proportions of fa l s e trisomy i n the disomic control tissues analyzed i n t h i s study ranged from 1 to 8.4% (see Appendix). Greater than two hybridization signals can appear 82 i n d i p l o i d nuclei by the s p l i t t i n g of one signal to make i t appear as a doublet. This i s thought to a r i s e from either premature chromatid separation during f i x a t i o n or c e l l s i n G2 phase of the c e l l cycle (Eastmond et a l . , 1995). Also, i n s u f f i c i e n t stringency of the post-hybridization wash can r e s u l t i n hybridization of the DNA probe to non-specific s i t e s i n the genome (Lomax et a l . , 1994). Extra signals may also appear as background fluorescence due to non-specific binding of fluorochrome-conjugated antibodies to nuclear or c e l l u l a r proteins fixed to the s l i d e . The proportion of somatic c e l l s with one signal for the chromosome of int e r e s t i n disomic controls ranged from 1.2 to 11.2% (see Appendix). These re s u l t s may be due to i n e f f i c i e n t penetration of the probe into the nucleus, high stringency post-hybridization wash, overlapping signals from two chromosomes and suboptimal hybridization conditions (Lomax et a l . , 1994; Eastmond et a l . , 1995). 4.3 Cytogenetic Analysis of Mosaic Conceptuses 4.3.a. Case 1 Conventional cytogenetic analysis of cultured chorionic plate showed that two out of four metaphases were 47,XY,+7 while cultured amnion showed f i v e d i p l o i d metaphases. These res u l t s suggest that this conceptus was affected with confined placental mosaicism since the amniotic ectoderm i s 83 derived from the same c e l l lineage as the f e t a l skin. From these r e s u l t s alone, i t was not possible to determine i f the fetus was indeed d i p l o i d since only amnion was analyzed. Therefore, molecular cytogenetic analysis was performed on formalin fixed - p a r a f f i n embedded f e t a l tissues. FISH analysis of f e t a l lung and kidney with the chromosome 7 s p e c i f i c probe showed that 7.2% and 2.2% of the nuclei exhibited three signals respectively. The upper range of the 95% confidence l i m i t for a diagnosis of trisomy was calculated to be 9.92 and 11.78% for the lung and kidney, respectively. Therefore the proportion of nuclei with three signals i n these tissues was not considered to be s i g n i f i c a n t . In order to confirm that the placenta was mosaic for trisomy 7, FISH analysis was performed on formalin fixed -p a r a f f i n embedded chorionic v i l l i from three s i t e s of the placenta. The proportion of nuclei with three signals exceeded the 95% confidence l i m i t of the upper reference l i m i t i n a l l three s i t e s . The l e v e l of trisomy i n the placenta was 11.45% for s i t e 1, 12.2% for s i t e 2 and 23.5% for s i t e 3. As these values are greater than the cut - off value for trisomy calculated to be 7.89%, s i g n i f i c a n t levels of mosaicism for trisomy 7 i n the placenta were confirmed. The d i s t r i b u t i o n of trisomic c e l l s i n the placenta was not determined since the trophoblast and v i l l u s stroma of the placenta could not be analyzed separately. The stroma of the placenta has been shown to be mosaic since 2/4 84 trisomic c e l l s were i d e n t i f i e d i n the cultured stroma by-conventional cytogenetics. However, the exact l e v e l of trisomic c e l l s i n the v i l l u s stroma could not be determined since the FISH analysis also included nuclei derived from the trophoblast c e l l lineage. If the trophoblast c e l l lineage was nonmosaic d i p l o i d , then high le v e l s of trisomy i n the v i l l u s stroma would have been d i l u t e d i n the FISH analysis. Since the embryological o r i g i n of the PGCs i s c l o s e l y related to the v i l l u s stroma, FISH analysis was performed on the t e s t i s of this gestation. The population of nuclei analyzed from th i s t e s t i s included both somatic and germ c e l l s . The proportion of nuclei with three signals was 2.5% i n the mosaic conceptus which was well below the cut - off value for trisomy, calculated to be 7.69%. These results suggest that the germ c e l l s of the t e s t i s were nonmosaic d i p l o i d . However, the p o s s i b i l i t y that there was a low l e v e l of germline mosaicism can not be ruled out because germ c e l l s constitute a small proportion of the number of c e l l s i n the f e t a l t e s t i s . Mendez and Emery (1979) found that the germ c e l l s constitute 5 to 15% of the seminiferous tubules i n the f e t a l gonad. There i s also a population of i n t e r s t i t i a l c e l l s surrounding the tubules i n the f e t a l gonad which p r o l i f e r a t e and occupy almost half of the