THREE CHROMOSOMES AND A BABY: CYTOGENETIC, BIOLOGICAL, AND CLINICAL ASPECTS OF THE TRISOMIC PLACENTA

by

PAUL JOHN YONG

B.Sc, The University of British Columbia, 1999

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

COMBINED DOCTOR OF PHILOSOPHY AND DOCTOR OF MEDICINE

in

THE FACULTY OF GRADUATE STUDIES

(Experimental Medicine)

THE UNIVERSITY OF BRITISH COLUMBIA April 2006

©Paul John Yong, 2006 Abstract

Trisomy - the presence of an extra chromosome - is found in one in four miscarriages, while 0.5-1% of ongoing pregnancies assessed by chorionic villus sampling have trisomy mosaicism with the abnormality predominantly or completely confined to the placenta

('confined placental mosaicism' (CPM)). Trisomy of chromosome 16, in particular, may be the most common chromosome abnormality at conception. In this thesis, ongoing trisomy CPM pregnancies, especially those involving trisomy 16, were first investigated to clarify the distribution of maternal-fetal and pediatric outcomes and to delineate the predictors of poorer outcome. CPM of trisomy 16 (CPM 16) was found to be associated with fetal growth restriction and malformation, as well as maternal preeclampsia, although long-term growth and development of the newborns was reassuring. The presence of amniotic fluid trisomy and uniparental disomy (UPD) increased risk of poorer outcome in CPM16 pregnancies. Also, the level of trisomy in the trophoblast, for both CPM 16 and other trisomy CPM, was the key placental lineage important for pregnancy outcome. Second, trisomic spontaneous abortions, especially trisomy 16 miscarriages, were studied to identify biological mechanisms in the pathogenesis of the trisomic placenta, such as trophoblast outgrowth and fibroblast protein kinase expression. Although trisomic trophoblast outgrowth was variable, and apparently normal in trisomy 15, there was a defect in outgrowth in trisomy 16 trophoblast. As well, protein kinase profiling of trisomy 16 fibroblasts showed both dosage-effects and amplified instability when compared to euploid fibroblasts. In conclusion, the trisomic placenta can have significant and varied effects on biological function and the clinical outcome of mother, fetus, and newborn. Table of Contents

Abstract ii

Table of Contents iii

List of Tables vii

List of Figures ix

List of Abbreviations xi

Acknowledgements xiii

Dedication xiv

Chapter 1 Introduction 1

1.1 Structure, embryology, and function of the human placenta 1 1.2 Cytogenetic terms and definitions 2 1.3 Confined placental mosaicism (CPM) 4 1.4 Epidemiology of trisomy and CPM during pregnancy 6 1.4.1 Trisomy in miscarriage 6 1.4.2 CPM in ongoing pregnancies 8 1.5 Research obj ectives 9 1.6 References 10

Chapter 2 Feto-placental growth in trisomy CPM 13

2.1 Note 13 2.2 Introduction 13 2.3 Methods 15 2.3.1 Trisomy CPM cases 15 2.3.2 Matched controls 17 2.3.3 Data analysis 17 2.2.3.1 Birth weight 17 2.2.3.2 Placental weight 18 2.2.3.3 Feto-placental (F-P) weight ratio 18 2.2.3.4 Determinants of placental weight and birth weight 18 2.2.4 Statistical analysis 19 2.4 Results 19 2.4.1 Clinical and cytogenetic data 19 2.4.2 Placental pathology 20 2.4.3 Birth weight 20 2.4.4 Placental weight 21 2.4.5 F-P weight ratio 21 2.4.6 Determinants of placental weight and birth weight 22 2.4.6.1 Sex of the fetus 22

iii 2.4.6.2 Involved chromosome 22 2.4.6.3 Level of trisomy in trophoblast, mesenchyme, chorion 22 2.4.6.4 Placental weight 25 2.5 Discussion 26 2.6 References 29

Chapter 3 Pathogenesis of CPM 16 pregnancies 40

3.1 Note 40 3.2 Introduction 40 3.3 Methods 42 3.3.1 CPM16 cases 42 3.3.2 Statistical analysis 44 3.4 Results 44 3.4.1 Pregnancy outcomes of CPM 16 44 3.4.2 Clinical outcomes of CPM 16 live births 44 3.4.3 Trisomy in amniotic fluid in CPM 16 live births 45 3.4.4 Ascertainment of CPM16 live births 46 3.4.5 Sex of the fetus in CPM 16 live births 47 3.4.6 CVS in CPM 16 live births 47 3.4.7 upd(16)mat in CPM 16 live births 48 3.4.8 Possible confounding 48 3.4.9 Gestational age at delivery in CPM16 live births 50 3.4.10 Intrauterine death and neonatal death in CPM16 50 3.5 Discussion 51 3.5.1 Clinical outcome of CPM 16 51 3.5.2 Amniotic fluid trisomy and CPM16 live births 51 3.5.3 Ascertainment and CPM 16 live births 52 3.5.4 Sex of the fetus and CPM 16 live births 52 3.5.5 CPM16andCVS 53 3.5.6 Intrauterine and neonatal death in CPM16 54 3.5.7 Evidence for imprinting on chromosome 16 54 3.6 References 57

Chapter 4 Preeclampsia and CPM 16 80

4.1 Note 80 4.2 Introduction 80 4.3 Methods 82 4.3.1 CPM16 cases 82 4.3.2 Matched controls 83 4.3.3 Definition of preeclampsia 83 4.3.4 Statistical analysis 83 4.4 Results 84 4.5 Discussion 85 4.6 References 87

iv Chapter 5 Postnatal follow-up of newborns from CPM 16 pregnancies 93

5.1 Note 93 5.2 Introduction 93 5.3 Methods 93 5.3.1 CPM16 cases 93 5.3.2 Statistical analysis 95 5.4 Results 95 5.4.1 Height 95 5.4.2 Weight 96 5.4.3 Development 96 5.5 Discussion 97 5.6 References 100

Chapter 6 Cytokeratin staining in villus cultures from miscarriage and CVS 110

6.1 Note 110 6.2 Introduction 110 6.3 Methods Ill 6.3.1 Tissue processing and culture Ill 6.3.2 Immunochemistry 112 6.3.3 Statistical analysis 113 6.4 Results 113 6.5 Discussion 113 6.6 References 115

Chapter 7 EVT outgrowth in trisomic miscarriage 119

7.1 Note 119 7.2 Introduction 119 7.3 Methods 121 7.3.1 Miscarriage cases 121 7.3.2 Tissue processing and culture 122 7.3.3 Irnmunochemistry 122 7.3.4 Karyotype confirmation 123 7.3.5 Statistical analysis 124 7.4 Results 124 7.5 Discussion 126 7.6 References 129

Chapter 8 Protein kinase profiling in trisomic miscarriage 138

8.1 Note 138 8.2 Introduction 138 8.3 Methods 140 8.3.1 Miscarriage cases 140 8.3.2 Tissue processing and culture 140 8.3.3 Immunochemistry and PCR 141 8.3.4 Protein kinase protein profiling 142

v 8.3.5 Protein kinase RNA profiling 143 8.3.6 Profiling data analysis 145 8.3.6.1 Confounding 145 8.3.6.2 Protein kinase expression at protein/RNA levels 146 8.3.6.3 Inter-individual variation in expression 146 8.3.7 Statistical analysis 147 8.4 Results 147 8.4.1 Protein kinase expression at the protein level 147 8.4.2 Protein kinase expression at the RNA level 148 8.4.3 Inter-individual variation in expression 148 8.5 Discussion 149 8.6 References 152

Chapter 9 Conclusion 161

9.1 References 164

Appendix A Ethics approval 167

Appendix B Extra references 168

Appendix C Protein kinases profiled in Chapter 8 179

vi List of Tables

Table 2.1 Clinical and cytogenetic data for the 69 trisomy CPM cases 31

Table 2.2 Clinical and cytogenetic data for the 69 trisomy CPM cases 34

Table 2.3 Findings for (local) cases where placental pathology was performed 36

Table 2.4 Clinical characteristics of the trisomy CPM cases and the matched controls 38

Table 3.1 Malformations among live births with survival beyond the neonatal period 60

Table 3.2 Association between the presence of trisomy in amniotic fluid and malformation 60

Table 3.3 Association between trisomy in amniotic fluid and trisomy in fetal tissues 60

Table 3.4 Association between upd(16)mat and malformation 60

Table 3.5 Associations between explanatory variables associated with birth weight.. 61

Table 3.6 Association between malformation and neonatal death 62

Table 3.7 Association between biased ascertainment and neonatal death 62

Table 3.8 Association between ascertainment by abnormal ultrasound and neonatal death 62

Table 3.9 Association between ascertainment by abnormal SS, and neonatal death 63

Table 3.10 Ascertainment and cardio-pulmonary malformations in neonatal deaths 64

Table 4.1 CPM16 cases meeting inclusion criteria. 89

Table 4.2 CPM16 cases meeting inclusion criteria 90

Table 4.3 Post-partum placenta cytogenetics 91

Table 4.4 Clinical features of the CPM16 cases and controls 92

Table 4.5 Clinical features of the CPM 16 cases with preeclampsia 92

Table 5.1 CPM16 cases meeting inclusion criteria 102

Table 5.2 Clinical data for included CPM 16 cases 103

Table 5.3 Follow-up data for length/height, weight and developmental outcome 104

Table 5.4 Association between trisomy in amniotic fluid and developmental delay 108 Table 5.5 Association between malformation and developmental delay 108

Table 6.1 CK7 and CK18 staining 116

Table 7.1 Association between gestational age and the presence of EVT outgrowths 131

Table 7.2 EVT outgrowth in euploid and trisomy 15 cases <10 weeks gestation 131

Table 7.3 EVT outgrowth in euploid and trisomy 16 cases <10 weeks gestation 131

Table 7.4 EVT outgrowth in euploid and all abnormal cases <10 weeks gestation 131

Table 8.1 Immunochemistry, and protein/RNA profiling of mesenchymal core cultures .155

Table 8.2 Protein levels of kinases with significant differences in protein expression 156

Table 8.3 RNA levels of kinases with significant differences in protein expression 157

Table 8.4 Coefficients of variation (CV) at the RNA and protein levels 158 List of Figures

Figure 2.1 Birth weight means for the chromosomes involved in the trisomy CPM cases 39

Figure 3.1 Distribution of gestational ages for CPM 16 live births 65

Figure 3.2 Distribution of birth weights for CPM16 resulting in live births 66

Figure 3.3 Distribution of the level of trisomy in amniotic fluid among CPM16 live births 67

Figure 3.4 Mean birth weight in the presence or absence of trisomy in amniotic fluid 68

Figure 3.5 Mean birth weight for unbiased and biased ascertainment 69

Figure 3.6 Mean birth weight for ascertainment: unbiased vs. abnormal serum screen... 70

Figure 3.7 Mean birth weight for ascertainment: unbiased vs. abnormal ultrasound 71

Figure 3.8 Mean birth weight for females and males 72

Figure 3.9 Mean birth weight for <100% and 100% trisomy on direct CVS 73

Figure 3.10 Mean birth weight for <100% and 100% trisomy on cultured CVS 74

Figure 3.11 Mean birth weight for bpd(16) and upd(16)mat 75

Figure 3.12 Mean gestational age for unbiased and biased ascertainment 76

Figure 3.13 Mean gestational age ascertainment: unbiased vs. abnormal ultrasound 77

Figure 3.14 Birth weights for live births and intrauterine deaths 78

Figure 3.15 Gestational ages for live births and neonatal deaths 79

Figure 5.1 Association between birth weight and developmental delay 109

Figure 6.1 CK7 and CK18 staining in CVS cultures 117

Figure 6.2 CK7 staining in JEG-3 cells (positive control) 118

Figure 7.1 Extravillus trophoblast (EVT) columns deriving from the chorionic villus 132

Figure 7.2 EVT outgrowths 133

Figure 7.3 Fibroblast-like outgrowths 133

Figure 7.4 Cytokeratin-7 staining of EVT outgrowths 134

ix Figure 7.5 Association between gestational age and EVT outgrowth 135

Figure 7.6 Proportion of explants with EVT outgrowths for euploid and trisomy 15 cases 136

Figure 7.7 Proportion of explants with EVT outgrowths for euploid and abnormal cases 137

Figure 8.1 ERK1 RNA and protein expression 159

Figure 8.2 Coefficient of variation (CV) of RNA and protein expression 160

x List of Abbreviations

AMA Advanced maternal age

ASD Atrial septal defect

BPD Biparental disomy bpd(16) Biparental disomy of chromosome 16

BCWH British Columbia Women's Hospital

BMI Body mass index

BW Birth weight

C&W Children's and Women's Health Centre of British Columbia

CV Coefficient of variation

DORV Double outlet right ventricle

Eu Euploid

EVT Extravillus trophoblast

ICM Inner cell mass

CGH Comparative genomic hybridization

CK7 Cytokeratin 7

CK18 Cytokeratin 18

CPM Confined placental mosaicism

CPM 16 Confined placental mosaicism involving trisomy 16

CVS Chorionic villus sampling

GA Gestational age

F-P Feto-placental

FISH Fluorescence in situ hybridization

HELLP Hemolysis, elevated liver enzymes, and low platelets IUD Intrauterine death

IUGR Intrauterine growth restriction

LB Live birth with survival beyond the neonatal period

MSAFP Maternal serum alpha-fetoprotein

MShCG Maternal serum human chorionic gonadotropin

ND Neonatal death

PDA Patent ductus arteriosus

PCR Polymerase chain reaction

PROM Premature rupture of membranes

RT-PCR Reverse transcription-polymerase chain reaction

SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SGA Small-for-gestational age

SUA Single umbilical artery

T16 Trisomy 16

T15 Trisomy 15

TA Termination of pregnancy

UPD Uniparental disomy upd(16)mat Maternal uniparental disomy of chromosome 16

VSD Ventricular septal defect

xii Acknowledgements

This thesis would not have been possible without the patience, understanding, assistance, and mentorship of the following people:

Dr. Wendy Robinson

Members of the Robinson laboratory

Dr. Deborah McFadden

Dr. Sylvie Langlois

Dr. Michael Whitlock

Irene Barrett

Dr. Colin MacCalman

Dr. Peter von Dadelszen

Dr. Anthony Chow

Dr. Lynn Raymond

Jane Lee

Dr. Norman Wong

Dr. Vince Duronio

Patrick Carew Dedication

This thesis is dedicated to the following people for their inspiration and support -

My parents (Peter and Rosalinda) and siblings (Rachelle, Brian, and Christina)

Dr. John Yun

Drs. Margaret and Robin Cottle, and the Christian Medical Dental Society (CMDS)

Fr. Gregory Smith

And all my friends

- You have blessed my life.

xiv 1 Introduction

Trisomy - the presence of an extra chromosome - has devastating effects on human pregnancy, leading to miscarriage, stillbirth, and children with syndromes of considerable morbidity and mortality. The classic example of the latter is trisomy of chromosome 21, which is associated with Down Syndrome. Although the association between trisomy and Down

syndrome has been known for over forty years, much remains to be known about the pathogenesis of trisomy in the human conceptus and its effects on the mother during pregnancy.

The purpose of this thesis is to provide insight into how trisomy in the placenta affects maternal-

fetal and pediatric health from cytogenetic, clinical and biological perspectives using two human

models: first-trimester miscarriages with trisomy, and ongoing pregnancies with trisomy

confined to the placenta (confined placental mosaicism). This introduction has two goals. First, background information will be provided on the structure, embryology and function of the

placenta; some cytogenetic terms and definitions; and confined placental mosaicism (CPM).

Second, the epidemiology of trisomic miscarriage and CPM in ongoing pregnancies will be

reviewed. Finally, the specific objectives of the thesis research will be described.

1.1 Structure, embryology, and function of the human placenta

The human conceptus is defined as the placenta, the gestational sac membranes, and the

embryo/fetus-proper, derived from a single-celled zygote. The membranes consist of the

amnion and the chorion laeve. The placenta consists of the chorionic plate, into which the

umbilical cord inserts, and a multitude of chorionic villi, which interact with the surrounding

maternal tissue of the uterine wall. The chorionic villi consist of an outer layer of trophoblast -

syncytiotrophoblast and cytotrophoblast - and an inner villus mesenchymal core. The

trophoblast is derived from the trophectoderm of the blastocyst, while the villus mesenchymal

1 core, chorionic plate, amnion, and chorion laeve are derived from the inner cell mass (ICM) of the blastocyst. The embryo/fetus-proper also develops from the ICM.

The placenta is a vital organ required for maintenance of normal pregnancy and the production of a healthy neonate, being involved in gas and heat transfer; pH and water haemostasis; metabolism and absorption; endocrine and exocrine synthesis and secretion; and in hematopoiesis and immunologic functions (Benirschke and Kaufmann 1995). Most of these functions involve in some way transfer across the trophoblast between maternal blood vessels and glands in the uterus and placental blood vessels in the villus mesenchymal core (Burton et al. 2001; Burton et al. 2002).

1.2 Cytogenetic terms and definitions

Trisomy refers to a karyotype with an extra autosomal chromosome (resulting in 47 chromosomes in total), as opposed to a normal euploid karyotype that has 2 sex chromosomes and 22 pairs of autosomal chromosomes. It can be classified by the timing of its origin relative to fertilization. The extra chromosome may be pre-zygotic or post-zygotic; that is, it may originate pre-fertilization during gametogenesis (oogenesis in the female and spermatogenesis in the male), or post-fertilization during embryonic development, respectively. Moreover, the extra chromosome can arise at different stages of gametogenesis or embryonic development. In gametogenesis, it can arise through an error in mitosis during any of the series of divisions from the primordial germ cell to the oogonium or spermatogonium, or through an error in meiosis I or meiosis II in the process of 'reduction division' from the (diploid) oogonium to the (haploid) secondary oocyte or from the (diploid) spermatogonium to the (haploid) secondary spermatocyte. In embryonic development, it can arise through an error in mitosis during any of the series of divisions from the initial single-celled embryo (i.e. the 'fertilized egg' or 'zygote') to the various stages of the multi-celled embryo such as the morula and blastocyst. If the third

2 copy arises during oogenesis or by duplication of the maternal copy during embryonic development, it is said to have a maternal origin; if the third copy arises during spermatogenesis or by duplication of the paternal copy during embryonic development, it is said to have a paternal origin.

It is important to emphasize that cytogenetic terms such as 'trisomy' more precisely describe the karyotype of a particular cell of a multi-celled individual, although often applied to the multi-celled individual itself. This distinction is critical because of the phenomenon of mosaicism, the presence of at least two cell lines with different karyotypes within an individual

(i.e. a feto-placental unit) derived from a single zygote. The etiology of mosaicism can be divided into two broad categories: 1) a euploid conceptus with post-zygotic origin of trisomy in a cell at some time during development, resulting in a mixture of trisomic and euploid cells; and

2) pre-zygotic origin of trisomy producing a trisomic conceptus, followed by loss of one of the three chromosomes in a cell at some time during development, also resulting in a mixture of trisomic and euploid cells (Kalousek and Vekemans 1996). The latter is also known as trisomy zygote rescue (Kalousek and Vekemans 1996).

Cytogenetic techniques have inherent limitations that can make the detection of mosaicism challenging in practice. For example, traditional (conventional) cytogenetic analysis requires metaphase chromosomes, which are generated by blocking dividing cells at metaphase.