t e s t i c u l a r volume at 14 to 18 weeks of development. After 18 weeks, the i n t e r s t i t i a l c e l l s degenerate u n t i l they occupy approximately 12% of the t e s t i s at b i r t h (Orvis et 85 a l . , 1988; Mancini et a l . , 1965). Therefore, due to the low proportion of germ c e l l s expected i n a fetus of 19 weeks development, such as Case 1, the a b i l i t y to detect trisomy i n the germ c e l l population by this molecular cytogenetic analysis i s compromised. Ideally, only germ c e l l s should be scored to detect chromosomal mosaicism i n the germline. However, i n this case the f e t a l germ c e l l s can not be distinguished from the surrounding somatic c e l l s by the cytogenetic method u t i l i z e d . An al t e r n a t i v e method to investigate the presence of trisomy i n male f e t a l germ c e l l s i s to perform FISH analysis on sections of p a r a f f i n embedded - formalin fi x e d t e s t i s mounted on a microscope s l i d e . The advantage to this technique when compared to preparing a c e l l suspension as i n the present study, i s that the morphology of the t e s t i s i s maintained and germ c e l l s can be stained with hematoxalin and eosin and i d e n t i f i e d . Then the p o s i t i o n of the germ c e l l s on the microscope s l i d e can be recorded before the c e l l s are processed for the FISH analysis. However, th i s technique requires thin sectioning of the gonad (5 to 7 microns) and can r e s u l t i n truncation of the nuclei under investigation and an underestimation of the number of chromosomes i n the nuclei (Kuchinka et a l . , 1595). 86 4.3.b. Case 2 Prenatal diagnosis performed on th i s pregnancy showed that 21/21 c e l l s were 47,XX,+16 from cultured v i l l i . Following termination, conventional cytogenetic analysis showed trisomy for 5/5 c e l l s i n cultured chorionic plate and 8/10 c e l l s for v i l l u s stroma, while the amnion was d i p l o i d i n the 5 metaphases analyzed. The findings from the conventional cytogenetic analyses suggested that this mosaic conceptus was affected with confined placental mosaicism. In order to explore the extent of chromosomal mosaicism i n the placenta, FISH analysis was performed on the v i l l u s stroma and trophoblast c e l l lineages from two s i t e s . Following enzymatic separation of the stroma from the chorionic v i l l i , FISH analysis revealed trisomy for chromosome 16 i n 50.4% (site 1) and 68.2% (site 2) of the n u c l e i . High leve l s of trisomy were also found i n both s i t e s of the trophoblast (79.6% and 72.4%). FISH analysis with a chromosome 16 s p e c i f i c probe was performed on the lung, kidney and brain since they represent the f e t a l endoderm, mesoderm and ectoderm, respectively. The proportion of nuclei with three signals for these tissues were 1.2%, 2.2% and 6.6%, respectively, which were less than the cut-off values for trisomy (8.82%, 6.17% and 7.06%). The cord blood showed 5.8% of the nuclei with three signals while the upper reference l i m i t of the 95% 87 confidence i n t e r v a l for detection of trisomy was 5.92%. Therefore no s i g n i f i c a n t levels of trisomy were observed i n any f e t a l somatic tissues analyzed. High lev e l s of trisomy 16 i n the placenta of a d i p l o i d fetus has been associated with a meiotic o r i g i n of the extra chromosome (Kalousek et a l . , 1993; Robinson et al-. , 1997). Molecular analysis of marker'D16S398 on chromosome 16 showed three a l l e l e s for the v i l l u s stroma, trophoblast and amniotic membrane. This indicated that the extra chromosome in the trisomic tissues arose from a meiotic nondisjunction event. PCR amplification of marker D16S423 indicated maternal UPD for th i s case since f e t a l tissues showed maternal contribution for the chromosome 16 marker while there was no contribution from either of the paternal a l l e l e s . The single a l l e l e for the f e t a l tissues indicated that the fetus was homozygous for th i s m i c r o s a t e l l i t e marker even though the mother was heterozygous. Since there was a meiotic o r i g i n of the extra chromosome, the reduction to homozygosity of th i s a l l e l e i n the fetus most l i k e l y arose from meiotic recombination occurring between th i s marker and the centromere of the chromosome. Meiotic recombination i n thi s region of the chromosome i s l i k e l y since marker D16S423 i s approximately 48.7 centimorgans away from the centromere (Buetow et a l . , 1994). The meiotic o r i g i n of the trisomic c e l l l i n e and maternal UPD i n f e t a l tissues suggest that the mosaic gestation arose from a trisomic zygote rescue. Further, the 88 finding of d i p l o i d c e l l