For conventional cytogenetic analysis of the villus cytotrophoblast, spontaneous cell divisions

(and therefore metaphases) are analyzed either directly or after a short-term (~1 day) incubation

(Eiben et al. 1990). Such direct or short-term preparations have the advantage of quicker results, but require very fresh tissue and produce chromosomes of relatively poorer quality

(Warburton 2000). For conventional cytogenetic analysis of other tissues (i.e. villus mesenchymal core, chorionic plate, chorion laeve, amnion, and embryonic/fetal tissues), dividing cells are generated by culturing the cells for at least one week. Such long-term culture

3 results in more high-quality metaphases, but has two major problems: 1) chromosomally abnormal cells may arise due to cell division in in vitro conditions ('culture artefact'); and 2) maternal cells from the uterus (in which the conceptus develops) may grow instead of or alongside placental or embryo/fetal cells ('maternal contamination'). Also, since both direct/short-term preparations and long-term culture techniques require cell divisions, conventional cytogenetics may select against chromosomally abnormal cells; that is, although a low level of mosaicism may be present in vivo, the abnormal cells may be unable to grow in vitro and thus may not be detected with conventional cytogenetic techniques. Furthermore, only a practical number of metaphases (e.g. 10-20) are examined for routine cytogenetic analysis; thus, a low level of mosaicism may not be detected simply by chance.

Any clinically relevant definition of mosaicism has to take into account the disadvantages of cell culture, while accepting that the probability of detecting mosaicism in a biological sample is dependent on the number of metaphases examined. One definition is the presence of at least two cell lines, with each present in at least two culture flasks set up from a particular tissue sample (Hsu et al. 1992). When only one cell of a given karyotype is found or when more than one cell is present but only in one flask, then the terms single-cell and multiple- cell pseudomosaicism, respectively, can be used. A finding of 100% trisomic or euploid cells could be referred to as 'full' or 'non-mosaic' trisomy or euploidy, respectively. In order to circumvent the problems of conventional cytogenetics, non-culture dependent methods of cytogenetic analysis can also be used, including fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and molecular methods such as PCR (Tonnies

2002).

1.3 Confined placental mosaicism (CPM)

One special case of mosaicism is confined placental mosaicism (CPM), which

4 describes the phenomenon of chromosomally abnormal cells confined to the placenta. It was first described by Dagmar Kalousek and Fred Dill at the University of British Columbia in 1983

(Kalousek and Dill 1983). CPM pregnancies provide a model to determine, independent of the fetus, the effects of a chromosome abnormality in the placenta on pregnancy outcome. In viable ongoing pregnancies, CPM is usually ascertained by first-trimester chorionic villus sampling

(CVS) (10-12 weeks gestation). The sampled chorionic villi are assessed by conventional cytogenetics following a direct/short-term preparation (reflecting the karyotype of villus cytotrophoblast) or long-term culture (reflecting the karyotype of the villus mesenchymal core).

The fetal karyotype is then assessed by second-trimester amniocentesis, which samples amniotic fluid cells originate from both the fetus proper (lung, urinary tact, skin) and from the amnion

(Hsu 2004). Cytogenetic analysis of the placenta and fetus can also be carried out at the end of pregnancy, whether after miscarriage, termination, or live birth. For live births, investigations of the newborn are limited to only a few cell types, usually peripheral blood lymphocytes or skin fibroblasts.

If trisomic cells are found in tissues of the embryo/fetus-proper, then strictly speaking, the trisomic cells are not confined to the placenta. However, some authors have considered cases with a low-level of trisomic cells in the embryo/fetus to still be CPM (Roland et al. 1994;

Robinson et al. 1997) because it is thought that trisomic cells had to be at least predominantly confined to the placenta for the conceptus to survive to term. Hence, CPM could be subdivided into those cases in which trisomic cells are completely or only predominantly confined to the placenta. With this definition, CPM could be initially ascertained by low-level trisomy at amniocentesis, followed by investigation of the placenta antenatally or post-partum. In contrast, some authors consider cases with even only low-level trisomy in embryonic/fetal tissues to be cases of generalized or true fetal mosaicism (Kalousek 1994). I use an encompassing definition of CPM that includes both completely and predominantly confined trisomy. However, because of technical limitations, a case that appears to be completely confined may actually have low- level fetal trisomy that went undetected during prenatal or postnatal investigations.

Nevertheless, when attempting to estimate the effect of trisomy in the placenta independent of the fetus, it is still more useful to consider apparently 'complete' CPM while accepting that an unspecified proportion in reality are probably only predominantly confined. In another classification, CPM is categorized by tissue distribution (Kalousek 1994): Type I (confined to villus trophoblast); Type II (confined to villus mesenchymal core); Type III (present in both lineages).

The effect of CPM on pregnancy outcome depends on several variables, including the chromosome involved in the trisomy, and the timing of chromosome gain or loss (Kalousek and

Vekemans 1996). For instance, it would be expected that the larger the chromosome and/or the more genes important for viability are present on the chromosome, the more adverse effects on pregnancy outcome. As well, the earlier the post-zygotic origin of trisomy or the later the trisomy zygote rescue, then the higher level and more extensive distribution of trisomic cells would be expected in the feto-placental unit, which presumably correlates with poorer pregnancy outcome. On the other hand, the level and distribution of trisomy may be also influenced by natural selection against trisomic cells and stochastic events during development, while some tissues may be more 'sensitive' to trisomy than others. Finally, uniparental disomy

(where both copies of a chromosome are inherited from the same parent) may be present in the fetus, particularly when the trisomy has a pre-zygotic origin (see Chapter 3).

1.4 Epidemiology of trisomy and CPM during pregnancy

1.4.1 Trisomy in miscarriage

Some pregnancies are 'lost' at the peri-implantation stage before they are detected clinically; that is, they are pre-clinical miscarriages. Although several studies have attempted to

6 quantify the proportion of conceptions that end in these pre-clinical losses, the landmark study was that of Wilcox et al. (1988) (Macklon et al. 2002). Wilcox et al. (1988) used a highly sensitive assay to measure maternal urine human chorionic gonadotropin (hCG), a marker of implantation, in a sample of women attempting to conceive. They also controlled for the detectable background urine hCG level by including a sample of control women. Their results suggest that about half of conceptions are lost, with 22% of conceptions ending as pre-clinical miscarriages and 31% ending as clinical miscarriages. It should be noted that it is likely that many fertilizations are lost even before conception (i.e. implantation), although a good estimate of this number is not available (Macklon et al. 2002). Unfortunately, cytogenetic data are not available for both pre-implantation losses and pre-clinical spontaneous abortions (Banzai et al.

2004).

Cytogenetic data are available for those clinically recognized pregnancies that end in spontaneous abortion. Approximately 50% of clinical miscarriages are associated with a chromosome abnormality, of which half are trisomies (Warburton 2000). However, the exact numbers greatly depend on both maternal age and gestational age (Hassold and Hunt 2001;

Warburton 1991). At British Columbia Women's Hospital (BCWH), 61% of clinical miscarriages are chromosomally abnormal, with the majority (44% of all miscarriages) associated with trisomy (D.E. McFadden and W.P. Robinson, unpublished data). The distribution of different trisomies among miscarriages is non-random; for example, the most common is trisomy of chromosome 16 (-10% of miscarriages at BCWH), while trisomy 1 miscarriage has only been reported three times in the literature (Hanna et al. 1997; Dunn et al.

2001; Banzai etal. 2004).

7 1.4.2 CPM in ongoing pregnancies

The largest study of CPM to date, the European Collaborative Research on Mosaicism in

CVS (EUCROMIC) (Hahnemann and Vejerslev 1997a), involved 64,053 CVS procedures with

98.1% (62,865) having conventional cytogenetics successfully performed on direct/short-term preparations (cytotrophoblast) and/or long-term cultures (mesenchymal core). 1.04% of all

CVS cases involved a mixture of chromosomally abnormal and euploid cells in at least one

placental lineage (trophoblast or mesenchyme), or between the lineages. It should be noted that

not all of these cases had investigation of fetal karoytype; and in an unspecified proportion, a

normal neonatal phenotype at birth was taken to be evidence of euploid karoytype. In an

additional 0.15% of all CVS cases, the placental lineage(s) investigated were fully abnormal,

while the fetus was found to be euploid. (The authors coined the term 'non-mosaic fetoplacental

discrepancy' to describe these latter cases, while I consider them to be CPM.) Combining both

figures gives a CPM incidence of 1.19% at CVS from EUCROMIC. A second publication from

EUCROMIC provided more cytogenetic details on the CPM cases involving autosomal trisomy,

which had an incidence of 0.69% at CVS (Hahnemann and Vejerslev 1997b). Among a subset

of these CPM cases that had both direct and cultured preparations, as well as strict exclusion of

trisomy from one or more fetal tissues (n = 172), 54% were Type I, 28% Type II, and 18% Type

III. Therefore, trisomy in the trophoblast lineage (Type I and Type III) was more frequent than

trisomy in the mesenchymal lineage (Type II and Type III). The distribution of different

trisomies among ongoing CPM pregnancies was also non-random, and differed from the

distribution among miscarriages. Notably, trisomy 7 was the most frequent CPM trisomy (19%

of all trisomies), while trisomy 16 was fifth most common (6%).

The clinical implications of prenatally diagnosed CPM are controversial. Some studies

found an increased risk of subsequent pregnancy loss (Hogge et al. 1986; Johnson et al. 1990;

Wapner et al. 1992; Wang et al. 1993), although most have not (Breed et al. 1991; Fryburg et al.

8 1993; Roland et al. 1994; Wolstenholme et al. 1994; Leschot et al. 1996). Similarly, with the exception of one unpublished study (DeLozier-Blanchet 1996), studies have not shown a higher rate of IUGR or small-for-gestational (SGA) newborns (Johnson et al. 1990; Breed et al. 1991;

Wapner et al. 1992; Fryburg et al. 1993; Roland et al. 1994; Wolstenholme et al. 1994; Leschot et al. 1996; Goldberg and Wohlferd 1997). A major issue is heterogeneity in methodology, including the definition of CPM (e.g. some studies considered a eumorphic infant as sufficient for CPM, without any fetal cytogenetic analysis) and the placental lineage analyzed (trophoblast and/or mesenchyme), as well as the inclusion of different chromosome abnormalities (e.g. autosomal trisomy versus structural aberrations and sex chromosome aneuploidy). CPM involving trisomy would be more likely to be associated with abnormal pregnancy outcome, given the severe phenotype of most trisomic pregnancies, from early spontaneous abortion to

Down syndrome. In particular, case series of trisomy 16 CPM (CPM 16) suggest that such pregnancies are particularly high-risk (Kalousek et al. 1993; Robinson et al. 1997).

1.5 Research objectives

The purpose of this thesis is to provide insight into how trisomy in the placenta affects maternal-fetal and pediatric health, through analysis of cytogenetic, biological, and clinical

aspects of ongoing trisomy CPM pregnancies and trisomic miscarriages. The objectives are: 1) to use ongoing CPM pregnancies, in particular those involving trisomy 16, to elucidate the role of the chromosome involved in the trisomy, the level and distribution of trisomic cells (in placental lineages and in amniotic fluid), and uniparental disomy (UPD) on maternal-fetal and pediatric outcomes; and 2) to use pregnancies ending in miscarriage, in particular those associated with trisomy 16, to investigate two biological mechanisms in the trisomic placenta: trophoblast outgrowth and mesenchymal core fibroblast protein kinase expression.

9 1.6 References

Banzai M, Sato S, Matsuda H, Kanasugi H (2004) Trisomy 1 in a case of a missed abortion. J Hum Genet 49:396-397

Breed AS, Mantingh A, Vosters R, Beekhuis JR, Van Lith JM, Anders GJ (1991) Follow-up and pregnancy outcome after a diagnosis of mosaicism in CVS. Prenat Diagn 11:577-580

Burton GJ, Hempstock J, Jauniaux E (2001) Nutrition of the human fetus during the first trimester—a review. Placenta 22 Suppl A:S70-77

Burton GJ, Watson AL, Hempstock J, Skepper JN, Jauniaux E (2002) Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 87:2954-2959

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12 2 Feto-placental growth in trisomy CPM1

2.1 Note

I wrote this chapter/manuscript and did the data organization and analysis, with the

following clarifications and exceptions. The trisomy CPM cases are from the ongoing UBC

study of trisomy mosaicism beginning in 1988. The original primary investigators were Dr. D.

Kalousek (Pathology and Laboratory Medicine) and Dr. S. Langlois (Medical Genetics).

Clinical data collection (e.g. birth weight, gestational age) and post-partum cytogenetic analysis

of the placenta and amnion were done in the Kalousek laboratory. During this period, I. Barrett

was the person primarily responsible in the Kalousek lab for the cytogenetic analysis and

weighing of the placenta. My supervisor, Dr. W. Robinson (Medical Genetics), is the current

primary investigator, and data from more recent cases have been collected and organized by her

laboratory. Matched controls were ascertained by myself from delivery records at BC Women's

Hospital under the supervision of Dr. P. von Dadelszen (Obstetrics and Gynaecology).

Placental pathology was collected by myself and reviewed with Dr. D. McFadden (Pathology

and Laboratory Medicine).

2.2 Introduction

Studies of outcomes such as fetal loss and RJGR in prenatally diagnosed CPM have been

equivocal (Chapter 1). A confounding factor in these studies is the type of chromosome

abnormality; for example, in general, autosomal trisomy is likely to be more high-risk than a

structural or sex chromosome abnormality. Also, although CVS can serve as an indicator of

trisomy in the first-trimester placenta, another strategy is to re-sample placenta post-partum

(Kalousek et al. 1991). The advantage of the latter is that multiple sites can be assessed to

1 A version of this chapter will be submitted for publication. Yong PJ, von Dadelszen P, Barrett IJ, McFadden DE, Kalousek DK, Robinson WP. Effect of the trisomic placenta on feto-placental growth.

13 provide a more accurate estimate of the level of trisomy, in cases where the distribution of placental trisomy is heterogeneous. Several studies have studied the degree of trisomy in the post-partum placenta from trisomy CPM pregnancies (Kalousek et al. 1996; Shaffer et al. 1996;

Robinson et al. 1997). In a series of 14 cases of CPM involving trisomy 7, Kalousek et al.

(1996) noted that the two small-for-gestational age (SGA) cases had higher levels of trisomy in the trophoblast compared to the other cases. In a series of 9 cases of CPM involving trisomy 2,

Shaffer et al. (1996) detected high levels of trisomy in the mesenchyme of the post-partum placenta in the two SGA cases compared to the other cases. And in a larger study with statistical analysis, Robinson et al. (1997) found that the level of trisomy in the trophoblast was significantly associated with 'poor outcome' (at least one of SGA, malformation, or intrauterine death) in a series of 44 trisomy CPM cases. Although these studies suggest that lower birth weight is associated with higher levels of trisomy in the placenta, none of them have directly statistically correlated birth weight with the level of trisomy. Furthermore, none of them have examined placental weight.

Placental weight is correlated with birth weight (Molteni et al. 1978; Williams et al.

1997; Sanin et al. 2001), although the direction of causation is not clear and is likely bi• directional. Although placental weight has not been investigated in trisomy CPM pregnancies, recent studies strongly support both lower birth weight (Stoll et al. 1998; Myrelid et al. 2002;

Frid et al. 2004) and lower placental weight (Stoll et al. 1998; Myrelid et al. 2002) in Down syndrome births.

I hypothesized that both placental and birth weight would be decreased in trisomy CPM, and that both weight measures would inversely correlate with the level of trisomy in the placenta. In this study, placental weight and birth weight were investigated in prenatally diagnosed trisomy CPM cases for 1) comparison to matched controls and a reference population; and 2) a statistical analysis of the relationship between placental and birth weight,

14 and the level of trisomy in various lineages of the placenta as well as the sex of the fetus and the involved chromosome.

2.3 Methods

2.3.1 Trisomy CPM cases

The study sample consisted of 69 cases of trisomy CPM singleton pregnancies resulting in a live birth from the ongoing study of trisomy mosaicism at the University of British

Columbia (UBC). The study was approved by the ethics committees of UBC and the Children's and Women's Health Centre of British Columbia (C&W) (Appendix A). Twenty-five cases

(36%) are local cases from C&W; the other 44 cases (63%) were referred to the ongoing study from other centres. The cases were collected during a 7-year time period (1988-1994) when

CPM placentas were weighed uniformly (see below) and the proportion of trisomic cells in the placenta was determined post-partum by conventional cytogenetics or FISH. Other inclusion criteria included (1) prenatal diagnosis via the detection of trisomic cells on CVS (by conventional cytogenetics); and (2) no trisomy in amniotic fluid cells, infant/fetal blood lymphocytes, and/or the amnion (by conventional cytogenetics, with a few exceptions by FISH or molecular (PCR) methods). The latter criterion was to enrich for cases where the trisomy is more likely to be completely confined to the placenta. Cases with concomitant polyploidy, sex chromosome aneuploidy, or structural chromosome abnormalities were excluded. In 63 of the

69 cases, the malformation status of the child was known: excluding digit and facial dysmorphism (as such data were not consistently reported), 92% (58/63) of the cases did not have malformations2. There was one case each of imperforate anus, hypospadias, hip dysplasia,

2Among the cases classified as normal were one case of familial benign megalencephaly, one case of familial hip dysplasia, and one case of mild hydronephrosis that resolved antenatally. 15 hydronephrosis, and 'possible' ventricular septal defect (VSD). In addition, there was one case of placental abruption (case 56).

Gestational age and birth weight were collected from UBC Medical Genetics medical records for the local cases and sent from collaborators for referred cases. Placental weights were determined by the C&W Pathology laboratory for the local cases and by I. Barrett in a research laboratory for the referred cases, but in the same manner: excess superficial blood was washed off, the cord and membranes trimmed, and then the 'trimmed' placentas weighed on the

same scale.

The chorionic plate and villus mesenchyme of the post-partum placenta were cultured

and analyzed by conventional cytogenetic analysis, in which 5-15 cells were examined from 1-3

sites. The trophoblast were isolated in a suspension after a variety of short-term enzymatic digests of chorionic villi, followed by FISH for the chromosome involved, typically on 500-

1000 nuclei from 1-3 sites (Lomax et al. 1994; Henderson et al. 1996). These digests involve collagenase or trypsin, and produce suspensions consisting of villus cytotrophoblast and

syncytiotrophoblast (DEM, personal communication). Since the cut-off determined by FISH on control samples (<10%) was determined by different methods during the time period (>3

standard deviations below the mean, or the procedure from Lomax et al. (1994)) and not all cases with FISH included control samples, all cases with <10% of nuclei with 3 signals were

simply all coded as '0%'; for cases with >10% trisomic nuclei, the actual percentage was recorded. For a given tissue in a particular placenta, the level of trisomy at all sampled sites were averaged to produce a mean level of trisomy for that tissue. Where available, data from placental pathology were also collected.

Some cytogenetic and/or clinical data from 28 of the cases were previously published

(Kalousek et al. 1991; Kalousek et al. 1993; Kalousek et al. 1996; Shaffer et al. 1996; Robinson

2 In 3 cases the trophoblast was assessed by direct/short-term culture followed by conventional cytogenetics et al. 1997; Kuchinka et al. 2001; Penaherrera et al. 2000). All data from the other 42 cases are

unpublished. The level of trisomy in the chorionic plate, villus mesenchyme, or trophoblast

may differ between this and previous studies because (1) data from different methodologies

have been used (e.g. only results from conventional cytogenetics were used for the chorionic

plate and mesenchyme in this study, while FISH was also considered in Robinson et al. (1997));

and (2) additional data have become available since the time of previous publications.

2.3.2 Matched controls

Matched controls from routine deliveries were used because adequate 'normal' controls

from Pathology were difficult to identify, as they had their own abnormalities that indicated an

examination by Pathology. For each trisomy CPM case (both local and referred), 2 controls

matched for maternal age (± 5 years) and for parity (0, 1, or > 2) were selected from deliveries

on the same date or the consecutive previous or next day by reviewing delivery records at BC

Women's Hospital (BCWH). For some trisomy CPM cases, no data for maternal age (n = 5) or

parity (n = 20) were available; in these cases, the controls were chosen randomly with respect to

maternal age or parity. Gestational age, placental weight, and birth weight were collected from

BCWH medical records. The placentas were weighed as per routine practice in Labour and

Delivery at BCWH: the placentas were weighed 'untrimmed', and superficial excess blood was

not washed clear before weighing.

2.3.3 Data analysis

2.3.3.1 Birth weight

Birth weight was compared directly between the trisomy CPM cases and matched

controls. Small-for-gestational age (SGA) infants (<10th centile) were identified by comparison

to published Canadian birth weight standards (Kramer et al. 2001).

17 2.3.3.2 Placental weight

Because the trisomy CPM placentas and matched control placentas were handled differently, the placental weight data were compared to similar reference populations in the literature. For the matched controls, the reference population consisted of gestational-age corrected mean placental weights from 29,902 singleton pregnancies collected from 1984-1991 in Detroit, in which placentas were untrimmed and not washed clear of excess blood

(Dombrowski et al. 1994). For the trisomy CPM cases, the reference population consisted of gestational age-corrected mean placental weights from 787 singleton pregnancies from 1993-

1995 in Providence (Rhode Island), in which cord and membranes were trimmed and 'excessive blood from the crevices' was removed (Pinar et al. 1996).

2.3.3.3 Feto-placental (F-P) weight ratio

F-P weight ratio was also calculated for the trisomy CPM cases and matched controls by dividing birth weight by placental weight. For the matched controls, F-P weight ratio was compared to gestational age-corrected mean F-P weight ratios from the reference population of

Dombrowski et al. (1994). For the trisomy CPM cases, the reference population was from

Heinonen et al. (2001), who produced gestational-age corrected mean F-P weight ratios for

15,047 singleton pregnancies collected from 1990-1999 in Kuopio (Finland). In Heinonen et

al. (2001) placentas were washed clear of excess blood, and although cord and membranes were not trimmed before weighing, they used a correction factor (trimmed weight = 0.854 x untrimmed weight) derived from >1500 placentas at their centre.

2.3.3.4 Determinants of placental weight and birth weight

The following factors potentially involved in the determination of placental weight and birth weight in the trisomy CPM cases were investigated: sex of the fetus, the chromosome

18 involved in the trisomy, and the level of trisomy in the placental lineages (trophoblast, villus

mesenchyme, and chorion). For these analyses, in order to both control for gestational age and

to maintain a continuous outcome variable, placental weight and birth weight were transformed

to number of standard deviations from the mean (z-scores) using gestational-age corrected

means and standard deviations for placental weight and birth weight measured from a single

population of newborns in Denver from Molteni et al. (1974).

2.3.4 Statistical analysis

Statistics were determined using SPSS-10.0 and the Vassar WebSite for Statistical

Computation (http://facultv.vassar.edu/~lowrv/VassarStats.html). Welch's approximate t-test

was used when there was inequality of variances, and the Mann-Whitney test and Spearman's

rank correlation were used when the assumption of normality was not met. Tests were 1-tailed

due to a priori evidence and rational mechanisms, unless otherwise noted. Means are reported ±

standard deviation.

2.4 Results

2.4.1 Clinical and cytogenetic data

Clinical and cytogenetic data for the 69 cases of trisomy CPM in this study are

summarized in Table 2.1 and Table 2.2. The following trisomies were present at CVS: trisomy

16 (n = 13), trisomy 7 (n = 10), trisomy 2 (n = 9), trisomy 12 (n = 6), trisomy 8 (n = 3), trisomy

9 (n = 4), trisomy 10 (n = 4), trisomy 15 (n = 3), trisomy 13 (n = 2), trisomy 17 (n = 2), trisomy

18 (n = 2), trisomy 22 (n = 2), trisomy 4 (n = 1), trisomy 11 (n = 1), trisomy 20 (n = 1), trisomy

21 (n = 1), and multiple trisomy (n = 5). In all cases of trisomy 7 where testing for the origin of

the chromosomes 7 in the child was performed (n = 5), the result was normal biparental

inheritance. None of the trisomy 15 cases (n = 3) had testing of origin. Trisomy was detected in

19 the chorion in 63% (35/56) of informative cases; in the mesenchyme in 69% (42/61); and in trophoblast in 33% (13/40) (Table 2.2).

2.4.2 Placental pathology

Of the 25 local trisomy CPM cases, placental pathology had been performed for 23 cases

(92%). Of these, 52% (12/23) had some sort of abnormality (Table 2.3). The frequency and range of abnormalities among these 23 local trisomy CPM cases were considered non-specific by a pediatric pathologist (D. McFadden). Placental pathology was not available for referred trisomy CPM cases or for matched controls.

2.4.3 Birth weight

Birth weight data for the trisomy CPM cases and matched controls are given in Table

2.4. Trisomy CPM birth weight was significantly lower compared to birth weight among matched controls (p = 0.001; Table 2.4). There was also a significantly higher rate of SGA infants in the trisomy CPM group (23%; 16/69) compared to the expected 10% (Binomial test, z-approximation, p < 0.001) and compared to the matched controls (8%; 11/138) (Fisher Exact test, p = 0.003).

In addition, maternal age was higher and sex ratio lower in the trisomy CPM group compared to the matched controls (Table 2.4). The differences in maternal age and sex ratio were expected given the well-established relationship between maternal age and trisomic pregnancy (Hassold and Hunt 2001) and the bias towards females in mosaic trisomies (see

Chapter 3). Maternal age was not significantly associated with birth weight among the trisomy

CPM cases or matched controls, and sex was not associated with birth weight in the trisomy

CPM cases. In contrast, males among the matched controls had heavier birth weights (t = 2.44, df = 136, p = 0.008), as also noted in surveys of the general population (Kramer et al. 2001).

20 Therefore, multiple linear regression was carried out with birth weight as the outcome variable,

and trisomy CPM, maternal age and sex of the fetus as explanatory variables. Neither sex nor

maternal age confounded the relationship between trisomy CPM and decreased birth weight

(data not shown).

2.4.4 Placental weight

Placental weight data are shown in Table 2.2. As expected, the matched controls had

heavier placentas because they were weighed untrimmed and without removing excess blood

(Table 2.4). Thus the placental weights were compared to similar reference populations. For

matched controls, 57 cases had placental weights below the means from Dombrowski et al.

(1994), while 81 had placental weights above the mean, which was borderline significant

(Binomial test, z-approximation, p = 0.05, 2-tailed). In contrast, for the trisomy CPM cases

placental weights were clearly lighter compared to the means from Pinar et al. (1996): 53 cases

had placental weights below the mean, while 16 had placental weights above the mean

(Binomial test, z-approximation, p < 0.001).

2.4.5 F-P weight ratio

F-P weight ratio data are shown in Table 2.2. The F-P weight ratios for the matched

controls are lower because their placentas were heavier compared to the trisomy CPM cases

(Table 2.4), and thus the F-P weight ratios were compared to reference populations. For

matched controls, 66 cases had F-P weight ratios below the means from Dombrowski et al.

(1994), while 72 had placental weights above the mean, which was not significantly different

from expected (Binomial test, z-approximation, p = 0.67, 2-tailed). Similarly, the distribution of

F-P weight ratios among the trisomy CPM cases was as expected when compared to means from

Heinonen et al. (2001): 34 cases had F-P weight ratios below and 35 cases above the mean.

21 2.4.6 Determinants of placental weight and birth weight

Possible determinants of placental weight and birth weight in the trisomy CPM cases include: sex of the infant, the chromosome involved in the trisomy, and the level of trisomy in the various placental tissues. To control for gestational age and to maintain a continuous outcome variable, placental and birth weight were converted to standard deviations (SDs) from the mean using the gestational age-corrected reference data of Molteni et al. (1978).

2.4.6.1 Sex of the fetus

Sex of the fetus was not associated with placental or birth weight (data not shown).

2.4.6.2 Involved chromosome

For the involved chromosome, there were sufficient sample sizes for trisomy 16 (n =

13), trisomy 7 (n = 10), and trisomy 2 (n = 9) to make comparisons between karyotypes; the other trisomies were grouped into 'other' (n = 37). Means and standard deviations for these 4 trisomic groups are illustrated in Figure 2.1. On bivariate comparisons (t-test), the only differences were between CPM involving trisomy 16 (CPM 16) and each of the other 3 groups for birth weight (p < 0.05), with CPM16 significantly lower in each comparison. Therefore, the

CPM 16 cases should be considered separately from all other CPM cases when considering birth weight. There were no significant associations with CPM 16 for placental weight as the outcome variable (data not shown).

2.4.6.3 Level of trisomy in trophoblast, mesenchyme, chorion

For bivariate comparisons (Spearman's correlation) with placental weight and birth weight, the level of trisomy in each lineage was coded to reduce the effect of random sampling error between cases (due to different sites of the placenta sampled, different number of sites

22 sampled, and different number of cells for cytogenetic analysis): 0% trisomy = '0'; 0-50% trisomy = T; 51-100% trisomy = '2'. The significant associations were between birth weight and the level of trisomic cells in the trophoblast (rho = -0.56, n = 40, p < 0.001), and between birth weight and the level of trisomic cells in the mesenchyme (rho = -0.28, n = 61, p = 0.014).

The negative direction of the coefficients indicates that in each instance a higher level of trisomy is associated with a lower birth weight. Since there were no associations with placental

weight, it was not surprising that the levels of trisomic cells in the trophoblast and mesenchyme

were also significantly negatively correlated with F-P weight ratio (data not shown).

The levels of trisomy in the trophoblast and mesenchyme were positively correlated with

each other (rho = 0.54, n = 35, p < 0.001), suggesting potential confounding in their associations

with birth weight. Furthermore, the level of trisomy in placental lineages may be confounded

by the chromosome involved; for example, the CPM 16 cases had a significantly higher level of

trisomy in the trophoblast compared to the other CPM cases (Mann-Whitney test, p < 0.005, 2-

tailed). Ideally, multiple linear regression should be carried out to characterize any independent

effects. However, no model could be developed that satisfied the assumptions of linear

regression.

Instead of multiple regression, associations between birth weight, and the level of

trisomy in the trophoblast and in the mesenchyme were analyzed for the CPM 16 cases and the

other CPM cases separately. In the CPM 16 group, both the trophoblast and mesenchyme were

significantly associated with lower birth weight (rho = -0.75, n = 10, p = 0.006; and rho = -0.79,

n = 11, p = 0.002); in the other CPM group, there was a trend towards an association between

the trophoblast and lower birth weight (rho = -0.26, n = 30, p = 0.083), but no evidence of an

association between the mesenchyme and birth weight (rho = -0.13, n = 50, p = 0.19). When the

level of trisomic trophoblast was categorized into an indicator variable (0% = '0', >0% = '1'),

the trend towards lower birth weight in the other CPM group was statistically significant (t =

23 1.74, df = 28, p = 0.046). Thus, trisomic trophoblast was associated with decreased birth weight in both the CPM 16 and other CPM groups (although the latter association was weaker), while the mesenchyme was associated with decreased birth weight only in the CPM16 group.

Furthermore, in the CPM 16 group, the level of trisomic trophoblast was highly significantly associated with the level of trisomy in the mesenchyme (rho = 0.90, n = 9, p < 0.001).

However, in the other CPM group, the level of trisomic trophoblast was not associated with the level of trisomic mesenchyme (rho = 0.22, n = 26, p = 0.15). When the levels of trisomy were categorized into indicator variables, there was still no association between trophoblast and mesenchyme in the other CPM group (Fisher Exact test, n -40, p = 0.25). Together, these results suggest that the trophoblast is the key tissue involved in the determination of birth weight in both CPM 16 and other CPM cases; and that the association between birth weight and the level of trisomy in the mesenchyme in the CPM 16 cases was spurious because of its correlation with the level of trisomic trophoblast.

To determine whether the presence of trisomy 16 (CPM 16) and the level of trisomy in the trophoblast had effects on birth weight independent of each other, associations were tested for CPM 16 at different levels of trisomic trophoblast. CPM 16 still showed decreased birth weight both when the level of trisomic trophoblast was '0%' (means: CPM16 = -0.34 ± 0.43 SD

(n = 3) vs. other CPM = 0.65 ± 1.03 SD (n = 24); Welch's approximate t = 3.08, df = 5.8, p =

0.012), and when the level of trisomic trophoblast was greater than 0% (means: CPM16 = -1.30

± 0.64 SD (n = 7) vs. other CPM = -0.20 ± 1.25 SD (n = 6); Welch's approximate t = 1.95, df =

7.2, p = 0.046). This suggests that the presence of CPM16 also has an effect on birth weight independent of the level of trisomy trophoblast. In other words, the presence of CPM 16 and the degree of trisomic trophoblast appear to have independent effects on birth weight.

24 2.4.6.4 Placental weight

Although sex of the fetus, the involved chromosome, nor the level of trisomy in the three placental lineages were associated with weight of the placenta, placental weight itself may be a determinant of birth weight in trisomy CPM cases. Placental weight and birth weight were found to be positively correlated in the trisomy CPM sample (r = 0.60, n = 69, p < 0.001), which replicates an established finding in the general population (Molteni et al. 1978; Williams et al.

1997; Sanin et al. 2001).

Thus, to determine whether CPM 16 and trisomic trophoblast mediate their effects on birth weight independent of placental weight, multiple linear regression was first carried out with birth weight as the outcome variable, and placental weight and the level of trisomic trophoblast as explanatory variables. The level of trisomic trophoblast had to be dichotomized into an indicator variable (0% = '0' and >0% = '1') because of heteroscedasticity (variance in the residuals decreased as the level of trisomic trophoblast increased). In the resulting model, both trisomic trophoblast (dichotomized) (b = -1.2, p < 0.001) and placental weight (b = 0.38, p

= 0.006) had significant independent effects. In other words, the level of trisomic trophoblast had an effect on birth weight independent of placental weight. Second, multiple linear regression was carried out with birth weight as the outcome variable, and placental weight and the involved chromosome as explanatory variables (CPM16 vs. other CPM). In the resulting model, both CPM 16 (b = -1.0, p = 0.002) and placental weight (b = 0.42, p < 0.001) had significant independent effects. Thus, the presence of CPM16 reduced birth weight independent of placental weight.

In summary, therefore, these results together suggest that the presence of trisomic trophoblast and trisomy 16 reduce birth weight independent of each other and of placental weight.

25 2.5 Discussion

In this study, placental weights and birth weights were lower in trisomy CPM cases compared to matched controls, as hypothesized. Surprisingly, the level of trisomy in the placenta was not associated with placental weight. However, the level of trisomic trophoblast

and the presence of trisomy 16 (CPM 16) decreased birth weight independent of each other and of placental weight, suggesting they affect placental function rather than a simply reducing placental growth. A non-specific range of abnormalities was noted on pathology examination of

the CPM placentas; in particular, there were was no pathognomic finding among CPM 16 placentas (Table 2.3). Thus routine placental pathology could not clearly identify the

mechanisms by which trisomy affects placental function; instead, it is likely that more in depth

cellular and physiological studies are required to determine how placental function is altered by

trisomy. For example, trisomic trophoblast could reduce birth weight by a defect in extravillus

trophoblast (EVT) invasion and remodelling of the spiral arteries resulting in poor utero•

placental perfusion (Wright et al. 2004) or a defect in syncytiotrophoblast formation and

function (Frendo et al. 2000). Trisomy 16 in the trophoblast, in particular, appears to have a

defect in EVT outgrowth (Chapter 7). In addition, F-P weight ratio was not altered in trisomy

CPM, indicating that placental weight and birth weight were reduced to a similar degree. Thus,

placental weight was likely decreased in trisomy CPM pregnancies due to the correlation

between placental and birth weight seen in the general population (Molteni et al. 1978; Williams

et al. 1997; Sanin et al. 2001). Together, these results imply the following model for the

pathogenesis of birth weight in trisomy CPM pregnancies: the presence of trisomy 16 and higher

levels of trisomic trophoblast decrease fetal growth through an alteration in placental function;

then, because of putative feto-placental signal(s) that regulate placental growth in the general

population and produce the correlation between placental and birth weights, placental growth is

similarly decreased.

26 One concern is that a number of factors known to be associated with birth weight, placental weight, and F-P weight ratio were not accounted for in this study because the clinical

data from referred cases were limited. For instance, F-P weight ratio is decreased in maternal

anemia (Williams et al. 1997), and in gestational diabetes (Lao et al. 1997). As well, Williams

et al. (1997) found the following variables had independent effects on birth weight: maternal

pre-pregnancy weight, maternal height, maternal body mass index, parity, smoking, anemia,

diabetes, weight gain, gestational age, female sex of the fetus, and ethnicity; for placental

weight, the following had independent effects: maternal pre-pregnancy weight, maternal height,

maternal BMI, parity, anemia, diabetes, weight gain, gestational age, and female sex of the

fetus, and ethnicity; and for F-P weight ratio, socio-economic status, maternal pre-pregnancy

weight, maternal BMI, smoking, anemia, gestational age, female sex of the fetus, and ethnicity

had independent effects. It is possible in theory that these variables - other than parity, which

was matched for - may not be equally distributed among the trisomy CPM cases and matched

controls, and could account for the relationships seen here. However, in contrast to maternal

age and sex of the fetus, which were expected to differ in the mothers of CPM pregnancies,

none of the variables discussed above have a theoretical reason to differ in CPM mothers.

Another concern is that placental weight is a crude marker, as much of what is measured is not

functional placental tissue and variability is induced by differences in handling prior to

measurement (Marais 1979; Lao and Wong 1996; Heinonen et al. 2001). Regardless, placental

weight is easy to measure and is weighed routinely after birth, which allows epidemiologic

studies of adequate sample size. Also, since placental weight is positively correlated with birth

weight, it is likely correlated with the 'functional' placental mass that modulates fetal growth.

In conclusion, lower placental weight and birth weight were present in trisomy CPM

pregnancies. Etiological factors include the level of trisomic trophoblast and the involvement of

trisomy 16, which independently affect placental function. Future investigations should include

27 identification of the specific functions that are altered in the trisomic placenta, and the mechanisms causing trisomic placental cells to function abnormally.