s i n the chorionic stroma suggests that the formation of the d i p l o i d c e l l l i n e occurred early i n development before the extraembryonic mesoderm f u l l y d i f f e r e n t i a t e d from the f e t a l tissues. A l t e r n a t i v e l y , the trisomic extraembryonic mesoderm may have arisen from the hypoblast while the d i p l o i d extraembryonic mesoderm d i f f e r e n t i a t e d from d i p l o i d epiblast c e l l s . The molecular r e s u l t from the amniotic membrane i s not in agreement with the conventional cytogenetic analysis. The sample of amnion for molecular analysis and conventional cytogenetics was taken from the amniotic sac which consists of ectoderm and mesoderm. The amniotic ectoderm i s derived from the same c e l l lineage which gives r i s e to f e t a l ectoderm, and the amniotic mesoderm i s derived from the extraembryonic mesoderm (Vogler, 1987). Therefore the t h i r d a l l e l e i n the m i c r o s a t e l l i t e analysis i s probably a r e s u l t of trisomy i n the amniotic mesoderm. The d i p l o i d r e s u l t obtained from the conventional cytogenetic analysis of the amnion sampled from the amniotic sac suggests that there was sel e c t i v e growth of d i p l o i d c e l l s i n culture. Another p o s s i b i l i t y i s that trisomic c e l l s grew i n culture but were missed i n the analysis since only 5 metaphases were analyzed. I d e n t i f i c a t i o n of oocytes for FISH analysis was possible i n th i s conceptus of 12 weeks gestational age because female meiosis i s i n i t i a t e d at 8 weeks of gestation. The meiotic oocytes have d i f f u s e chromatin and are much 89 larger than somatic c e l l s . The molecular cytogenetic analysis of these oocytes showed that 26.0% were trisomic for chromosome 16. This i s i n agreement with our hypothesis that the extraembryonic mesoderm contributes to the formation of the secondary yolk sac endoderm and i n doing so i s involved i n the formation of the PGCs. This i s also i n agreement with the twinning hypothesis proposed by Kalousek (1993). In this scenario, the trisomic zygote rescue occurred i n a f r a c t i o n of the embryonic progenitor c e l l s at the blastocyst stage. The trisomic embryonic progenitors gave r i s e to a nonviable trisomic twin and d i p l o i d c e l l s contributed to the chromosomally normal embryo. The extraembryonic mesoderm i s formed from trisomic c e l l s of hypoblast o r i g i n and d i p l o i d c e l l s from the pri m i t i v e streak. Thus, since the extraembryonic mesoderm was mosaic, i t contributed trisomic c e l l s to the development of the secondary yolk sac and subsequently the PGCs. Another p o s s i b i l i t y i s that the hypoblast c e l l lineage was trisomic i n th i s gestation and contributed to the formation of the primary and secondary yolk sac as well as to the extraembryonic mesoderm, as suggested by Enders and King (1988) and Bianchi et a l . (1993). If we consider that the PGCs d i f f e r e n t i a t e from the endoderm of the secondary yolk sac, then i t i s expected that they are at r i s k of being trisomic as well. It i s also possible that the human PGCs are derived from the epiblast c e l l s , as i s the case i n the rodent 90 (Bianchi et a l . , 1993). Clonal analysis of epiblast c e l l s by i n j e c t i o n of a f i x a b l e fluorescent dye followed by fate mapping has shown that the clones which gave r i s e to the PGCs were derived from the same region of the epiblast which contributed to the formation of the extraembryonic mesoderm (Lawson and Hage, 1994). In the mouse, the PGCs migrate into the endoderm of the yolk sac and then migrate back into the germinal ridge ( i b i d . ) . Perhaps the epiblast of case 2 became mosaic with a d i p l o i d and trisomic c e l l l i n e subsequent to the trisomic zygote rescue. The c e l l s of the epiblast which gave r i s e to the f e t a l somatic tissues were a l l d i p l o i d due to selection forces or by chance, and both d i p l o i d and trisomic c e l l s contributed to the formation of the extraembryonic mesoderm and PGCs. Such a hypothesis suggests that the precursor populations for major tissue types are l o c a l i z e d to s p e c i f i c regions i n the epiblast. Embryological studies by manipulation of mouse embryos have constructed fate maps of the epiblast to depict the geographical organization of the progenitors of various embryonic tissues. For example, c e l l s i n the d i s t a l part of the epiblast eventually give r i s e to the neuroectoderm and surface ectoderm of the early somite stage embryo (Quihlan et a l , 1995; Parameswaran and Tarn, 1995). The c e l l s i n the l a t e r a l epiblast midway along the proximal d