28 2.6 References

Dombrowski MP, Berry SM, Johnson MP, Saleh AA, Sokol RJ (1994) Birth weight-length ratios, ponderal indexes, placental weights, and birth weight-placenta ratios in a large population. Arch Pediatr Adolesc Med 148:508-512

Frendo JL, Vidaud M, Guibourdenche J, Luton D, Muller F, Bellet D, Giovagrandi Y, Tarrade A, Porquet D, Blot P, Evain-Brion D (2000) Defect of villous cytotrophoblast differentiation into syncytiotrophoblast in Down's syndrome. J Clin Endocrinol Metab 85:3700-3707

Frid C, Drott P, Otterblad Olausson P, Sundelin C, Anneren G (2004) Maternal and neonatal factors and mortality in children with Down syndrome born in 1973-1980 and 1995- 1998. Acta Paediatr 93:106-112

Hassold T, Hunt P (2001) To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2:280-291

Heinonen S, Taipale P, Saarikoski S (2001) Weights of placentae from small-for-gestational age infants revisited. Placenta 22:399-404

Henderson KG, Shaw TE, Barrett U, Telenius AH, Wilson RD, Kalousek DK (1996) Distribution of mosaicism in human placentae. Hum Genet 97:650-654

Kalousek DK, Howard-Peebles PN, Olson SB, Barrett JJ, Dorfmann A, Black SH, Schulman JD, Wilson RD (1991) Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism. Prenat Diagn 11:743-750

Kalousek DK, Langlois S, Barrett I, Yam I, Wilson DR, Howard-Peebles PN, Johnson MP, Giorgiutti E (1993) Uniparental disomy for chromosome 16 in humans. Am J Hum Genet 52:8-16

Kalousek DK, Langlois S, Robinson WP, Telenius A, Bernard L, Barrett IJ, Howard-Peebles PN, Wilson RD (1996) Trisomy 7 CVS mosaicism: pregnancy outcome, placental and DNA analysis in 14 cases. Am J Med Genet 65:348-352

Kramer MS, Piatt RW, Wen SW, Joseph KS, Allen A, Abrahamowicz M, Blondel B, Breart G (2001) A new and improved population-based Canadian reference for birth weight for gestational age. Pediatrics 108:E35

Kuchinka BD, Barrett IJ, Moya G, Sanchez JM, Langlois S, Yong SL, Kalousek DK, Robinson WP (2001) Two cases of confined placental mosaicism for chromosome 4, including one with maternal uniparental disomy. Prenat Diagn 21:36-39

Lao TT, Wong WM (1996) Placental ratio and intrauterine growth retardation. Br J Obstet Gynaecol 103:924-926

29 Lomax BL, Kalousek DK, Kuchinka BD, Barrett IJ, Harrison KJ, Safavi H (1994) The utilization of interphase cytogenetic analysis for the detection of mosaicism. Hum Genet 93:243-247

Marais WD (1979) Placental size at birth. S Afr Med J 55:153

Molteni RA, Stys SJ, Battaglia FC (1978) Relationship of fetal and placental weight in human beings: fetal/placental weight ratios at various gestational ages and birth weight distributions. J Reprod Med 21:327-334

Myrelid A, Gustafsson J, Ollars B, Anneren G (2002) Growth charts for Down's syndrome from birth to 18 years of age. Arch Dis Child 87:97-103

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong SL, Lewis S, Bruyere H, Howard- Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X- chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446

Pinar H, Sung CJ, Oyer CE, Singer DB (1996) Reference values for singleton and twin placental weights. Pediatr Pathol Lab Med 16:901-907

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard- Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927

Sanin LH, Lopez SR, Olivares ET, Terrazas MC, Silva MA, Carrillo ML (2001) Relation between birth weight and placenta weight. Biol Neonate 80:113-117

Shaffer LG, Langlois S, McCaskill C, Main DM, Robinson WP, Barrett IJ, Kalousek DK (1996) Analysis of nine pregnancies with confined placental mosaicism for trisomy 2. Prenat Diagn 16:899-905

Stoll C, Alembik Y, Dott B, Roth MP (1998) Study of Down syndrome in 238,942 consecutive births. Ann Genet 41:44-51

Williams LA, Evans SF, Newnham JP (1997) Prospective cohort study of factors influencing the relative weights of the placenta and the newborn infant. Bmj 314:1864-1868

Wright A, Zhou Y, Weier JF, Caceres E, Kapidzic M, Tabata T, Kahn M, Nash C, Fisher SJ (2004) Trisomy 21 is associated with variable defects in cytotrophoblast differentiation along the invasive pathway. Am J Med Genet A 130:354-364

30 Table 2.1 Clinical and cytogenetic data for the 69 trisomy CPM cases

Case Previous Study Local or Chr Sex Anomalies AF Bid Am Publications case # Referred % % % 1 88.7 R 13 F None 0 0 0 2 2 R 7 M None 0 3 3 R 7 M None 0 4 4 R 10 M None 0 0 5 5 R 15 F None 0 0 6 6 R 16 M None 0 0 0 7 10 R 15 M None ? 0 8 12 R 18 F None ? 0 0 9 Robinson et al. 89.20 R 8 M 0 0 0 (1997) case 89.20 10 15 L 12 F None 0 0 0 11 16 R 12 M ? 0 12 90.13 R 12 M None ? 0 13 90.29 R 12 F None ? 0 0 14 90.75 L 13 F None 0 0 15 90.40 L 2 F None 0 16 Kalousek et al. 90.87 R 7 F None ? 0 0 (1996) case 1 17 Kalousek et al. 90.90 R 16 M None 0 0 (1993) case 5 Johnson et al. (1993) caseB Robinson et al. (1997) case 90.90 Penaherrera et al. (2000) case 90.90 18 91.7 L 8/ F None 0 0 8,21 19 Kalousek et al. 91.10' L 16 F None 0 0 0 (1993) case 6 20 Shaffer et al. 91.43 L 2 F None 0 0 0 (1996) case 7 21 91.24 R 16 F Hip 0 0 ? dysplasia 22 Kalousek et al. 91.33 R 7 M None 0 0 (1996) case 2 Robinson et al. (1997) case 91.33 23 91.47 R 2 F None 0 24 Shaffer et al. 91.54 L 2 F None 0 0 (1996) case 3 25 Kalousek et al. 91.55 R 16 F None 0 0 0 (1993) case 7 Robinson et al. (1997) case 91.55 Penaherrera et al. (2000) case 91.55 26 Robinson et al. 91.56 R 22 F None 0 0 (1997) case 91.56 27 91.63 R 12 F None ? 0 0 28 Robinson et al. 91.69 L 10 M None 0 (1997) case 91.69

31 Case Previous Study Local or Chr Sex Anomalies AF Bid Am Publications case # Referred % % % 29 Kalousek et al. 91.71 R 16 F None 0 0 0 (1993) case 3 Johnson et al. (1993) caseC Robinson et al. (1997) case 91.71 Penaherrera et al. (2000) case 91.71 30 91.79 R 9 F None ? 0 0 31 Kalousek et al. 91.80 R 7 F None 0 0 (1996) case 5 Robinson et al. (1997) case 91.80 32 91.85 L 8 F None 0 0 33 91.87 L 21 M None 0 0 34 91.88 R 15,21 M None 0 0 35 Robinson et al. 91.89 R 10 F None ? 0 0 (1997) case 91.89 36 91.93 R 18 F None ? 0 0 37 91.94 L 2,18,1 M None 0 8 38 Robinson et al. 91.96 R 9 M None 0 0 (1997) case 91.96 39 92.11 R 7 M None ? 0 0 40 92.14 L 10 M Hydro• 0 0 0 nephrosis 41 92.20 R 20 F None ? 0 0 42 Robinson et al. 92.21 R 10,12 M None ? 0 0 (1997) case 92.21 43 92.24 R 15 F None ? 0 0 44 Kalousek et al. 92.25 R 16 M Imp anus 0 0 0 (1993) case 4 Robinson et al. (1997) case 92.25 45 Shaffer et al. 92.29 L 2 F None 0 (1996) case 4 Robinson et al. (1997) case 92.29 46 Kalousek et al. 92.48 L 16 F Mild 0 0 (1993) case 8 hypospadias Robinson et al. (1997) case 92.48 47 Kalousek et al. 92.49 R 16 F None 0 0 0 (1993) case 9 Robinson et al. (1997) case 92.49 48 Kuchinka et al. 92.55 L 4 F None 0 (2000) case CPM4-2 49 92.56 L 13,18 F None 0 50 Kalousek et al. 92.58 L 7 M None 0 0 (1996) case 6 51 Shaffer et al. 92.59 R 2 M 0 0 (1996) case 8 52 92.77 R 17 F None 0 0 Case Previous Study Local or Chr Sex Anomalies AF Bid Am Publications case # Referred % % % 53 92.81 R 2 F None 0 0 0 54 92.92 R 11 F None ? 0 0 55 Robinson et al. 93.1 R 2 M 0 (1997) case 93.1 56 Robinson et al. 93.7 L 22 F None 0 0 (1997) case 93.7 57 Robinson et al. 93.53 L 12 M None 0 (1997) case 93.53 58 93.54 R 9 M None 0 0 0 59 93.83 R 2 F None 0 0 0 60 Robinson et al. 93.134 R 16 F None 0 0 0 (1997) case 93.134 61 93.177 L 17 F None 0 0 62 94.20 L 8 F None 0 63 94.26 L 16 F 0 64 94.35 R 16 M None 0 0 65 94.76 L 9 F None 0 0 66 94.82 L 7 F 0 0 67 Kalousek et al. 94.85 R 7 M None 0 0 (1996) case 8 Robinson et al. (1997) case 94.85 68 94.105 R 16 F Possible 0 0 VSD 69 Kalousek et al. 94.107 L 7 F None 0 0 (1996) case 14 Informative cases 69 69 69 69 63 53 42 58 Summary L=25 M None = 58 statistics R = 44 =26 (92%)

"Study case #" = case number for the ongoing study of trisomy mosaicism at UBC. "Chr" = chromosome involved in the trisomy. "AF" = % trisomic cells in amniotic fluid, assessed by amniocentesis. "Bid" = % trisomic cells in fetal or infant blood, assessed by cordocentesis or peripheral blood sampling. "Am" = % trisomic cells in the amnion, sampled post-partum. "?" = tissue assessed, but results not available. Full references in Appendix AI.

33 Table 2.2 Clinical and cytogenetic data for the 69 trisomy CPM cases

Case Gest Placental Birth Feto-placental Chorion Mesen Troph Age weight weight (F-P) weight % % % (weeks) (g) (g) ratio 1 40 550 3370 6.13 0 0 36 2 38 392 2556 6.52 4 50 16 3 40 345 2784 8.07 73 32 0 4 40 513 3238 6.31 13 100 - 5 38 365 2414 6.61 0 10 - 6 40 434 3689 8.50 0 47 - 7 37 587 3481 5.93 - 0 - 8 41 394 3481 8.84 0 0 - 9 37 540 3120 5.78 - 20 - 10 37 425 2530 5.95 100 90 0 11 41 451 3518 7.80 - 0 0 12 36 411 3377 8.22 0 0 - 13 40 509 3831 7.53 33 3 - 14 40 325 3420 10.52 27 18 - 15 38 435 2700 6.21 11 2 - 16 36 411 2336 5.68 0 0 - 17 41 495 3008 6.08 25 28 0 18 40 575 3445 5.99 58 91 17 19 36 405 2660 6.57 20 0 - 20 38 390 2970 7.62 0 0 - 21 40 480 2681 5.59 20 80 63 22 40 652 3482 5.34 40 84 0 23 35 367 2700 7.36 0 0 - 24 41 520 3555 6.84 8 0 - 25 40 446 3320 7.44 0 0 0 26 38 376 2010 5.35 73 75 22 27 40 595 4284 7.20 0 0 - 28 40 470 3940 8.38 0 53 0 29 38 203 1960 9.66 87 100 100 30 37 617 3717 6.02 33 28 0 31 40 511 3717 7.27 67 47 0 32 40 520 2985 5.74 - - 0 33 37 385 2620 6.81 11 16 - 34 40 512 3774 7.37 0 0 35 40 628 3859 6.14 40 20 0 36 40 482 3944 8.18 0 10 0 37 40 570 3650 6.40 - 0 0 38 40 246 3292 13.38 83 40 0 39 40 414 3348 8.09 0 0 0 40 39 500 3210 6.42 - 0 - 41 38 748 3575 4.78 7 23 - 42 41 444 4120 9.28 100 80 0 43 39 485 2638 5.44 0 33 - 44 35 269 1800 6.69 100 - 100 45 42 535 3377 6.31 - - 0 46 36 430 2280 5.30 - 100 61 47 31 277 1045 3.77 100 100 80 48 38 390 3175 8.14 - - - 49 38 450 3200 7.11 60 32 -

34 Case Gest Placental Birth Feto-placental Chorion Mesen Troph Age weight weight (F-P) weight % % % (weeks) (g) (g) ratio 50 39 365 3700 10.14 - - 0 51 36 497 3119 6.28 100 7 0 52 40 484 3250 6.71 0 0 0 53 40 526 2936 5.58 27 20 - 54 39 287 3235 11.27 0 0 - 55 39 450 2923 6.50 18 3 0 56 33 280 1820 6.50 - 100 19 57 40 583 4040 6.93 100 50 0 58 38 297 2270 7.64 20 43 - 59 39 576 3263 5.66 7 5 0 60 38 442 2817 6.37 13 0 0 61 40 385 3300 8.57 0 20 - 62 40 572 4520 7.90 - - - 63 36 245 1960 8.00 - - - 64 39 520 2800 5.38 0 73 35 65 41 360 3121 8.67 7 50 0 66 40 474 3500 7.38 0 - 0 67 40 393 3460 8.80 27 40 14 68 36 480 2014 4.20 87 93 78 69 40 470 3885 8.27 0 13 0 Total* cases 69 69 69 69 56 61 40 Mean 38.7±2.0 452+106 3118±662 7.09±1.63 28.6±35.4 31.6+34.8 16.0±29.5 {range} {31-42} {203-748} {1045- {3.77-13.38} {0-100} {0-100} {0-100} 4520}

'Chorion' = % trisomic cells in the chorion, sampled post-partum. 'Mesen' = % trisomic cells in the chorionic villus mesenchyme, sampled post-partum. 'Troph' = % trisomic cells in the trophoblast, sampled post-partum. For the mesenchyme and chorion, "0%" = no trisomic cells on conventional cytogenetics. For the trophoblast, 0-10% on FISH were all coded as "0%" (see text). "-" = not performed.

35 Table 2.3 Findings for (local) cases where placental pathology was performed

Case Chromosome Placental pathology

1 13 2 7 3 7 4 10 5 15 6 16 7 15 8 18 9 8 10 12 Normal 11 12 12 12 13 12 14 13 Normal 15 2 Normal 16 7 17 16 18 8/8,21 Lymphocytic villitis 19 16 Normal: Minimal focal microscopic findings not considered sufficiently extensive to be of any clinical significance (focally increased perivillus fibrin, slight variation in villus maturity, focal congestion, a few villi which are hypervascular, a suggestion of very focal non-specific vessel villitis) 20 2 Normal 21 16 22 7 23 2 24 2 Early acute chorioamniotis 25 16 26 22 27 12 28 10 Focal old mural thrombus 29 16 30 9 31 7 32 8 1) Placental infarcts; 2) Trophoblastic cysts; 3) Deciduitis of membranes, focal; 4) Accessory lobe placenta. 33 21 1) Decidual necrosis and inflammation - membranes and maternal surface. 34 15,21 35 10 36 18 37 2,18,18 Normal 38 9 39 7 40 10 1) Infarct with decidual necrosis and thrombosis (abruption), old, placental margin; 2) Hemosiderin deposition, macrophages, membranes. 41 20 42 10,12 43 15

36 44 16 45 2 1) Focal villitis of undetermined etiology. 2) Solitary intervillus thrombus. 46 16 1) Perivillus fibrinosis; 2) Hemorrhage, old, extraplacental membranes. 47 16 48 4 Normal 49 13,18 Normal 50 7 Normal 51 2 52 17 53 2 54 11 55 2 56 22 1) Increased intervillus fibrin; 2) Hemorrhage with early organization is consistent with placental abruption. 57 12 1) Perivillus fibrin deposition with no evidence of infarction. 58 9 59 2 60 16 61 17 Normal 62 8 Normal 63 16 1) Decreased fetal vascularization of chorionic surface; 2) Focal mural thrombosis fetal vessels. 3) Focal villus immaturity. 64 16 65 9 Focally prominent fibrinosis. 66 7 67 7 69 16 Normal Total # with abnormalities 12/23 (52%)

37 Table 2.4 Clinical characteristics of the trisomy CPM cases and the matched controls

Trisomy CPM cases Matched controls Significance

mean n Mean n Maternal age 37.8±3.5 years 64 35.3±4.4 years 138 p < 0.001 Parity 1.14+1.06 49 1.06+1.07 138 n.s. , Gestational age 38.7±2.0 weeks 69 38.7+1.8 weeks 138 n.s. Sex of the fetus 26 M : 43 F 69 79 M : 59 F 138 p = 0.006 Birth weight 3118±662g 69 3435±586g 138 p = 0.001 Placental weight 77% < mean 69 41% < mean 138 p< 0.001; n.s. F-P weight ratio 49% < mean 69 48% < mean 138 n.s.; n.s.

Placentas were handled differently before weighing for the trisomy CPM cases and matched controls. Therefore, placental weight and F-P weight ratio for the CPM cases and matched controls were instead compared to gestational age-specific means from published reference populations with similar handling procedures (see text). Shown is the proportion of CPM cases and matched controls that were below the gestational age-corrected means from the reference populations, with the p-values when compared to the expected 50% < mean for the CPM cases and controls, respectively. For gestational age and birth weight, the t-test was used; for maternal age, Welch's approximate t-test was used because of unequal variances; for parity, Mann- Whitney test (z-approximation) was used because of non-normality; and for sex of the fetus, the Fisher Exact test was used. All tests were 1-tailed except for parity and gestational age.

38 Figure 2.1 Birth weight means for the chromosomes involved in the trisomy CPM cases

1.001

0.50-

0.00-

-0.50"

-1.00"

Other +7 +2 +16

Trisomy

Mean ± standard deviation. The trisomy 16 group was significantly different from each of the other 3 trisomic groups (see text).

39 3 Pathogenesis of CPM16 pregnancies4

3.1 Note

I wrote this chapter/manuscript and did the data organization and analysis, with the

following clarifications and exceptions. The CPM16 cases are from the ongoing UBC study collected since 1988. Uniparental disomy testing was performed initially by Dr. S. Langlois,

and since 1994 in the lab of Dr. W. Robinson. The results in this Chapter are slightly different

from the publications (see footnote) because (1) sample size is increased; (2) live births are

separated from neonatal/intrauterine deaths; (3) birth weight is corrected for gestational age

using more recent data from (Kramer et al. 2001); and (4) the role of ascertainment is

investigated. However, the major conclusions remain the same.

3.2 Introduction

Trisomy 16 may be the most frequent chromosome abnormality at conception

(Wolstenholme 1995). Among all clinically recognized pregnancies, it has an incidence of

-1.5% (Hassold and Jacobs 1984). Although most trisomy 16 embryos are spontaneously

aborted or are noted to have arrested development between 8-15 weeks gestation, some embryos

survive and those pregnancies are candidates for prenatal diagnosis (Benn 1998). It has been

estimated that approximately 34 per 100,000 chorionic villus sampling (CVS) analyses detect

trisomy 16 (Wolstenholme 1995), while Benn (1998) notes that a recent estimate for

amniocentesis has not been reported. In these surviving embryos, the trisomy 16 cells are

virtually always completely or predominantly confined to the placenta (CPM), with only one

case of apparently non-mosaic (full) trisomy 16 in the fetus diagnosed at CVS (1994).

4 A version of this chapter has been published. 1) Yong PJ, Marion SA, Barrett IJ, Kalousek DK, Robinson WP. 2002. Evidence for imprinting on chromosome 16: the effect of uniparental disomy on the outcome of mosaic trisomy 16 pregnancies. Am J of Med Genet 112(2): 123-32. 2) Yong PJ, Barrett IJ, Kalousek DK, Robinson WP. 2003. Clinical aspects, prenatal diagnosis and pathogenesis of trisomy 16 mosaicism. J Med Genet 40(3): 175-82.

40 Almost all CPM 16 cases originate from a trisomy 16 zygote as a consequence of a maternal meiosis I nondisjunction, followed by trisomy zygote rescue (Robinson et al. 1997).

When a trisomy 16 conceptus is rescued, one of the two maternal chromosomes or the paternal chromosome can be lost. If the former occurs, the result is biparental disomy 16 (bpd(16)) or a chromosome 16 inherited from each parent; if the latter occurs, the result is maternal uniparental disomy 16 (upd(16)mat) or both chromosomes inherited from the mother (Engel 1980; Spence et al. 1988). Uniparental disomy (UPD) could have a distinct phenotypic effect if imprinted genes (i.e. genes whose expression depends on whether they are inherited from the mother or father) exist on chromosome 16. Imprinting on human chromosome 16 has been proposed based on orthology with imprinted regions in the mouse (Searle et al. 1989). Although reports of malformation in cases of upd(16)mat have raised the possibility of imprinting (Ledbetter and

Engel 1995), studies of upd(16)mat have neither conclusively supported or excluded imprinting effects (Kotzot 1999). In fact, in an earlier review, Kalousek and Barrett (1994) noted that the degree of trisomy 16 in the placenta seemed to correlate with the growth restriction of a chromosomally normal fetus independent of UPD status. Besides upd(16)mat and the level of trisomy in the post-partum trophoblast (Chapter 2), other factors potentially contributing to the pathogenesis of CPM 16 are the level of trisomy in amniotic fluid (by amniocentesis) and in the amnion (sampled after delivery), as well as ascertainment bias and sex of the fetus.

I hypothesized that CPM 16 pregnancies would tend to be growth restricted with significant rates of malformation, and that the risk of these outcomes will be associated with each of the following predictive factors: upd(16)mat, degree/distribution of trisomy in amniotic fluid (by amniocentesis) and the placenta (by CVS), ascertainment, and sex of the fetus. In this study, statistical analysis was performed on data from a large series (n = 173) of published and unpublished CPM 16 cases with the purpose of (1) summarizing the prenatal and perinatal outcome of CPM 16 pregnancies; and (2) evaluating the predictive value of the factors for

41 measures of pregnancy outcome. The identification of important predictive factors will aid genetic counseling after prenatal diagnosis and will elucidate factors that are involved in the pathogenesis of CPM 16 during pregnancy.

3.3 Methods

3.3.1 CPM16 cases

The study sample (n = 173 cases) consists of CPM 16 pregnancies diagnosed prenatally by CVS or amniocentesis with or without molecular testing for the UPD status of chromosome

16 (Appendix AI). Rare cases with paternal origin of the trisomy (n = 2), partial trisomy (n =

3), and concomitant aneuploidy (n = 1) were excluded in order not to confound the analysis

(Appendix AI). Some cases are from an ongoing study of CPM at the University of British

Columbia (UBC) (n = 69), which consists of cases referred from other centres (n = 63) and

cases initially ascertained at the Children's and Women's Health Centre of British Columbia

(C&W) (n = 6). Some data from most of these cases have been published previously, and there

is overlap with cases published by other research groups (Appendix AI). The study was

approved by the ethics committees of UBC and C&W (Appendix A). Informed consent was

obtained from parents to provide clinical data and for determination of their child's UPD status.

UPD testing was carried out as described (Robinson et al. 1997). Clinical information was sent

by the referring physician either directly or by filling out a questionnaire; in those cases where

prenatal diagnosis occurred in Vancouver, clinical information was also gathered from medical

records at the UBC Department of Medical Genetics. An additional 104 cases are from other

published reports to date. Using the review of CPM 16 cases by Benn (1998) as a starting point,

data were verified from the original sources and care was taken to eliminate duplicated cases

(i.e. those published in two separate reports).

42 Data were collected on the following variables: (1) pregnancy outcome (i.e. live birth, intrauterine death or termination of pregnancy); (2) sex of the fetus; (3) gestational age at delivery; (4) malformation detected in the fetus/neonate/infant ("malformation" used as general term independent of etiology, including possible disruptions and deformations); (5) fetal/neonatal weight at pregnancy outcome (in standard deviations from the Canadian population mean corrected for gestational age and sex (Kramer et al. 2001)); (6) ascertainment

(e.g. advanced maternal age, abnormal serum screen or ultrasound, etc.); (7) UPD status; (8) percent trisomy in amniotic fluid on amniocentesis (by conventional cytogenetics); and (9) presence or absence of trisomy in tissues of the fetus. For variable (9), data from conventional cytogenetics, FISH or molecular methods (PCR) were taken into account; if results were contradictory (i.e. one was positive for trisomy, the other negative), then the fetal tissue was coded as positive. Molecular detection of trisomy in fetal tissues was only considered for cases from the UBC study where detailed information on chromosome 16 markers was available, and cases from other published reports where there was an explicit statement that PCR showed or excluded trisomy.

It should be emphasized that the study sample may be biased towards cases with poorer outcomes, since such cases are more likely to be ascertained (e.g. due to anomalies observed on ultrasound), referred for research purposes, and/or submitted for publication. Therefore, purely descriptive statistics should not be considered estimates of the actual values in the general population, but are intended as descriptions of the study sample specifically. Statistical associations between variables are more likely to be unbiased. Furthermore, it should be noted that there was variation in the quantity and quality of data available among the cases (e.g. for descriptions of malformations).

43 3.3.2 Statistical analysis

Statistical analysis was carried out using SPSS 10.0 and the VassarStats Web Site for

Statistical Computation (http://faculty.vassar.edu/lowry/VassarStats.html). Tests were 1-tailed due to a priori evidence or rational mechanisms, unless otherwise noted. Welch's approximate t-test was utilized when there was inequality of variances. Means are reported ± standard

deviation.

3.4 Results

3.4.1 Pregnancy outcomes of CPM16

There were 166 cases informative for pregnancy outcome. Even with an expected bias

towards poor outcome, the majority of cases (62%) resulted in live births with survival beyond

the neonatal period. Eleven percent of pregnancies ended in intrauterine death (IUD) and 5% in

neonatal death, while 22% of the pregnancies were electively terminated. For the descriptive

statistics and statistical associations below, only the live births (with survival beyond the

neonatal period) were included. Intrauterine and neonatal deaths were analyzed separately

below, to allow comparison to the live births with survival beyond the neonatal period.

3.4.2 Clinical outcome of CPM16 live births

Figure 3.1 shows the distribution of gestational ages at delivery for the live births (n = 77

informative cases): the average gestational age was 36.0 ± 3.2 weeks. The distribution of birth

weights (number of standard deviations from the mean) for the live births is illustrated in Figure

3.2. Virtually all birth weights (93%) were below the general population mean birth weight (i.e.

0 standard deviations), with an average birth weight of -1.76 ± 1.08 standard deviations from

the general population mean. Forty two % of live births had at least one malformation (37 of 89

cases informative for malformation status). With the exception of one case with only a minor

44 anomaly (asymmetric nipples), all cases had at least one major malformation. Some malformations were present in 6 or more live births (i.e. in > 6% of informative cases) and were considered more likely to have a true association with CPM 16: VSD5 12% (n = 11), ASD6 10%

(n = 9), and hypospadias 27% (n = 7, of 26 informative male cases). All of these malformations were significantly more frequent when compared to the corresponding frequencies among neonates in the general population (Table 3.1, p < 0.0001).

3.4.3 Trisomy in amniotic fluid in CPM16 live births

Fifty-six percent of the live births with amniocentesis (49/87) had 0% trisomy detected in amniotic fluid. For those positive for trisomy (>0%) in amniotic fluid, the distribution was skewed towards low-level mosaicism (Figure 3.3), although the highest level detected was 93% trisomy. For statistical analyses, the level of trisomy in amniotic fluid was coded into a binary variable: 0% trisomy = '0'; >0% trisomy = T.

The presence of trisomy in amniotic fluid was associated with lower birth weight (t =

3.39, df = 62, p = 0.0005; Figure 3.4) and higher risk of malformation (Fisher Exact test, n = 82, p = 0.017; RR = 1.79; Table 3.2). Considering only cases where amniotic fluid was positive for trisomy, the percent trisomy was not associated with either birth weight or malformation (data not shown). Therefore, the simple presence of trisomy in amniotic fluid (as sampled by amniocentesis), but not the actual level above 0%, is predictive of outcome. It should be noted, however, that there was considerable variability in outcomes even for a given result in amniotic fluid. Figure 3.4 shows that there is a large standard deviation for birth weight among both cases negative for trisomy in amniotic fluid (std dev = 1.02) and cases positive for trisomy in amniotic fluid (std dev = 1.00). In addition, Table 3.2 shows that although the risk of

5 Excluding 1 case of tetralogy of Fallot and 3 cases of endocardial cushion defect/atrioventricular canal. 6 Excluding 3 cases of endocardial cushion defect/atrioventricular canal.

45 malformation increases when trisomy is present in amniotic fluid, the risk of malformation is still considerable (33%) even when no trisomy is detected.

This variability raises the question of how well the level of trisomy in amniotic fluid cells reflect trisomy in 'true' fetal tissues. Fetal tissues (other than umbilical cord and amnion) were examined in 59 live births; most of these were analyzed after birth, although findings from cordocentesis were also included. The most common tissues studied were blood (n = 55) and skin (n = 18). The presence of trisomy in amniotic fluid was associated with the presence of trisomy in fetal tissues (Fisher exact test, n = 52, p = 0.042; RR = 5.77; Table 3.3). However, for the 28 cases positive for trisomy in amniotic fluid, 75% (21/28) were negative in fetal tissues

(Table 3.3), which reflects the patchy distribution of trisomy in fetal tissues and the limited cytogenetic investigations in live born infants.

3.4.4 Ascertainment of CPM16 live births

Ascertainment was divided into two types: 1) 'unbiased' (e.g. AMA, past or family history of chromosome abnormality, investigation of Mendelian or biochemical disorder); and

2) 'biased' (abnormal serum screen, and abnormal ultrasound including altered fetal development or amniotic fluid level). Cases with biased ascertainment may have poorer outcome, as an abnormal serum screen or ultrasound indicates underlying pathology. Half of live births had an unbiased ascertainment (n = 36); and half had biased ascertainment (n = 35), of which 22 had abnormal serum screen and 13 had abnormal ultrasound findings. Abnormal ultrasound findings included IUGR (n = 4), single umbilical artery (n = 2), IUGR + dolichocephaly (n = 1), IUGR + hydropic placenta (n = 1), IUGR + oligohydramnios (n = 1),

IUGR + polydramnios + thick cystic placenta (n = 1), large and thick placenta with echogenic regions (n = 1), large sonolucent and cystic placenta (n = 1), and nuchal edema (n = 1). Biased ascertainment had a significant association with lower birth weight (Welch's approximate t =

46 2.85, df = 35.4, p = 0.0035; Figure 3.5), but not with higher risk of malformation (Fisher Exact test, n = 65, p = 0.13; data not shown). The significant association with lower birth weight still held when biased ascertainment was separated into abnormal serum screen (Welch's approximate t = 2.63, df = 41.9, p = 0.006; Figure 3.6) or abnormal ultrasound finding (Welch's approximate t = 2.80, df = 32.0, p = 0.005; Figure 3.7).

3.4.5 Sex of the fetus in CPM16 live births

The sex ratio of the live births was 0.48 (32 males and 67 females), which is

significantly different from the expected ratio of 1.07 calculated from prenatal controls by

Huether et al. (1996) (Binomial test, z-approximation, n = 99, p < 0.001). This deviation in sex ratio towards females confirms a previous observation made by Benn (1998). The male sex had

a significant association with lower birth weight (t = 2.35, df = 70, p = 0.011; Figure 3.8), but no

association with increased risk of malformation (Fisher Exact test, n = 84, p = 0.26; data not

shown).

3.4.6 CVS in CPM16 live births

In Chapter 2, the level of trisomy in the trophoblast of the post-partum placenta was

associated with birth weight. Here, the level of trisomy on direct CVS (villus cytotrophoblast)

and cultured CVS (villus mesenchyme) was investigated because they provide a 'snapshot' of

the first-trimester placenta, and a sufficient number of cases were available for a statistical

analysis. Since most cases had 100% trisomy on either CVS, these cases were compared to

those with <100% trisomy. Full (100%) trisomy in direct CVS was significantly associated with

lower birth weight (t = 4.08, df = 27, p < 0.001; Figure 3.9), but not with higher risk of

malformation (Fisher Exact test, n = 33, p = 0.067; data not shown). Similarly, the level of

trisomy in cultured CVS was associated with lower birth weight (t = 1.89, df = 27, p = 0.035;

47 Figure 3.10), but not with risk of malformation (Fisher Exact test, n = 35, p = 0.13; data not

shown).

3.4.7 upd(16)mat in CPM16 live births

If chromosome loss occurs randomly during trisomy rescue, one-third of CPM 16 should

have upd(16)mat. Forty two-percent (22/53) of live births had upd(16)mat, which is not

significantly different from the expected 33% (Binomial test, z-approximation, n = 53, p =

0.13). Upd(16)mat was significantly associated with both lower birth weight (t = 2.10, df = 42,

p = 0.021; Figure 3.11), and higher risk of malformation (Fisher Exact test, n = 49, p = 0.020;

RR = 1.78; Table 3.4). Again, it is emphasized that there is variability even within categories,

with 43% of bpd(16) having a malformation (Table 3.4) and birth weight standard deviations of

1.08 and 1.04 for bpd(16) and upd(16)mat, respectively (Figure 3.11). In addition, a wide range

of malformations were present among upd(16)mat cases, with no particular malformation seen

significantly more frequently compared to the bpd(16) cases (data not shown).

Since cases of upd(16)mat with particularly poor outcome may be more likely to be

reported in the literature, the t-test for association between upd(16)mat and birth weight was

repeated using only cases from the UBC study. The frequency of upd(16)mat in the UBC study

was also 42% (15/36). Among cases in the UBC study, upd(16)mat was still associated with

both lower birth weight (t = 1.72, df = 29, p = 0.049; data not shown) and higher risk of

malformation (Fisher Exact test, n = 33, p = 0.027; RR = 2.26; data not shown). Thus, any

publication bias towards upd(16)mat cases with poor outcome appears to be minimal.

3.4.8 Possible confounding

Thus far, the following variables have shown significant associations with birth weight:

trisomy in amniotic fluid, biased ascertainment (both abnormal serum screen and abnormal

48 ultrasound), male sex of the fetus, trisomy on direct and cultured CVS, and upd(16)mat.

Therefore, the effect of one of these variables on birth weight may be confounded by any of the other variables. Ideally, multiple linear regression would be utilized to determine if there are independent effects; however, sample size was too small to accommodate so many explanatory

variables. Thus, bivariate associations were sought for all the explanatory variables (Table 3.5).

The only significant associations were between the following: (1) trisomy in amniotic fluid and biased ascertainment; (2) trisomy in amniotic fluid and ascertainment by abnormal serum

screen, specifically; and (3) trisomy in direct CVS and trisomy in cultured CVS. Therefore,

confounding between the variables is unlikely except for between trisomy in amniotic fluid and

biased ascertainment (notably, abnormal serum screen), and between direct and cultured CVS.

On the other hand, the effect of placental trisomy (CVS) may be independent of fetal trisomy

(amniocentesis), and vice versa; and, further, the effect of upd(16)mat may be independent of

both placental and fetal trisomy. A multiple linear regression model that satisfied the

assumptions for regression - for birth weight as the outcome variable and trisomy on

amniocentesis and abnormal ascertainment as explanatory variables - could not be constructed.

Stratification of ascertainment - dividing cases with unbiased ascertainment and cases with

ascertainment by abnormal serum screening to see if trisomy amniotic fluid retains its effect on

birth weight in both conditions - was limited by small sample size. Similarly, neither multiple

linear regression nor stratification could be carried out for direct and cultured CVS.

In contrast, only two variables, trisomy in amniotic fluid and upd(16)mat, had significant

associations with higher risk of malformation. Therefore, multiple logistic regression modeling

was carried out. Both explanatory variables had independent effects on birth weight (data not

shown).

49 3.4.9 Gestational age at delivery in CPM16 live births

None of the explanatory variables was significantly associated with gestational age at delivery, except for biased ascertainment (t = 1.92, df = 58, p = 0.030; Figure 3.2), which was associated with lower gestational age. In particular, abnormal ultrasound (t = 2.14, df = 37, p =

0.020; Figure 3.13), but not abnormal serum screen (Welch's approximate t = 1.32, df = 34.6, p

= 0.098; data not shown), was significantly associated with lower gestational age at delivery.

3.4.10 Intrauterine death and neonatal death in CPM16

To clarify the nature of the intrauterine and neonatal deaths, associations were determined between each outcome and birth weight, malformation, trisomy in amniotic fluid, ascertainment, upd(16)mat, sex of the fetus, and gestational age at delivery (neonatal deaths only). Risk of intrauterine death was significantly associated only with lower birth weight (t =

2.67, df = 76, p = 0.005; Figure 3.14). Risk of neonatal death was associated with lower gestational age (t = 3.25, df = 83, p = 0.001; Figure 3.15), presence of malformation (Fisher

Exact test, n = 97, p = 0.001; RR = infinity; Table 3.6), and biased ascertainment (Fisher Exact test, n = 79, p = 0.043; RR = 6.17; Table 3.7). Abnormal ultrasound had a significant association with neonatal death (Fisher Exact test, n = 54, p = 0.030; RR = 8.71; Table 3-8), while abnormal serum screen had a non-significant trend (Fisher Exact test, n = 62, p = 0.18;

RR = 4.44; Table 3-9). Abnormal ultrasound findings included IUGR (n = 1), olighydramnios

(n = 1), left kidney agenesis + probable atrioventricular canal (n = 1), IUGR + dilated cerebral ventricles and single umbilical artery + a small placental haematoma that disappeared 2 weeks later (n = 1).

The lack of association of outcome with trisomy in amniotic fluid, upd(16)mat, or sex of the fetus indicates that these (potentially) antenatal variables cannot predict intrauterine or neonatal death. Figure 3.15 indicates that gestational age is a poor predictor for neonatal death,

50 with the exception of very pre-term deliveries (< 28 weeks). Similarly, for the prediction of neonatal death, presence of malformation has a positive predictive value of only 18%, but a negative predictive value of 100% (Table 3-6); ascertainment by abnormal ultrasound has a positive predictive value of only 24%, but a good negative predictive value of 97% (Table 3.8).

Table 3.10 describes the ascertainment and cardio-pulmonary malformations for each neonatal death, which includes 3 cases with pulmonary hypoplasia (of which 1 was associated with oligohydramnios), 1 case with a rare congenital tracheal narrowing, and 1 case with only one coronary artery that arose from the pulmonary artery trunk. Sample size was insufficient to perform logistic regression modelling or stratification analysis.

3.5 Discussion

3.5.1 Clinical outcome of CPM16

Although this sample is likely biased towards poorer outcome, the majority of prenatally

diagnosed CPM 16 pregnancies resulted in live births with survival beyond the neonatal period.

The distribution of gestational ages for all the live births suggests that CPM 16 pregnancies may be at higher risk for preterm delivery (Figure 3.1). In addition, consistent with the hypothesis,

virtually all birth weights for the live births were below the gestational age-mean (Kramer et al.

2001) (Figure 3.2). This indicates that some level of below-average growth is a nearly universal phenomenon in CPM16. Malformations that are likely to have a true association with CPM16

are VSD, ASD and hypospadias (Table 3.1). As hypothesized, birth weight and malformation

showed associations with most of the predictive factors.