i s t a l axis give r i s e mostly to embryonic mesoderm while the proximal epiblast c e l l s contribute to the extraembryonic mesoderm and PGCs (Parameswaran and Tam, 1995). These re s u l t s are 91 consistent with the observations made by Lawson and Hage m which proximal epiblast c e l l s i n the mouse gave r i s e to both extraembryonic mesoderm and PGCs. The trisomic r e s u l t found i n oocytes of th i s mosaic gestation suggests that there i s a si m i l a r o r i g i n of the extraembryonic mesoderm and PGCs. However, since the exact stage of development during which the trisomic zygote rescue took place i n th i s mosaic gestation i s not known, these res u l t s do not support one of the above hypotheses over another. 4.3.c. Case 3 Conventional cytogenetic analysis was previously performed on th i s gestation and published by Kalousek et a l . (1993). Direct preparation from the trophoblast c e l l lineage showed that 9/9'cells were trisomic for chromosome 16 as were 47/47 c e l l s from cultured chorionic stroma. Conversely, 15 c e l l s from cultured amnion, skin, kidney and spleen as well as 63 c e l l s from blood were found to be d i p l o i d by conventional cytogenetic analysis. Molecular analysis on blood of the fetus revealed that i t was UPD for the maternal copies of chromosome 16. Therefore Kalousek et a l . (1993) c l a s s i f i e d t h i s gestation with CPM type III and suggested that i t arose from trisomic zygote rescue. FISH analysis with a chromosome 16 s p e c i f i c probe was performed on formalin fixed - p a r a f f i n embedded archival 92 placenta, kidney, lung and oocytes. The FISH analysis f a i l e d on several attempts for the kidney and three s i t e s from the placenta while i t was successful for the lung and oocytes. Although analysis of formalin fixed - p a r a f f i n embedded tissues i s essential for the study of cases several years old, FISH analysis on such tissues can become problematic. For example, the tissues may not have been fixed i n formalin for several days a f t e r the pregnancy was terminated or the tissues may not have been fix e d i n formalin for a s u f f i c i e n t period of time. As a r e s u l t , the genomic DNA may have degenerated, making the FISH analysis d i f f i c u l t . The lung and oocytes showed 3.2% and 5.0% of nuclei with three signals while the cut-off values from the disomic controls were 9.92% and 6.87% respectively. The data provided by Kalousek et a l . (1993), indicated that cultured amniotic f l u i d , post-termination amniotic membrane, skin, kidney, spleen and blood showed only d i p l o i d c e l l s while a l l c e l l s from d i r e c t analysis of chorionic v i l l i , culture of chorionic plate and v i l l i showed trisomy i n a l l c e l l s analyzed. Together these results provide strong evidence that the placenta has high lev e l s of trisomy 16 i n trophoblast and stroma while the fetus i s d i p l o i d . Unlike case 2, the oocytes of th i s gestation had no s i g n i f i c a n t l e v e l s of trisomy 16. However, i n t h i s FISH analysis, the a b i l i t y to detect trisomy i n the oocytes was compromised by the paired chromosomes i n zygotene and pachytene stages of 93 meiosis. Since technical d i f f i c u l t i e s prevented the a b i l i t y to perform two - colour FISH with an i n t e r n a l control on the p a r a f f i n embedded - formalin fixed oocytes, one - colour FISH with a chromosome 16 s p e c i f i c probe was used. Thus i t was not possible to select oocytes which had unpaired chromosomes for FISH analysis. In order to explore the effects that paired homologues may have had on the a b i l i t y to detect trisomy i n oocytes, FISH analysis was performed on formalin f i x e d - p a r a f f i n embedded oocytes from a trisomy 18 fetus. As shown i n Table 6.9 i n the Appendix, the chromosome 18 s p e c i f i c DNA probe showed one signal i n 29%, two signals i n 36% and three signals i n 39% of the oocytes analyzed, even though i t i s expected that 100% of the oocytes are trisomic for chromosome 18. The re s u l t s from th i s trisomic fetus are consistent with those obtained by Cheng et a l . (1995). They studied the p a i r i n g c h a r a c t e r i s t i c s of homologous chromosomes i n oocytes from trisomy 18 fetuses. FISH analysis with a chromosome 18 s p e c i f i c paint indicated that the homologues i n the pachytene stage of meiosis were arranged i n three possible configurations: 1) a l l three chromosomes synapsed to form one thick hybridization signal; 2) two of the chromosomes synapsed while one remained unpaired; and 3) a l l three chromosomes remained unpaired and were v i s u a l i z e d as three separate univalents. Thus, the