3.5.2 Amniotic fluid trisomy and CPM16 live births

The presence of trisomy in amniotic fluid as assessed by amniocentesis was associated

with lower birth weight (Figure 3.4) and increased risk of malformation (Table 3.2), suggesting

51 that fetal trisomy is an important determinant of outcome. However, trisomy in amniotic fluid was associated with biased ascertainment (namely, by abnormal serum screen), and it was not possible to delineate whether the effect of amniotic fluid trisomy was independent of ascertainment by abnormal serum screen. More abnormal serum screen values may reflect more abnormal placental function (see below).

3.5.3 Ascertainment and CPM16 live births

The association between ascertainment by abnormal serum screen and birth weight suggests that the processes giving rise to increased MShCG and/or MS AFP in CPM 16 (Benn

1998), such as altered secretion, post-translational modification or clearance of the proteins in the placenta (Frendo et al. 2004), may be associated with processes that cause poorer fetal growth (Morssink et al. 1996). Previous studies have demonstrated associations between idiopathic elevated MS AFP and/or MShCG and poor pregnancy outcomes such as IUGR (e.g.

(Chandra et al. 2003; Lepage et al. 2003). However, as noted, it was not possible to determine whether an abnormal serum screen has an effect independent of trisomy in amniotic fluid.

Although ascertainment by abnormal ultrasound was associated with lower gestational age, the difference in magnitude (36.8 vs. 34.8, on average) may not be clinically significant except for the lower bounds of the distributions.

3.5.4 Sex of the fetus and CPM16 live births

The sex ratio in the study sample (0.48) was biased towards females as initially noted in

Benn (1998), and was significantly different from the expected sex ratio calculated from prenatal controls (Huether et al. 1996). It was also different from the sex ratio in trisomy 16 spontaneous abortions (1.00) (Hassold et al. 1983), providing evidence for greater frequency of trisomy rescue in female trisomy 16 embryos or for selection against male CPM 16 embryos post-rescue. This is similar to a report of a significant excess of females in prenatally diagnosed trisomy 21 mosaicism (sex ratio = 0.72) compared to an excess of males in prenatally diagnosed non-mosaic trisomy 21 (Hook et al. 1999). The same phenomenon for prenatally diagnosed trisomy 21 mosaicism was also reported by Huether et al. (1996), who also found a significant excess of females in prenatally diagnosed trisomy 18 mosaicism (sex ratio = 0.52) and a trend towards excess females in prenatally diagnosed trisomy 13 mosaicism (sex ratio = 0.76) compared to prenatal controls. In addition, it is interesting that males had lower birth weights than females, providing more evidence for selection against CPM 16 males.

3.5.5 CPM16 and CVS

It has previously been shown that the clinical outcome of trisomy apparently confined to the placenta was strongly associated with the level of trisomy in the term trophoblast but not with the level of trisomy on CVS (Robinson et al. 1997). However, the data were confounded by the inclusion of a mixture of trisomies involving different chromosomes and different origins. In this study sample, full (100%) trisomy on direct CVS (cytotrophoblast) was found to be associated with low birth weight. Cytotrophoblast function is important for implantation and for differentiation to the hormone secreting syncytiotrophoblast and invasive extravillus cytotrophoblast (see Chapters 1 and 7). Full trisomy on cultured CVS (chorionic villus mesenchyme) was also found to be associated with low birth weight. Given the results in

Chapter II, this is likely due to its association with the level of trisomy in direct CVS, thus reflecting the level of trisomic trophoblast. This is supported by the fact that the p-value for the association between direct CVS and birth weight was more statistically significant than that for cultured CVS: p < 0.001 versus p = 0.035, respectively.

Interestingly, the level of trisomy in direct CVS and cultured CVS was not associated with the presence of trisomy in amniotic fluid. This observation suggests that the effects of

53 trisomy in the placenta (the trophoblast, specifically) on birth weight is independent of trisomy in fetal tissues, which supports the model of placental trisomy causing an alteration in placental function that negatively affects fetal growth (Chapter 2).

3.5.6 Intrauterine and neonatal death in CPM16

As only birth weight was significantly associated with intrauterine death, there were no useful variables identified for its prediction. In contrast, risk of neonatal death was associated

with lower gestational age, higher risk of malformation, and biased ascertainment (in particular,

abnormal ultrasound). Malformation and abnormal ultrasound had poor positive predictive

values (18% and 24%) but good negative predictive values (100% and 97%). Thus, while

normal ultrasounds are reassuring, malformation or other abnormal ultrasound finding does not

predict neonatal death.

3.5.7 Evidence for imprinting on chromosome 16

Maternal uniparental disomy for chromosome 16 (upd(16)mat) had an effect on birth

weight and risk of malformation, independent of the presence of trisomy in the placenta and

fetus as evaluated by CVS and amniocentesis, respectively. It is therefore hypothesized that

upd(16)mat predisposes to growth restriction and malformation as a result of one or more

imprinted gene(s) on chromosome 16. Conceptually, these gene(s) may be imprinted in the

fetus or placenta, or both. In addition, it is conceivable that individual cases of growth

restriction or malformation may be caused by isodisomy, which would result in the fetus being

homozygous for a deleterious recessive mutation on chromosome 16 that is heterozygous in the

mother. This probably does not account for the growth restriction seen in the study, however,

since it is unlikely that deleterious recessive alleles are heterozygous in a sufficient number of

mothers to cause the consistent pattern of growth restriction observed in upd(16)mat cases. It

54 cannot be ruled out that isodisomy accounts for the slightly higher rate of malformation among upd(16)mat cases. However, most cases of upd(16)mat are heterozygous for the majority of chromosome 16 and malformations are observed in some cases even in the absence of any detectable region of isodisomy (W. Robinson, unpublished data).

Currently, there are no known imprinted genes on chromosome 16. While Searle et al.

(1989) noted that an imprinted region of mouse chromosome 11 has orthology with human chromosome 16, the imprinted genes since identified in this region have orthologues on other human chromosomes (Morison and Reeve 1998). Specifically, the orthologues of GrblO/Megl and U2afl-rsl are located on human chromosomes 7pl2 and 21q22.2, respectively. The Mouse

Genome Database of the Jackson Laboratory lists seven genes that are located within 4cM of the imprinted mouse chromosome 11 loci and have orthologues on human chromosome 16 (but are not known to be imprinted): (1) N-methylpurine-DNA glycosylase (Mpg); (2) epidermal growth factor receptor, related sequence (Egfr-rs); (3) hemoglobin a, adult chain 1 (Hba-al); (4) hemoglobin a, adult chain 2 (Hba-a2); (5) hemoglobin X, a-like embryonic chain in Hba complex (Hba-x); (6) proximal locus to the hemoglobin a chain complex (Phg); and (7) a globin regulatory element containing gene (Mare). The human orthologues of murine genes

Mpg, Hba-al, Hba-a2, Hba-x, and Mare have been pinpointed to 16pl3.3, while that of murine

Egfr-rs has been narrowed tol6pter-pl3. The human orthologue of Egfr-rs (Kielman et al.

1993; Kielman et al. 1996) is of particular interest. Egfr-rs is expressed in the preimplantation and early postimplantation mouse embryo, and may be implicated in the development of both the trophectoderm and ICM of the blastocyst (Hendrey et al. 1995). Additional evidence for imprinting at 16pl3.3 comes from Wyszynski and Panhuysen (1999), who studied recurrent alcoholism in families and found paternal effects at two markers located at 16pl3.3 (D16S475 and D16S2622). The region orthologous to human chromosome 16pl3.3 is only 4cM from the

55 imprinted gene U2afl-rsl in the mouse. This clustering may indicate a functional interaction that produces imprinting in the orthologous region. For example, two regions in humans where imprinted genes are known to be clustered and have functional interactions are 1 lpl5 and

15qll-ql3 (Morison and Reeve 1998). Tilghman (1999) reviewed models that may account for the short-range (IGF2 and H19in llpl5) and the long-range (SNRPN and genes up to 1 Mb

away in 15ql l-ql3) interactions between imprinted genes in these clusters. Thus, it appears that

a good candidate region for imprinted gene(s) on chromosome 16 resides in pl3.3.

The results of this study raise the question of whether testing for UPD is indicated after prenatal diagnosis of CPM 16. In this regard it is important to emphasize that although upd(16)mat cases are on average more growth restricted than those with bpd(16), there is

substantial overlap in the distribution of birth weights (Figure 3.11). Moreover, although

upd(16)mat fetuses may be at higher risk for major malformation, most observed malformations

are not life threatening and there is no evidence for higher rates of intrauterine or neonatal death.

Knowledge of UPD status is unlikely to change management of CPM16 pregnancies, and thus

prenatal testing is not recommended.

56 3.6 References

Association of Clinical Cytogeneticists Working Party on Chorionic Villi in Prenatal Diagnosis (1994) Cytogenetic analysis of chorionic villi for prenatal diagnosis: an ACC collaborative study of U.K. data. Prenat Diagn 14:363-379

Benn P (1998) Trisomy 16 and trisomy 16 Mosaicism: a review. Am J Med Genet 79:121-133

Chandra S, Scott H, Dodds L, Watts C, Blight C, Van Den Hof M (2003) Unexplained elevated maternal serum alpha-fetoprotein and/or human chorionic gonadotropin and the risk of adverse outcomes. Am J Obstet Gynecol 189:775-781

Dragani TA, Peissel B, Zanesi N, Aloisi A, Dai Y, Kato M, Suzuki H, Nakashima I (2000) Mapping of melanoma modifier loci in RET transgenic mice. Jpn J Cancer Res 91:1142- 1147

Engel E (1980) A new genetic concept: uniparental disomy and its potential effect, isodisomy. Am J Med Genet 6:137-143

Frendo JL, Guibourdenche J, Pidoux G, Vidaud M, Luton D, Giovangrandi Y, Porquet D, Muller F, Evain-Brion D (2004) Trophoblast production of a weakly bioactive human chorionic gonadotropin in trisomy 21-affected pregnancy. J Clin Endocrinol Metab 89:727-732

Hassold T, Quillen SD, Yamane JA (1983) Sex ratio in spontaneous abortions. Ann Hum Genet 47 Pt 1:39-47

Hassold TJ, Jacobs PA (1984) Trisomy in man. Annu Rev Genet 18:69-97

Hendrey J, Lin D, Dziadek M (1995) Developmental analysis of the Hba(th-J) mouse mutation: effects on mouse peri-implantation development and identification of two candidate genes. Dev Biol 172:253-263

Hook EB, Cross PK, Mutton DE (1999) Female predominance (low sex ratio) in 47,+21 mosaics. Am J Med Genet 84:316-319

Huether CA, Martin RL, Stoppelman SM, D'Souza S, Bishop JK, Torfs CP, Lorey F, May KM, Hanna JS, Baird PA, Kelly JC (1996) Sex ratios in fetuses and liveborn infants with autosomal aneuploidy. Am J Med Genet 63:492-500

Kalousek DK, Barrett I (1994) Confined placental mosaicism and stillbirth. Pediatr Pathol 14:151-159

Karason A, Gudjonsson JE, Upmanyu R, Antonsdottir AA, Hauksson VB, Runasdottir EH, Jonsson HH, Gudbjartsson DF, Frigge ML, Kong A, Stefansson K, Valdimarsson H, Gulcher JR (2003) A susceptibility gene for psoriatic arthritis maps to chromosome 16q: evidence for imprinting. Am J Hum Genet 72:125-131

57 Kielman MF, Smits R, Devi TS, Fodde R, Bernini LF (1993) Homology of a 130-kb region enclosing the alpha-globin gene cluster, the alpha-locus controlling region, and two non- globin genes in human and mouse. Mamm Genome 4:314-323

Kielman MF, Smits R, Hof I, Bernini LF (1996) Characterization and comparison of the human and mouse Distl/alpha-globin complex reveals a tightly packed multiple gene cluster containing differentially expressed transcription units. Genomics 32:341-351

Kotzot D (1999) Abnormal phenotypes in uniparental disomy (UPD): fundamental aspects and a critical review with bibliography of UPD other than 15. Am J Med Genet 82:265-274

Kramer MS, Piatt RW, Wen SW, Joseph KS, Allen A, Abrahamowicz M, Blondel B, Breart G (2001) A new and improved population-based Canadian reference for birth weight for gestational age. Pediatrics 108:E35

Ledbetter DH, Engel E (1995) Uniparental disomy in humans: development of an imprinting map and its implications for prenatal diagnosis. Hum Mol Genet 4 Spec No: 1757-1764

Lepage N, Chitayat D, Kingdom J, Huang T (2003) Association between second-trimester isolated high maternal serum maternal serum human chorionic gonadotropin levels and obstetric complications in singleton and twin pregnancies. Am J Obstet Gynecol 188:1354-1359

Morison EVI, Reeve AE (1998) A catalogue of imprinted genes and parent-of-origin effects in humans and animals. Hum Mol Genet 7:1599-1609

Morssink LP, Sikkema-Raddatz B, Beekhuis JR, De Wolf BT, Mantingh A (1996) Placental mosaicism is associated with unexplained second-trimester elevation of MShCG levels, but not with elevation of MSAFP levels. Prenat Diagn 16:845-851

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard- Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927

Searle AG, Peters J, Lyon MF, Hall JG, Evans EP, Edwards JH, Buckle VJ (1989) Chromosome maps of man and mouse. IV. Ann Hum Genet 53 ( Pt 2):89-140

Spence JE, Perciaccante RG, Greig GM, Willard HF, Ledbetter DH, Hejtmancik JF, Pollack MS, O'Brien WE, Beaudet AL (1988) Uniparental disomy as a mechanism for human genetic disease. Am J Hum Genet 42:217-226

Tilghman SM (1999) The sins of the fathers and mothers: genomic imprinting in mammalian development. Cell 96:185-193

Wolstenholme J (1995) An audit of trisomy 16 in man. Prenat Diagn 15:109-121

58 Wyszynski DF, Panhuysen CI (1999) Parental sex effect in families with alcoholism. Genet Epidemiol 17 Suppl 1:S409-413

59 Table 3.1 Malformations among live births with survival beyond the neonatal period

Malformation % (frequency) in % of neonates in general Significance3 study sample population VSD 12%(llb) 0.448%; 5% [*] p< 0.0001; p = (Hoffman and Kaplan 2002) 0.005 ASD 10% (9b) 0.106% p< 0.0001 (Hoffman and Kaplan 2002) Hypospadias 27% (7C) 0.8% p < 0.0001 (Gallentine et al. 2001) aBinomial test: n = # of informative cases, k = frequency in study sample, p[expected] = % of neonates in general population. bOf 90 live birth cases informative for malformation status. cOf 26 male live birth cases informative for malformation status. See Appendix AI for full references.

Table 3.2 Association between the presence of trisomy in amniotic fluid and malformation

Malformation No Yes Total Risk Relative risk Trisomy in >0% 15 21 36 58% 1.79 amniotic fluid 0% 31 15 46 33%

Presence (>0%) and absence (0%) of trisomy in amniotic fluid as assessed by amniocentesis. Fisher Exact test, n = 82, p = 0.017.

Table 3.3 Association between trisomy in amniotic fluid and trisomy in fetal tissues

Trisomy in fetal tissues No Yes Total Risk Relative risk Trisomy in >0% 21 7 28 25% 6.00 amniotic fluid 0% 23 1 24 4%

Presence (>0%) and absence (0%) of trisomy in amniotic fluid as assessed by amniocentesis. Fisher Exact test, n = 52, p = 0.042.

Table 3.4 Association between upd(16)mat and malformation

Malformation No Yes Total Risk Relative risk upd(16)mat 5 16 21 76% 1.78 bpd(16) 16 12 28 43%

Fisher Exact test, n = 49, p = 0.020.

60 Table 3.5 Associations between explanatory variables associated with birth weight

bpd(16) upd(16)mat Amniotic fluid >0% 11 8 p = 0.61 0% 18 13 Ascertainment Biased 11 10 p = 0.62 Unbiased 10 9 Ascertainment Serum screen 7 4 p = 0.42 Unbiased 10 9 Ascertainment Ultrasound 4 6 p = 0.40 Unbiased 10 9 Sex of fetus M 8 7 p = 0.42 F 22 14 Direct CVS 100% 13 10 p = 0.46 <100% 3 1 Cultured CVS 100% 11 9 p = 0.13 <100% 4 0 Trisomy in amniotic fluid 0% >0% Ascertainment Biased 13 17 p = 0.017 Unbiased 24 9 Ascertainment Serum screen 7 14 p = 0.005 Unbiased 24 9 Ascertainment Ultrasound 6 3 p = 0.51 Unbiased 24 9 Sex of fetus M 12 13 p = 0.15 F 36 21 Direct CVS 100% 22 3 p = 0.47 <100% 3 1 Cultured CVS 100% 27 2 p = 0.39 <100% 4 1 Ascertainment Unbiased Biased Sex of fetus M 11 10 p = 0.53 F 25 25 Direct CVS 100% 9 12 p = 0.098 <100% 5 1 Cultured CVS 100% 18 8 p = 0.45 <100% 5 1 Ascertainment Unbiased Serum screen Sex of fetus M 11 8 p = 0.43 F 25 14 Direct CVS 100% 9 4 p = 0.23 <100% 5 0 Cultured CVS 100% 18 4 p = 0.42 <100% 5 0 Ascertainment Unbiased Ultrasound Sex of fetus M 11 2 p = 0.25 F 25 11 Direct CVS 100% 9 8 p = 0.21 <100% 5 1 Cultured CVS 100% 18 4 p = 0.72 <100% 5 1 Sex of the fetus F M Direct CVS 100% 19 8 p = 0.32 <100% 7 1 Cultured CVS 100% 23 6 p = 0.29 <100% 5 3 Direct CVS <100% 100% Cultured CVS 100% 1 25 p = 0.002

<100% 5 4

Significance values from Fisher Exact test, without correction for multiple comparisons.

Table 3.6 Association between malformation and neonatal death

Neonatal death No Yes Total Risk Relative risk Malformation Yes 37 8 45 18% infinity No 52 0 52 0%

Fisher Exact test, n = 97, p = 0.001.

Table 3.7 Association between biased ascertainment and neonatal death

Neonatal death No Yes Total Risk Relative risk Ascertainment Biased 35 7 42 16.7% 6.17 Unbiased 36 1 37 2.7%

Fisher Exact test, n = 79, p = 0.043.

Table 3.8 Association between ascertainment by abnormal ultrasound and neonatal death.

Neonatal death No Yes Total Risk Relative risk Ascertainment U/S 13 4 17 24% 8.71 Unbiased 36 1 37 3%

"U/S" = ultrasound. Fisher Exact test, n = 97, p = 0.001.

62 Table 3.9 Association between ascertainment by abnormal SS, and neonatal death

Neonatal death No Yes Total Risk Relative risk Ascertainment SS 22 3 25 12% 4.44 Unbiased 36 1 37 3%

"SS" = serum screen. Fisher Exact test, n = 62, p = 0.18.