p a i r i n g c h a r a c t e r i s t i c s of homologous chromosomes decreased the a b i l i t y to detect trisomy i n oocytes from Case 3, and there i s a p o s s i b i l i t y 94 that low leve l s of trisomy may have been undetectable using this analysis. In order to i l l u s t r a t e the advantage of using an int e r n a l control to select for oocytes i n which the homologous chromosomes were not paired, FISH analysis was performed on fresh oocytes from a trisomy 18 fetus. Since the oocytes were not formalin fixed - p a r a f f i n embedded, two colour FISH was used successfully to v i s u a l i z e centromeric s p e c i f i c probes for chromosomes 18 and 16. In th i s analysis the oocytes were scored for the number of signals displayed by the chromosome 18 s p e c i f i c probe only i f the i n t e r n a l control, chromosome 16, showed two hybridization signals. As shown i n Table 6.11 i n the Appendix, 4% of the oocytes showed one signal, 17% showed two signals and 71% showed three signals for chromosome 18. A comparison of these res u l t s with those obtained from the formalin fixed -p a r a f f i n embedded oocytes above, shows the advantage of performing FISH analysis on fresh oocytes. One approach which may be useful for future studies of aneuploidy i n oocytes would be to use a cosmid DNA probe s p e c i f i c for DNA sequences close to the centromere. Since cosmid probes are t y p i c a l l y smaller than the commercial alpha s a t e l l i t e centromeric probes, smaller hybridization signals may allow for accurate i d e n t i f i c a t i o n of the number of homologous chromosomes which are paired. The lack of trisomic oocytes i n Case 3 suggest that not a l l cases diagnosed with CPM type III w i l l show germline 95 mosaicism. This can occur for several d i f f e r e n t reasons. The p o s i t i o n of.the trisomic c e l l s i n the mosaic gestation may be the most important factor. For example, the trisomic extraembryonic mesoderm may have contributed to the formation of the PGCs i n case 2 but not case 3. Developmental age of the fetus may also play a r o l e . It i s possible that case 3 showed no evidence of trisomic oocytes as a r e s u l t of selection due to a developmental disadvantage of oocytes with an extra chromosome 16. The pregnancy from case 2 was terminated at 12 weeks while case 3 was terminated at 25 weeks gestation. During development, the majority of oocytes undergo apoptosis so that the number declines from 7 000 000 at 18 weeks to 400 000 by 28 weeks of development. It i s possible that trisomic oocytes from case 3 were selected against during t h i s developmental process. 4.4 C l i n i c a l Implications of t h i s Study The trisomic oocytes i d e n t i f i e d i n case 2 of t h i s study suggests that individuals who are c l a s s i f i e d with CPM type III w i l l also have chromosomal mosaicism i n t h e i r germ c e l l s . Chromosomal mosaicism i d e n t i f i e d i n the germ c e l l s may a f f e c t the fecundity of an in d i v i d u a l i n several ways. F i r s t l y , the aneuploid germ c e l l s may not be capable of developing into f e r t i l e ova/spermatozoa. Depending on the proportion of aneuploid germ c e l l s i n the gonad, th i s may 96 lead to i n s u f f i c i e n t ova/spermatozoa production and thus i n f e r t i l i t y . For example, the majority of males affected with trisomy 21 are i n f e r t i l e and are unable to produce sperm or have a marked decrease i n spermatogenesis. (Stearns et a l . , 1960). Another p o s s i b i l i t y i s that the germ c e l l s are capable of completing development and f e r t i l i z a t i o n ; as i s the case i n females affected with Down's syndrome. These individuals are able to conceive children with a normal karyotype, but are at a greater r i s k of producing aneuploid conceptuses which are expected to abort (Neri, 1984). In individuals affected with germ c e l l chromosomal mosaicism, the proportion of trisomic germ c e l l s w i l l influence the individuals fecundity i n the reproductive years. A high proportion of aneuploid germ c e l l s i s expected to decrease an individual's a b i l i t y to conceive normal children, while a low proportion of aneuploid germ c e l l s may have l i t t l e or no detrimental e f f e c t s . Although t h i s study suggests that a diagnosis of CPM type III i s associated with germ c e l l mosaicism, i t i s l i m i t e d i n the a b i l i t y to predict the incidence of germ c e l l mosaicism i n cases diagnosed with CPM type I I I . The f a i l u r e to f i n d a s i g n i f i c a n t l e v e l of trisomy i n case 3 indicates that germ c e l l mosaicism may not occur i n a l l cases diagnosed with CPM type I I I . Further studies must be performed i n order to determine the empirical r i s k of germline mosaicism i n individuals diagnosed with CPM types II and I I I . This study has shown that trisomy for 97 chromosome 16 can p e r s i s t i n the germ c e l l s u n t i l at least 12 weeks of gestation. However, older fetuses and individuals of reproductive age must be studied to determine the effects of chromosomal mosaicism on the developmental dynamics of the germ c e l l s . 4.5. Concluding Remarks This study was undertaken to explore the p o s s i b i l i t y that mosaic conceptuses diagnosed with CPM types II and/or III are associated with germline mosaicism. Three mosaic gestations which were ascertained as a re s u l t of suspected CPM were analyzed using interphase FISH i n order to determine the tissue s p e c i f i c d i s t r i b u t i o n of chromosomal mosaicism. The molecular cytogenetic analysis proved to be a useful technique since nondividing tissues including formalin fixed - p a r a f f i n embedded archival tissues were amenable to cytogenetic analysis. This method allowed for analysis of hundreds of nuclei which increased the a b i l i t y to detect mosaicism and avoided the r i s k of tissue culture bias which can be experienced with conventional cytogenetic analysis. For case 1, the d i s t r i b u t i o n of trisomy 7 i n the placenta was not determined since the v i l l u s stroma and cytotrophoblast of the placenta could not be analyzed separately. The FISH analysis showed s i g n i f i c a n t (11 to 23%) but low levels of trisomy 7 i n the placenta; however, 98 the s p e c i f i c l e v e l of mosaicism i n the v i l l u s stroma could not be determined. The a b i l i t y to detect trisomy i n the germ c e l l s of t h i s fetus was compromised by the i n a b i l i t y to d i s t i n g u i s h gonocytes from somatic c e l l s i n the t e s t i s . Since the proportion of germ c e l l s i n the prepubertal t e s t i s i s s i g n i f i c a n t l y less than 15% then lev e l s lower than 100% of trisomy i n the germ c e l l s would be d i f f i c u l t to detect. Cases 2 and 3 of t h i s study were more informative than case 1 for two reasons: 1) the fetuses were female and thus the oocytes were distinguishable from somatic c e l l s of the ovary; and 2) high levels of trisomy for chromosome 16 were observed i n the stroma of the placenta by FISH and/or conventional cytogenetic analysis. Molecular cytogenetic analysis of case 2 showed s i g n i f i c a n t levels of trisomy i n both placental c e l l lineages and the oocytes while s i g n i f i c a n t levels were not detected i n the f e t a l somatic c e l l s . This finding i s i n agreement with the hypothesis that mosaicism detected i n the stroma of the placenta i s associated with germ c e l l mosaicism. However, the f a i l u r e to detect germl c e l l mosaicism i n case 3 indicates that t h i s observation does not hold true for every i n d i v i d u a l diagnosed with CPM type I I I . To further explore the c l i n i c a l implications of t h i s study, many more mosaic conceptuses diagnosed with confined placental mosaicism must be analysed. Cytogenetic analysis of germ c e l l s from fetuses of several gestational ages as well as adults with a hi s t o r y of CPM type III w i l l provide 99 information about the emperical r i s k of germ c e l l mosaicism and on the developmental pote n t i a l of aneuploid germ c e l l s . The re s u l t s from case 2 also suggest that the developmental o r i g i n of the PGCs i s the same as the v i l l u s stroma. We propose that the extraembryonic mesoderm i s involved i n the repair of the ruptured yolk sac at 14 days of human development to form the secondary yolk sac. The extraembryonic mesoderm may therefore, contribute to the formation of the PGCs which are f i r s t observed i n the endoderm of the secondary yolk sac at 4 weeks of development. As a re s u l t of the lack of current knowledge about the developmental o r i g i n of the PGCs and the extraembryonic mesoderm i n humans, several hypotheses concerning early embryologic development can not be distinguished. The res u l t s from this study do not favor one postulate over another to explain the developmental events involved i n the formation of the PGCs for several reasons: 1) the p o s s i b i l i t y of selection against mutant c e l l l i n e s during human development; 2) the exact timing of the mutational event which gave r i s e to the d i p l o i d c e l l l i n e i n cases 2 and 3 i s not known; and 3) the exact c e l l which f i r s t gave r i s e to the d i p l o i d c e l l l i n e i n early embryogenesis can not be traced. A more informative method to es t a b l i