63 Table 3.10 Ascertainment and cardio-pulmonary malformations in neonatal deaths

Case GA Ascertainment Cardio-pulmonary malformations

Hsu et al. (1998) 29wks High MSAFP coarctation of aorta, VSD case 3 95.28 38wks AMA partial anomalous pulmonary venous return, large ASD, congenital tracheal narrowing Devi et al. (1992; 33wks High MSAFP pulmonary hypoplasia 1993) (5.25 MoM) Garber et al. 36wks High MShCG muscular VSD, large ASD, single coronary (1994) case 2; artery that arose form pulmonary artery trunk Hsu et al. (1998) case 2 Sanchez et al. 25wks 23wks: IUGR, SUA, no malformation (1997) dilated cerebral ventricles, placental haematoma

24wks: IUGR, SUA, hypokinesia, enlarged cisterna magna, dilation of both cerebral laternal venticles, hypoplasia of corpus callosum Abu-Amero et 28wks High MShCG no malformation; al. (1999) case 2; (14-17 MoM) persistent hyaline membrane formation Vaughan et al. (1994) case 2; 17wks (following Moore et al. abnormal screen): (1997) case 16 IUGR, SUA

Repeat scans at 20, 23, 25 wks showed appropriate growth

28wks: no growth for 3 wks Watson et al. 35wks AMA pulmonary hypoplasia (1988) Oligohydramnios Referred from M 32-33 26wks: presented with DORV, posterior subpulmonic VSD, PDA, wks vaginal bleeding and hypoplastic left ventricle, tubular hypoplasia lower abdominal of proximal aorta ending in a preductal cramping; ultrasound coarctation, aberrant right subclavian artery, showed agenesis of left retroesophageal vascular ring, hypoplastic kidney and probable left pulmonary artery, ASD secundum, atrioventricular canal persistent left superior vena cava to coronary sinus, right ventricular hypertrophy, anterior descending artery arising from right coronary total anomalous pulmonary venous return to ductus venosus, pulmonary hypoplasia with left sided diaphragmatic hernia

"AMA" = advanced maternal age; "MSAFP" = maternal serum alpha-fetoprotein; "MShCG' maternal serum human chorionic gonadotropin; "SUA" = single umbilical artery. Figure 3.1 Distribution of gestational ages for CPM16 live births

12-

81 Count

30 32 34 36 38 40 42

Gestational age (weeks) Figure 3.2 Distribution of birth weights for CPM16 resulting in live births

Birth weight Figure 3.3 Distribution of the level of trisomy in amniotic fluid among CPM16 live births

Count

r 20 30 40 50 60 70 80

% trisomy in amniotic fluid

Excluding 49 cases with 0% trisomy and 8 cases positive for trisomy but for which the percentage was not known).

67 Figure 3.4 Mean birth weight in the presence or absence of trisomy in amniotic fluid

-1.00'

BW •2.00 -!

-3.00'

0% >0%

Level of trisomy on amniocentesis

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.02 and 1.00, respectively. The sample sizes were 41 and 23, respectively, t = 3.39, df = 62, p = 0.0005.

68 Figure 3.5 Mean birth weight for unbiased and biased ascertainment.

-1.0CH

TP-45

BW

-2.00- #•22

-3.00"

Unbiased Biased

Ascertainment

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.30 and 0.59, respectively. The sample sizes were 27 and 30, respectively. Welch's approximate t = 2.85, df = 35.4, p = 0.0035.

69 Figure 3.6 Mean birth weight for ascertainment: unbiased vs. abnormal serum screen

-1.001

BW

-2.00"

-3.00-

Unbiased Serum screen

Ascertainment

Mean birth weight (BW) ± 1 standard deviation. The standard deviations were 1.30 and 0.72, respectively. The sample sizes were 27 and 18, respectively. Welch's approximate t = 2.63, df = 41.9, p = 0.006.

70 Figure 3.7 Mean birth weight for ascertainment: unbiased vs. abnormal ultrasound

-1.00H

BW

-2.00-

-3.00"

Unbiased Ultrasound

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.30 and 0.32, respectively. The sample sizes were 27 and 12, respectively. Welch's approximate t = 2.80, df = 32.0, p = 0.005.

71 Figure 3.8 Mean birth weight for females and males

-1.00"

BW -2.00"

-3.00-

female male

Mean birth weight (BW) ± standard deviation. The standard deviations were 0.99 and 1.15, respectively. The sample were 48 and 24, respectively, t = 2.35, df = 70, p = 0.011.

72 Figure 3.9 Mean birth weight for <100% and 100% trisomy on direct CVS

1.00H

0.00 H

BW -1.00H

-2.00-

<100% trisomy 100% trisomy

Direct CVS

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.05 and 0.86, respectively. The sample sizes were 3 and 26, respectively, t = 4.08, df = 27, p < 0.001.

73 Figure 3.10 Mean birth weight for <100% and 100% trisomy on cultured CVS

0.00 H

-1.00-i BW

-2.00-1

<100% trisomy 100% trisomy

Cultured CVS

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.24 and 1.02, respectively. The sample sizes were 4 and 25, respectively, t = 1.89, df = 27, p = 0.035.

74 Figure 3.11 Mean birth weight for bpd(16) and upd(16)mat

-1.001

BW

-2.00'

-3.00'

bpd(16) upd(16)mat

Mean birth weight (BW) ± standard deviation. The standard deviations were 1.08 and 1.04, respectively. The sample sizes were 25 and 19, respectively, t = 2.10, df = 42, p = 0.021.

75 Figure 3.12 Mean gestational age for unbiased and biased ascertainment

40.0 H

38.0'

Weeks 36.0 •

34.0-1

32.0'

Unbiased Biased

Ascertainment

Mean gestational age ± standard deviation. The standard deviations were 2.8 and 3.4, respectively. The sample sizes were 27 and 33, respectively, t = 1.92, df = 58, p = 0.030.

76 Figure 3.13 Mean gestational age ascertainment: unbiased vs. abnormal ultrasound

38.0'

36.0' Weeks

34.01

32.0 •

Unbiased Ultrasound

Ascertainment

Mean gestational age ± standard deviation. The standard deviations were 2.8 and 2.1, respectively. The sample sizes were 27 and 12, respectively, t = 2.14, df = 37, p = 0.020. Figure 3.14 Birth weights for live births and intrauterine deaths

1.00H

o.oo "i

-1.00-

BW

-2.00 H

-3.00-

-4.00 H

Live birth Intrauterine death

The mean birth weights (BW) ± standard deviation were -1.76±1.08 and -2.99±1.05, respectively. The sample sizes were 72 and 6, respectively, t = 2.67, df = 76, p = 0.005. Figure 3.15 Gestational ages for live births and neonatal deaths

Weeks

Live birth Neonatal death

The mean gestational ages ± standard deviation were 36.0±3.2 and 32.0±4.4). The sample sizes were 77 and 8, respectively, t = 3.25, df = 83, p = 0.001.

79 4 Preeclampsia and CPM167

4.1 Note

I wrote this chapter/manuscript and did the data organization and analysis, with the following clarifications and exceptions. The CPM 16 cases in this study are from the same ongoing UBC study as previously described. For those CPM 16 cases ascertained locally, I reviewed Medical Genetics and BC Women's Hospital medical records for additional maternal clinical data under the supervision of Dr. S. Langlois (Medical Genetics) and Dr. P. von

Dadelszen (Obstetrics and Gynaecology). As well, I collected matched controls from BC

Women's Hospital medical records under the supervision of Dr. P. von Dadelszen. For referred

CPM 16 cases, maternal clinical data were collected by Dr. D. Kalousek and Dr. W. Robinson.

4.2 Introduction

Preeclampsia is a maternal syndrome during pregnancy characterized by hypertension, decreased systemic organ perfusion related to vasoconstriction due to increased sensitivity to vasopressors, loss of endothelial integrity resulting in fluid leak, and intravascular coagulation

(Roberts and Lain 2002). It is the number one cause of maternal mortality in developed countries and increases perinatal mortality five-fold (Roberts and Lain 2002). The only 'cure' is delivery of the placenta, indicating this maternal syndrome has a placental origin. The incidence of preeclampsia is approximately 3-5% (Roberts and Cooper 2001), depending on the definition and the population. There are numerous risk factors for preeclampsia, including nulliparity, advanced maternal age, previous pregnancy with preeclampsia, family history of preeclampsia, and increased placental mass/demand (e.g. hydatidiform mole and multi-fetal pregnancy)

(Dekker 1999). Preeclampsia has been classically associated with abnormal placentation; in

7 A version of this chapter will be submitted for publication. Yong PJ, von Dadelszen P, Langlois S, Barrett IJ, Kalousek DK, Robinson WP. Preeclampsia and confined placental mosaicism for trisomy 16.

80 particular, poor invasion and remodeling of the uterine spiral arteries by the extravillus trophoblast (EVT) (see Chapter 7). However, similar poor invasion and remodeling have been seen in intrauterine growth restriction (IUGR) without preeclampsia (Khong et al. 1986), placental abruption (Dommisse and Tiltman 1992), and even in pre-term premature rupture of membranes (pPROM) (Kim et al. 2002) and in pre-term labour with intact membranes (Kim et al. 2003). Thus, poor placentation does not appear to be sufficient to cause preeclampsia. There are likely other maternal and feto-placental factors that contribute to the risk of developing the syndrome. It may well be that some cases of preeclampsia have a primarily maternal cause, other cases a primarily feto-placental cause, and still others a combination of maternal and feto• placental causes (Cross 2003). Animal models have shown that a purely maternal and a purely feto-placental initiating event can cause preeclampsia, suggesting that the human syndrome may consist of distinct pathogenic subtypes (Cross 2003).

In 1987, a higher risk of preeclampsia was reported in a series of trisomy 13 pregnancies compared to trisomy 18, trisomy 21 and control pregnancies (Boyd et al. 1987). Subsequently, there have been a number of reports of preeclampsia in trisomy 13 pregnancies (Feinberg et al.

1991; Boyd et al. 1995; Heydanus et al. 1995), a series of trisomy 13 pregnancies showing the

same association (Bower et al. 1987), and a large case-control study that confirmed the findings of Boyd and colleagues (Tuohy and James 1992). Estimates for the incidence of preeclampsia in trisomy 13 pregnancies (that do not miscarry or are not terminated) range from 22% (Bower et al. 1987) to 44% (Tuohy and James 1992). Boyd et al. (1987) found no association between preeclampsia and trisomies 18 and 21, and Tuohy and James (1992) found no association with trisomy 18. A decreased risk of preeclampsia has been found in trisomy 21 (Zhang et al. 2004).

These findings suggest a chromosome 13-specific effect in preeclampsia.

I hypothesized that CPM 16 would be significantly associated with a higher risk of preeclampsia, and that the risk would correlate with UPD and placental trisomy. Thus, CPM 16

81 cases were reviewed to determine the frequency of preeclampsia, and to evaluate factors contributing to the risk of preeclampsia. This will improve genetic counseling after prenatal diagnosis of CPM of trisomy 16 (CPM 16) and add insight into the clinical significance of trisomy 16 confined to the placenta.

4.3 Methods

4.3.1 CPM16 cases

CPM 16 cases from the ongoing study of trisomy mosaicism at the University of British

Columbia (UBC), involving cases ascertained locally and referred from other centres, were

included in this study if there was (1) prenatal diagnosis of trisomy 16 via CVS or

amniocentesis; (2) continuation of the pregnancy past 20 weeks gestation (because preeclampsia

rarely occurs before then); (3) sufficient clinical data to include or exclude a diagnosis of

preeclampsia; and (4) the availability of DNA for molecular determination of UPD status of

chromosome 16 in the fetus (i.e. bpd(16) or upd(16)mat). Exclusion criteria were confirmed

paternal origin of the trisomy 16 or concomitant aneuploidy or polyploidy in addition to trisomy

16, since such cases are rare and would confound the analysis. The study was approved by the

ethics committees of UBC and the Children's and Women's Health Centre of British Columbia

(Appendix A). As research into CPM has generally focused on fetal outcomes, clinical data for

preeclampsia varied between cases. For example, in the local cases blood pressure levels and

other pertinent findings were available from medical records to independently make or exclude

a diagnosis of preeclampsia. In referred cases, data were generally provided by the referring

party completing research study forms. These forms asked the referrer to list any pregnancy

complications, or to indicate 'yes' or 'no' to a list of pregnancy complications such as

hypertension, and then to elaborate on clinical details. For the subset of cases where the post-

82 partum placenta was available, the level of trisomy in the chorionic plate, chorionic villus mesenchyme, and trophoblast was determined as described in Chapter 2.

4.3.2 Matched controls

For each CPM 16 case, 2 matched controls were ascertained from British Columbia's

Women's Hospital (BCWH) delivery records according to the following criteria: same date of delivery or closest consecutive or previous date of delivery, matched for maternal age (± 5 years) and parity (0, 1 or >2). Gestational age at delivery and sex of the infant were also noted.

Then, the corresponding BCWH medical records for each control were reviewed for data regarding blood pressure and other clinical features of preeclampsia.

4.3.3 Definition of preeclampsia

Preeclampsia was defined by the criteria for clinical diagnosis in the 2000 guidelines of the Australasian Society for the Study of Hypertension in Pregnancy (Brown et al. 2000b;

Brown et al. 2000a). Briefly, a diagnosis of preeclampsia was made in the presence of hypertension (>140 mm Hg systolic or >90 mm Hg diastolic), plus one additional feature of proteinuria, low platelets or elevated liver enzymes, but not edema or IUGR. The Australasian guidelines do include IUGR, but it is not useful as a criterion in CPM 16 pregnancies which tend to be growth restricted (Yong et al. 2003).

4.3.4 Statistical analysis

Statistical analyses were performed using SPSS 10.0 and the VassarStats Web Site for

Statistical Computation (http://faculty.vassar.edu/lowry/VassarStats.html). Unless otherwise noted, p-values are 1-tailed due to a priori evidence or rational mechanisms.

83 4.4 Results

Nineteen CPM 16 cases met the study criteria (Tables 4.1, 4.2,4.3). Twenty-six percent

(5/19) of the CPM16 pregnancies were preeclamptic (Table 4.3), while 5% (2/38) of matched controls were preeclamptic (Fisher Exact test, p = 0.035; RR = 5.00). Table 4.4 compares possible confounders between the CPM16 cases and the matched controls: parity, maternal age, gestational age at delivery, and sex of the infant. Only gestational age was significantly different because pre-term delivery is common in CPM16 pregnancies. However, this factor would result in a difference that is conservative because the longer gestational ages among controls should allow near-term preeclampsia to develop, while CPM 16 pregnancies may be delivered for other indications (e.g. IUGR, fetal distress) before clinical features of preeclampsia appear.

The CPM 16 cases were tested for associations between preeclampsia and UPD status of chromosome 16 or level of trisomy in placental lineages post-partum (Table 4.3). The frequency of upd(16)mat was similar among preeclamptic and non-preeclamptic cases 60%

(3/5) and 54% (7/13), respectively. The preeclamptic cases all had trends towards higher levels of trisomic cells in the three placental lineages compared to non-preeclamptic cases, although sample size was small and none reached statistical significance: chorionic plate (mean = 98.3% vs. 51.8%; Welch's approximate t = 1.89, df = 3.0, p = 0.08), villus mesenchyme (73.0% vs.

33.3%; t = 1.01, df = 5, p = 0.18), and trophoblast (75.7% vs. 63.4%; t = 0.50, df = 6, p = 0.32).

Table 4.3 shows that higher levels of trisomy across the placental lineages were seen in both the preeclamptic CPM16 cases (with the exception of the villus mesenchyme of 92.95) and in some non-preeclamptic CPM16 cases (e.g. 91.71 and 92.25), while lower levels of trisomy across the lineages were only seen in non-preeclamptic cases (e.g. 91.55 and 91.10).

The CPM16 cases with preeclampsia had a wide range of clinical findings (Table 4.5).

Three of the 4 preeclamptic male CPM 16 cases had hypospadias, in contrast to 1 of the 3 non-

84 preeclamptic male CPM16 cases. Among the other male CPM16 live births in the ongoing

UBC study of trisomy mosaicism,' which did not meet this study's criteria, 18% (3/19) had hypospadias (Yong et al. 2003). The frequency of hypospadias in the preeclamptic male

CPM 16 cases (3/4) was significantly increased when compared to all the other male CPM 16 cases in the UBC study (4/22) (p = 0.047, RR = 4.13).

4.5 Discussion

As hypothesized, the risk of preeclampsia in CPM 16 pregnancies (26%) was increased

compared to controls and should be considered during management of pregnancies after prenatal

diagnosis of CPM 16. This association indicates a chromosome 16-specific effect on risk of

preeclampsia, in addition to the known chromosome 13-specific effect. One possible cause of

the association between CPM 16 and preeclampsia is an increase in placental size, thereby

increasing the inflammatory load. However, CPM 16 placentas were smaller when compared to

a reference population (Chapter 2). Boyd et al. (1987) also excluded placental weight as an

etiological factor in trisomy 13.

Consistent with the hypothesis, preeclamptic CPM16 cases showed a trend towards

higher levels of trisomic cells in the placenta (chorionic plate, villus mesenchyme and

trophoblast). Specifically, both the preeclamptic CPM 16 cases and some non-preeclamptic

CPM 16 cases had higher levels of trisomy in the three placental lineages, while low levels of

trisomy were seen only in the non-preeclamptic cases. Therefore, placental trisomy could

predispose to preeclampsia, but requires other maternal-fetal factors for development of the full

syndrome. A possible mechanism for this predisposition to preeclampsia in CPM 16

pregnancies with high levels of placental trisomy is abnormal extravillus trophoblast (EVT)

growth and function. EVT outgrowth may be decreased in trisomy 16 (Chapter 7); however,

EVT defects have also been identified in trisomy 21 (Wright et al. 2004), which is associated

85 with reduced risk of preeclampsia (Zhang et al. 2004). It should be noted that the risk of preeclampsia was not increased in upd(16)mat, suggesting that no imprinted genes on chromosome 16 are related to preeclampsia in CPM 16 pregnancies.

Three of the 4 male CPM 16 cases with preeclampsia had hypospadias. Since hypospadias has consistently been found in male survivors of homozygous oc-thalassemia

(Abuelo et al. 1997; Dame et al. 1999; Fung et al. 1999) it has been proposed that a gene associated with hypospadias exists on 16pl3.3 that is affected by the nearby oc-globin gene deletions (Dame et al. 1999). The expression of this putative gene may be abnormal in trisomy

16, predisposing to hypospadias. Alternatively, homozygous a-thalassemia survivors may have hypospadias because of hypoxia secondary to the high affinity of Bart's hemoglobin (Fung et al.

1999). Preeclampsia is associated with utero-placental hypoxia (Roberts and Lain 2002), which could thus also predispose to hypospadias. In a recent study of 8,894 cases in a malformation database, an association between gestational hypertension (including preeclampsia) and presence of malformation was found, which on multivariate analysis proved to be due specifically to hypospadias, other anomalies of the penis, and 'multiple congenital abnormalities' (syndromes affecting the face, limbs, and stature) (Vesce et al. 1997).

In conclusion, CPM 16 is at higher-risk for preeclampsia. Future studies should assess the incidence of CPM 16 in a series of idiopathic preeclamptic pregnancies. In particular, trisomy CPM may be implicated in preeclampsia that is pre-term, multi-systemic in presentation, and/or associated with hypospadias.