s h the o r i g i n of the PGCs i n humans would be to construct fate maps by label i n g precursor c e l l s of early embryos and following t h e i r developmental patterns. However, since such 100 experiments are unethical we must gain insight into the developmental o r i g i n of the human PGCs using more i n d i r e c t approaches. One method would be to develop fate maps i n embryos of monkeys by labeling precursor c e l l s and following t h e i r developmental patterns. Insight into the developmental o r i g i n of the PGCs can also come from the study of human conceptions which are affected with chromosomal mosaicism. Many more conceptuses diagnosed with types II and III CPM must be studied to document the presence of aneuploid c e l l s i n the d i f f e r e n t embryologic c e l l lineages and correlate that information to the presence of germ c e l l mosaicism. 101 5. References Baker T.G. (1963) A quantitative and c y t o l o g i c a l study of germ c e l l s i n human ovaries. Journal of C e l l Sciences. 2 = 213-224 Barton S.C, Surani M.A.H. and Norris M.L. (1984) Role of paternal and maternal genomes i n mouse development. Nature 311:374-376 Bianchi D.W., Wilkins-Haug L.E., Enders A.C. and Hay E.D. 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American Journal of Human Genetics 55:74-81 Worton R.G. and Stern R.A. (1984) A Canadian collaborative study of mosaicism i n amniotic f l u i d c e l l cultures. Prenatal Diagnosis 4:131-144 110 6. Appendix 6.1. Raw Data for Mosaic Conceptuses Table 6.1: FISH analysis of tissues from Case 1 using centromeric DNA probe s p e c i f i c for chromosome 7. Tissue Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Lung 4.2 88.0 7.2 0.6 501 Kidney 4.6 93.2 2.2 0 501 Placenta Site 1 5.2 82 .8 11.4 0.6 500 Placenta Site 2 3.6 83 .6 12.2 0.6 500 Placenta Site 3 4.0 70.7 23.5 1.8 601 Testis 6.1 91.2 2.5 2.7 708 N = t o t a l number of nuclei scored Table 6.2: FISH analysis of tissues from Case 2 using centromeric DNA probe s p e c i f i c for chromosome 16. Tissue Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Brain 7 85.6 6.6 0.8 501 Kidney 5.2 93 .6 1.2 0 500 Lung 8.2 89.4 2.2 1.0 500 Blood 6 87.4 5.8 0.8 501 Trophoblast Site 1 2 24 72.4 1 500 Trophoblast Site 2 1.4 18.0 79.6 1.2 504 Stroma Site 1 0.6 29.8 68.2 1.4 500 Stroma Site 2 1.0 48.2 50.4 0.4 1148 Oocytes 7.5 64.8 26 1.7 227 N = t o t a l number of nuclei scored Table 6.3: FISH analysis of tissues from Case 3 using centromeric DNA probe s p e c i f i c for chromosome 16. Tissue Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Brain 7 85.6 6.6 0.8 501 Kidney 5.2 93 .6 1.2 0 500' N - t o t a l number of nuclei scored 6.2 Raw data of FISH analysis performed on control samples for formalin f i x e d - p a r a f f i n embedded tissues. Table 6.4: FISH analysis of disomic kidney control samples using a centromeric DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N prob e 91335 5.1 92 .3 2.6 0 507 16 96100 7.8 90.2 2.0 0 511 16 96126 6.4 92 . 0 1.4 0.2 502 16 9628 3.5 94.2 2.3 0 517 16 96144 10.4 80.6 8.4 0.6 500 16 96182 11.2 92.0 1.8 0 500 7 96178 9.8 79.6 7.3 0.8 510 7 96210 3.6 78.6 7.4 1.0 501 7 91111 3.2 87.0 3.2 0.4 500 7 93326 2.2 95.0 2.4 0 500 7 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 11.78 114 Table 6.5: FISH analysis of disomic lung control samples using a centromeric DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N prob e 96139 11.2 81.6 6 1.2 500 7 96182 9.8 82.4 7.4 0.4 500 7 93187 3 . 6 94.2 2.2 0 500 7 9628 3.2 94.6 2.2 0 500 7 96100 2.2 95.8 2.0 0 500 7 91335 3.8 92.6 2.8 0.2 503 16 96126 4.4 91.4 2.4 0.8 501 16 93187 5.4 88.8 5.2 0.4 501 16 9673 8.8 89.2 2.0 0 501 16 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 9.92 115 Table 6.6: FISH analysis of disomic controls for placental samples using a centromeric DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N prob e 93173 11.4 82 . 8 5.4 0.4 500 7 96182 10.2 85.4 3.8 0.6 500 7 96193 9.2 86.8 3.5 0.5 197 7 96151 8.0 88.1 3 . 6 0.3 327 7 96173 2.1 95.4 2.1 0.4 512 16 96182 3.8 90.6 4.8 0.8 501 16 96151 6.4 87.4 6.0 0.2 500 16 96144 5.8 90.3 3.7 0.2 513 16 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 7.89 116 Table 6.7: FISH analysis of formalin f i x e d - p a r a f f i n embeded disomic testes using a centromeric DNA probe s p e c i f i c for chromosome 7. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N 9628 4.6 92 . 6 2.6 0.2 500 96126 7.1 90.6 2.3 0 512 93187 6.4 90 . 6 2.8 0.2 501 93326 6.1 90.8 2.9 0.2 511 93140 6.2 88.6 5.2 0 501 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 7.69 Table 6.8: FISH analysis of formalin fixed - p a r a f f i n embeded disomic oocytes using a centromeric DNA probe s p e c i f i c for chromosome 16. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 . 2 3 4 N 9660 31.4 67.0 2.9 0 102 95399 22 71. 6 5.0 1.4 141 96151 26.7 66.3 5.0 2.0 101 95400 36.6 57 .4 5.0 1.0 101 9661 20.0 77 .3 2 .7 0 75 9639 23 .0 71.0 5.0 1.0 100 95317 33 . 0 63 .0 3.0 1.0 100 9196 34.0 61.0 4.0 1.0 100 95272 22.0 74.0 3.0 1.0 100 95252 31.0 65.0 4.0 0 100 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 6.87 Table 6.9: FISH analysis of formalin f i x e d - p a r a f f i n embeded oocytes from a trisomic 18 conceptus using a centromeric DNA probe s p e c i f i c for chromosome 18. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe 9790 29 36 39 6 100 18 N = t o t a l number of nuclei scored 119 6.3 Raw data for FISH analysis performed on fresh control samples prepared by the squash preparation. Table 6.10: FISH analysis of disomc fresh oocytes using a chromosome s p e c i f i c centromeric DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 -N Probe (Internal control) 9722 7 89 . 0 4 . 0 0 200 16 (18) 9650 13 .5 83 . 0 3.5 0 200 16(18) 9694 14.5 80.5 4.5 0.5 200 16(18) 96112 7.5 90.0 2.5 0 200 16(18) 976 8.3 90.7 0.5 0.5 193 16(18) 9689 6.2 89.7 3.9 0.2 517 X(8) 9659 4.0 94.2 1.8 0 502 X(8) 9683 5.4 92 .4 2.2 0 501 X(8) N= t o t a l number of oocytes scored Cut - o f f value for trisomy = 7.06 120 Table 6.11: FISH analysis of a squash preparation of an oocyte from a trisomy 18 syndrome fetus. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe (Internal control) 9733 4 17 71 8 100 18(16) N= t o t a l number of nuc l e i scored 6.4 Raw data for FISH analysis of touch preparations of disomic fresh control tissues. Table 6.12: FISH analysis of disomic fresh kidney using a chromomsome s p e c i f i c DNA centromeric probe. Patient Number Proportion of Nuclei with Indicated Number of Singnals 1 2 3 4 N Probe 9662 3.9 94.3 1.8 0 441 16 9522 2.7 93 .8 3.3 0.2 600 18 9643 4.0 92 .0 3.0 0 505 18 9645 12 86.8 1.2 0 500 2 N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 6.17 121 Table 6.12: FISH analysis of disomic lung touch preparations using a chromosome s p e c i f i c DNA centromeric probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe 96115 4.2 94.8 1.0 0 500 16 9643 5.1 89.5 5.4 0 600 18 9612 5.0 93 . 0 2.0 0 500 2 9659 7.2 93 .8 3.2 0 500 18 9645 9.4 84.6 5.8 0.2 500 7 N= t o t a l number of nuc l e i analyzed Cut - o f f value for trisomy = 8.82 12 Table 6.13: FISH analysis of disomic fresh b r a i n using a centromeric chromosome s p e c i f i c DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe 9659 8.8 89.0 2.2 0 504 18 9685 3.0 92 .4 4.4 0.2 502 16 96115 3.4 94.4 2.2 0 502 16 96106 3.3 95.2 1.5 0 522 16 9694 2.0 91.6 5.8 0.6 500 16 9657 6.0 93 .0 1.0 0 501 16 9682 5.2 92 .4 2.4 0 500 16 9676 5.2 92 . 8 1.8 0.2 500 16 N = t o t a l number of nu c l e i scored Cut - o f f value for trisomy = 7.57 123 Table 6.14: FISH analysis of disomic cord blood using a chromosome s p e c i f i c centromeric DNA probe. Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe 96106 2.0 93 .2 4.6 0.2 504 16 9712 3.6 93 .6 2.2 0.6 502 16 9721 6.8 89 . 8 3.4 0.2 503 16 96112 3.0 94.2 2.8 0 500 16 9655 5.2 92 . 6 1.8 0.4 502 16 9722 6.3 90.4 3.3 0 511 16 9657 7.0 89.6 3.0 0.4 503 18 9658 5.0 90.7 4.0 0.4 505 18 976 5.8 92.6 1.6 0 501 16 N = t o t a l number of nu c l e i scored Cut - o f f value for trisomy = 5.92 124 6.5 FISH analyis of fresh enzymatically digested disomic placenta. Table 6.15: FISH analysis using a centromeric DNA probe s p e c i f i c for chromosome 16, of disomic trophoblast i s o l a t e d from chorionic v i l l i . Patient Number Proportion of Nuclei with Indicated Number of Signals 1 2 3 4 N Probe 9694 3.1 95 .1 1.8 0 512 16 9722 2.5 94.7 2.1 0.6 512 16 96115 3.5 94 . 9 1.2 0.4 492 16 9662 1.2 85 . 0 3.8 0 503 16 9612 3.5 95 .1 1.2 0.2 511 16 9645 3.2 89.4 7.2 0.2 500 18 9650 8.8 89 . 0 2 . 0 0.2 500 18 9659 3 92.0 5.0 0 504 18 N = t o t a l number of nuclei scored Cut - off value for trisomy = 6.59 125 Table 6.16: FISH analysis using a chromosome s p e c i f i c centromeric DNA probe, of fresh stromal c e l l s i s o l a t e d from chorionic v i l l i . Patient Number Proportion of Nuclei 1 2 3 4 N Probe 9645 1.6 89.5 3.7 0 600 18 9486* 3 95.8 1.2 0 500 16 94107* 10.8 87 .0 1.0 0.4 500 16 9259* 9.6 87.6 1.8 1.0 500 18 9454* 6.2 91.6 1.6 0.4 500 16 94105* 2 . 0 93 .4 3.8 0 500 7 * Laboratory established control data. N = t o t a l number of nuclei scored Cut - o f f value for trisomy = 6.59 126
Thesis/Dissertation
eng
Genetics
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Graduate
An Investigation of conceptuses with placental chromosomal mosaicism for the presence of germline mosaicism
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