86 4.6 References

Abuelo DN, Forman EN, Rubin LP (1997) Limb defects and congenital anomalies of the genitalia in an infant with homozygous alpha-thalassemia. Am J Med Genet 68:158-161

Bower C, Stanley F, Walters BN (1987) Pre-eclampsia and trisomy 13. Lancet 2:1032

Boyd PA, Lindenbaum RH, Redman C (1987) Pre-eclampsia and trisomy 13: a possible association. Lancet 2:425-427

Boyd PA, Maher EJ, Lindenbaum RH, Hoogwerf AM, Redman C, Crocker M (1995) Maternal 3; 13 chromosome insertion, with severe pre-eclampsia. Clin Genet 47:17-21

Brown MA, Hague WM, Higgins J, Lowe S, McCowan L, Oats J, Peek MJ, Rowan JA, Walters BN (2000a) The detection, investigation and management of hypertension in pregnancy: executive summary. Aust N Z J Obstet Gynaecol 40:133-138

Brown MA, Hague WM, Higgins J, Lowe S, McCowan L, Oats J, Peek MJ, Rowan JA, Walters BN (2000b) The detection, investigation and management of hypertension in pregnancy: full consensus statement. Aust N Z J Obstet Gynaecol 40:139-155

Cross JC (2003) The genetics of pre-eclampsia: a feto-placental or maternal problem? Clin Genet 64:96-103

Dame C, Albers N, Hasan C, Bode U, Eigel A, Hansmann M, Brenner R, Bartmann P (1999) Homozygous alpha-thalassaemia and hypospadias—common aetiology or incidental association? Long-term survival of Hb Bart's hydrops syndrome leads to new aspects for counselling of alpha-thalassaemic traits. Eur J Pediatr 158:217-220

Dekker GA (1999) Risk factors for preeclampsia. Clin Obstet Gynecol 42:422-435

Dornmisse J, Tiltman AJ (1992) Placental bed biopsies in placental abruption. Br J Obstet Gynaecol 99:651-654

Feinberg RF, Kliman HJ, Cohen AW (1991) Preeclampsia, trisomy 13, and the placental bed. Obstet Gynecol 78:505-508

Fung TY, Kin LT, Kong LC, Keung LC (1999) Homozygous alpha-thalassemia associated with hypospadias in three survivors. Am J Med Genet 82:225-227

Heydanus R, Defoort P, Dhont M (1995) Pre-eclampsia and trisomy 13. Eur J Obstet Gynecol Reprod Biol 60:201-202

Khong TY, De Wolf F, Robertson WB, Brosens I (1986) Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for- gestational age infants. Br J Obstet Gynaecol 93:1049-1059

87 Kim YM, Bujold E, Chaiworapongsa T, Gomez R, Yoon BH, Thaler HT, Rotmensch S, Romero R (2003) Failure of physiologic transformation of the spiral arteries in patients with preterm labor and intact membranes. Am J Obstet Gynecol 189:1063-1069

Kim YM, Chaiworapongsa T, Gomez R, Bujold E, Yoon BH, Rotmensch S, Thaler HT, Romero R (2002) Failure of physiologic transformation of the spiral arteries in the placental bed in preterm premature rupture of membranes. Am J Obstet Gynecol 187:1137-1142

Roberts JM, Cooper DW (2001) Pathogenesis and genetics of pre-eclampsia. Lancet 357:53-56

Roberts JM, Lain KY (2002) Recent Insights into the pathogenesis of pre-eclampsia. Placenta 23:359-372 ^

Tuohy JF, James DK (1992) Pre-eclampsia and trisomy 13. Br J Obstet Gynaecol 99:891-894

Vesce F, Farina A, Giorgetti M, Jorizzo G, Bianciotto A, Calabrese O, Mollica G (1997) Increased incidence of preeclampsia in pregnancies complicated by fetal malformation. Gynecol Obstet Invest 44:107-111

Wright A, Zhou Y, Weier JF, Caceres E, Kapidzic M, Tabata T, Kahn M, Nash C, Fisher SJ (2004) Trisomy 21 is associated with variable defects in cytotrophoblast differentiation along the invasive pathway. Am J Med Genet A 130:354-364

Yong PJ, Barrett IJ, Kalousek DK, Robinson WP (2003) Clinical aspects, prenatal diagnosis, and pathogenesis of trisomy 16 mosaicism. J Med Genet 40:175-182

Zhang J, Christianson RE, Torfs CP (2004) Fetal trisomy 21 and maternal preeclampsia. Epidemiology 15:195-201

88 Table 4.1 CPM16 cases meeting inclusion criteria

PE Case Source References Origin Y 91.14 Local Kalousek et al. (1993) case 2 Kalousek et al. (1993) Robinson et al. (1997) Unpublished Penaherrera et al. (2000) Y 92.48 Local Kalousek et al. (1993) case 8 Unpublished Robinson et al. (1997) Y 93.94 Referred Schneider et al. (1996) Schneider et al. (1996) Robinson et al. (1997) Y 94.116 Referred Robinson et al. (1997) Unpublished Y 92.95 Local Robinson et al. (1997) Unpublished Hsu et al. (1997) case XIV-11 N 91.55 Referred Kalousek et al. (1993) case 7 Unpublished Robinson etal. (1997) Penaherrera et al. (2000) N 91.71 Referred Kalousek et al. (1993) case 3 Unpublished Johnson et al. (1993) case C Robinson et al. (1997) Penaherrera et al. (2000) N 92.25 Referred Kalousek et al. (1993) case 4 Unpublished Robinson et al. (1997) N 93.103 Referred Schwinger et al. (1989) case 5 Unpublished Wolstenholme (1995) Robinson et al. (1997) N 93.73 Referred Verpet al. (1989) Unpublished Penaherrera et al. (2000) N 93.93 Referred Kennerknecht and Terinde (1990) Unpublished N 95.28 Referred Robinson et al. (1997) Unpublished Penaherrera et al. (2000) N 98.234 Referred Penaherrera et al. (2000) Unpublished N 99.143 Local Penaherrera et al. (2000) Unpublished N 91.10 Local Kalousek et al. (1993) case 6 Unpublished N 93.132 Referred Woo et al. (1997) Woo et al. (1997) N 16-51 Referred Unpublished Unpublished N 16-55 Referred Unpublished Unpublished N 16-54 Referred Unpublished Unpublished

"PE" = preeclampsia. "References" = some data previously published in these references. "Origin" = origin of data regarding preeclampsia. Full references in Appendix AI. Table 4.2 CPM16 cases meeting inclusion criteria

PE Case Paritv Mat age Outcome Sex GA

Y 91.14 1 27 TA F 25 Y 92.48 0 40 LB M 36 Y 93.94 1 34 LB M 39 Y 94.116 0 41 LB M 33 Y 92.95 1 36 LB M 36 N 91.55 1 35 LB F 40 N 91.71 1 37 LB F 38 N 92.25 1 34 LB M 37 N 93.103 3 39 LB M 37 N 93.73 1 35 LB F 37 N 93.93 0 21 LB M 37 N 95.28 1 41 ND F 38 N 98.234 0 34 LB F 38 N 99.143 1 29 TA F 22 N 91.10 0 44 LB F 36 N 93.132 1 42 LB F 33 N 16-51 1 36 LB F 35 N 16-55 0 31 LB F 34 N 16-54 0 33 LB F 37

"PE" = preeclampsia. "TA" = termination of pregnancy; "LB" = live birth with survival beyond the neonatal period; "ND" = neonatal death. "GA" = gestational age in weeks.

90 Table 4.3. Post-partum placenta cytogenetics

Post-partum placenta cytogenetics PE Case BW Malf UPD Chorion Mesenchyme Trophoblast Y 91.14 410g N UPD 100% 100% - Y 92.48 2280g Y BPD 100% 70% Y 93.94 1935g Y UPD - - - Y 94.116 1498g N BPD 100% 88% 61% Y 92.95 1559g Y UPD 95% 4% 96% N 91.55 3319g N BPD 0% 0% 0% N 91.71 1960g N BPD 87% 100% 100% N 92.25 1650g Y UPD 100% - 85% N 93.103 2750g N UPD - - - N 93.73 2315g N BPD - - - N 93.93 2210g Y BPD - - - N 95.28 2000g Y UPD - - - N 98.234 2578g Y BPD - - 54% N 99.143 ? Y BPD - - 78% N 91.10 2660g N BPD 20% 0% N 93.132 1620g Y UPD - - - N 16-51 1530g Y UPD - - - N 16-55 1278g Y UPD - - - N 16-54 ? Y UPD - - -

"BW" = birth weight. "Malf = malformation. "UPD" = uniparental disomy for chromosome 16 (upd(16)mat); "BPD" = biparental disomy for chromosome 16 (bpd(16)). Percentages are averaged over sites sampled. '?' = not available.

91 Table 4.4 Clinical features of the CPM16 cases and controls

CPM (n = 19) Controls (n = 38) Sig

Preeclampsia 26% (5/19) 5% (2/38) p = 0.035 Parity 0.74 + 0.74 0.71 ±0.65 n.s. Maternal age 35.2 ± 5.6 yrs 33.9 ±5.1 yrs n.s. Gestational age 35.2 ±4.6 wks 39.8 ± 1.3 wks p < 0.001 Sex ratio 0.58 (7 M; 12 F) 0.65 (15 M; 23F) n.s.

Means ± standard deviations are provided for parity, maternal age, and gestational age. Statistics: Fisher Exact test (pre-eclampsia, sex ratio), 2-sample t-test (maternal age), Welch's approximate t-test (gestational age), Mann-Whitney test (parity).

Table 4.5 Clinical features of the CPM16 cases with preeclampsia

CPM16 case Clinical features of preeclampsia 91.14 Severe hypertension and proteinuria at approximately 21 weeks gestation. BP ranged to 180/130 with +4 to +5 proteinuria, but only mild headaches, no hyperreflexia and mild edema. Hypertension did not respond to antihypertensive therapy and decision made to terminate pregnancy at 25 weeks. Eumorphic female fetus. 92.48 Uncomplicated until 35 weeks, except for peripheral edema 2 months prior. Blood pressure ranged from to 150/100, with low platelets and elevated liver enzymes, but no proteinuria. Male fetus delivered at 36 weeks gestation with hypospadias 93.94 Intermittent hypertension, proteinuria and edema from 35 to 39 weeks. Male fetus delivered at 39 weeks with hypospadias, left hydronephrosis, and 5th finger clinodactyly. 94.116 Severe preeclampsia and HELLP syndrome. Date of onset not provided. Eumorphic male fetus delivered at 33 weeks. 92.95 'Toxemia' and low platelets. Date of onset not provided. Male fetus delivered at 36 weeks with hypospadias. 5 Postnatal follow-up of newborns from CPM16 pregnancies1

5.1 Note

I wrote this chapter/manuscript and did the data organization and analysis, with the following clarifications and exceptions. These CPM 16 cases are from the ongoing UBC study of trisomy mosaicism described previously. Long-term follow-up data have been collected by

Dr. D. Kalousek, Dr. S. Langlois, and Dr. W. Robinson.

5.2 Introduction

Genetic counselling after prenatal diagnosis of CPM is difficult in part because there has been no systematic study of long-term outcome of neonates from such pregnancies. I hypothesized that neonates from CPM 16 pregnancies will demonstrate good catch-up growth and developmental outcome, since the trisomy is completely or predominantly confined to the placenta. In this Chapter, the postnatal growth and development of CPM 16 cases at least one year after birth were described, and risk factors for abnormal growth or development were identified.

5.3 Methods

5.3.1 CPM16 cases

There were 27 cases of follow-up after prenatal diagnosis of CPM 16. Table 5.1 provides the origin of the postnatal data, either from published studies from other centres, published studies from our centre, and/or unpublished data from the ongoing UBC study of trisomy mosaicism. The inclusion criterion was prenatal diagnosis of trisomy 16 at CVS or

8 A version of this chapter has been accepted for publication. Langlois S, Yong PJ, Yong S-L, Barrett IJ, Kalousek DK, Miny P, Exeler R, Morris K, Robinson WP (2006) Postnatal follow-up of prenatally diagnosed trisomy 16 mosaicism. Prenat Diagn.

93 amniocentesis, and the presence of postnatal follow-up data at one year of age or more for length/height, weight, and/or developmental outcome. The study was approved by the ethics committees of UBC and C&W (Appendix A).

To qualify for analysis of catch-up growth in height or weight, the birth length or weight

had to be small-for-gestational age (SGA). SGA has several definitions; the definition of the

International SGA Advisory Board was chosen, which is less than 2 standard deviations below

the gestational age-corrected mean (~3rd percentile) (Lee et al. 2003). Otherwise, the case was

denoted as 'not-applicable' (N/A). Catch-up growth in length or weight was defined as a length

or weight above 2 standard deviations below the mean at some point of postnatal life. Standard

deviations from the mean for length and weight at birth were calculated manually by using

gestational age-specific means and standard deviations (Usher and McLean 1969). Standard

deviations from the mean for postnatal length and weight were determined using the 2000

growth charts from the Centers for Disease Control (CDC), which represent United States

surveys from 1963 to 1994 (the most recent being the joint National Center for Health Statistics

(NCHS) and CDC Third National Health and Nutrition Examination Survey (NHANES ni))

(Ogden et al. 2002). Standard deviations were calculated using the CDC Epi Info software

program, available for download at http://www.cdc.gov/epiinfo, which can calculate percentiles

and z-scores (standard deviations from the mean) using the 2000 CDC growth charts. When

doing so, corrections in postnatal age were made for the gestational age at delivery. In a few

cases, only percentiles (and not raw data for height or weight) were provided by the referring

source.

For developmental outcome, postnatal age was corrected for gestational age at delivery.

Most cases had an explicit statement from the referring source regarding 'normal' versus

'delayed' development. In one case (case 8), no explicit statement was made regarding speech

development, but delay was assumed because the child was undergoing speech therapy. For a

94 few cases no diagnostic statement was available regarding 'normal' or 'delayed' development, although the age at which the child reached certain milestones was provided. These children were evaluated using the Denver II Developmental Assessment.

The following additional data were collected on the 27 cases (Table 5.2): 1) % trisomic cells in amniotic fluid at amniocentesis; 2) uniparental disomy (UPD); 3) malformations; 4) gestational age at delivery; 5) sex of the infant; and 6) ascertainment ('biased' = abnormal ultrasound or serum screen; 'unbiased' = advanced maternal age (AMA), investigation of biochemical or Mendelian disorders, history of aneuploidy pregnancy, etc.).

5.3.2 Statistical analysis

Statistical analyses were performed using SPSS 10.0 and the VassarStats Web Site for

Statistical Computation (http://faculty.vassar.edu/lowry/VassarStats.html). Unless otherwise noted, p-values are 1-tailed due to a priori evidence or rational mechanisms.

5.4 Results

Table 5.2 shows the percent trisomy at amniocentesis, UPD status, malformations, gestational age at delivery, sex of the infant, and ascertainment. Table 5.3 describes the follow- up data for growth in length/height and weight, and for developmental outcome.

5.4.1 Height

There were 17 cases informative for height (Table 5.2). Ten of the cases (59%) had birth lengths above -2 standard deviations (SD) from the mean, and therefore were not applicable to assessment of catch-up growth. Seven cases (41%) had birth lengths below -2 SD, of which all

(100%) demonstrated catch-up growth in height.

95 5.4.2. Weight

There were 18 cases informative for weight (Table 5.2). Nine of the cases (50%) had birth weights above -2 SD, and therefore were not applicable to assessment of catch-up growth.

Nine cases (50%) had birth lengths below -2 SD, of which 7/9 (78%) demonstrated catch-up growth in weight. The 2 cases without catch-up growth were cases 13 and 16. It should be noted that case 13 had congenital hypothyroidism which may account for the lack of catch-up growth in weight, although the child did exhibit catch-up growth in height and normal development, suggesting adequate treatment.

5.4.3 Development

All 27 cases were informative for developmental outcome, of which 21/27 (78%) had normal development. The 6 cases with evidence of developmental delay were cases 1, 3, 8, 12,

15, and 17. The presence of evidence of developmental delay was significantly associated with trisomy (>0%) in amniotic fluid by amniocentesis (Fisher Exact test, p = 0.038; Table 5.4). The risk of developmental delay when trisomy was present in amniotic fluid was 36% (5/14) and 0%

(0/11) when trisomy was absent in amniotic fluid. Risk of developmental delay was significantly associated with presence of malformation (Fisher Exact test, p = 0.035; Table 5.5).

The risk of developmental delay in the presence of malformation was 38% (6/16), and 0%

(0/10) in the absence of malformation.

Risk of developmental delay was significantly associated with lower birth weight (t =

1.76, df = 21, p = 0.047; Figure 5.1). The mean birth weight and standard deviation in cases with developmental delay was -2.66 ± 1.52 (n = 5) and in cases with normal development -1.60

± 1.10 (n = 18). Figure 1 shows that the birth weight distributions for cases with normal development and cases with delayed development are overlapping, indicating that birth weight is not a clinically useful predictor of developmental delay.

96 Multiple logistic regression was performed with developmental delay as the outcome variable, and trisomy in amniotic fluid and malformation as explanatory variables. (Birth

weight could not be incorporated as an explanatory variable because it was not significantly

associated with developmental delay at the 2-tailed level.) The regression results showed that

the effects of trisomy in amniotic fluid and malformation on risk of developmental delay were

independent of each other (data not shown).

In order to assess the possible confounding of birth weight, bivariate associations were

sought with trisomy in amniotic fluid and malformation. The presence of trisomy in amniotic

fluid was associated with lower birth weight (t = 2.71, df = 19, p = 0.007), but the presence of

malformation was not (t = 1.32, df = 20, p = 0.10). This suggests that the association between

developmental delay and lower birth weight may be confounded by the presence of trisomy in

amniotic fluid (or vice versa). There were no statistically significant associations between

developmental delay, and the level of trisomy above 0% at amniocentesis, UPD status, sex of

the infant, method of ascertainment, or gestational age at delivery.

5.5 Discussion

The findings in this study support the hypothesis of generally positive long-term

outcome after prenatal diagnosis of CPM16. Of SGA infants from CPM16 pregnancies, the

majority showed catch-up growth in height (100%) and weight (78%). Therefore, the prognosis

for postnatal growth in infants with severe growth restriction related to CPM 16 appears to be

quite good. The majority of infants (78%) also showed normal developmental outcome. The

association between developmental delay and the presence of trisomy on amniocentesis suggests

that amniotic fluid cells are an indicator of low-level mosaicism in the infant with implications

for development. Although this association was confounded by birth weight, it is likely that

mosaicism in the fetus/infant accounts for both developmental delay and lower birth weight

97 (Chapter 3); however, it cannot be ruled out that low birth weight itself may contribute to developmental delay, for example secondary to neonatal complications (Gutbrod et al. 2000).

The association between developmental delay and malformation suggests that the presence of

fetal malformation may also be indicator of low-level mosaicism in the infant. Of the 5/6

infants with developmental delay that had malformations, 3 of the 5 had heart malformations

(Tables 5.2 and 5.3): case 1 (VSD), case 8 (ASD and 'other' cardiac anomalies), and case 15

(DORV, PDA, endocardial cushion defect). Although the developmental outcome of infants

with congenital heart disease (CHD) is usually normal, those requiring open heart surgery may

be at increased risk for delay (Limperopoulos et al. 2002). Cases 8 and 15 did undergo surgery

for heart anomalies. In case 15, cardiac surgery occurred within the first year of life, and

therefore before the onset of speech delay; in case 8, the onset of developmental delay was not

available. It should also be emphasized that neither the level of trisomy above 0%, uniparental

disomy (UPD), nor gestational age at delivery was associated with developmental delay.

Multiple logistic regression results suggested that if malformation is a marker of low-

level trisomy in the infant, then it is a different and independent marker compared to the

presence of trisomy in amniotic fluid by amniocentesis. Interestingly, Greally et al. (1996)

reported that a CPM 16 infant with a hypoplastic aortic isthmus was found to have trisomic cells

by FISH on aortic tissue sampled during surgery. However, another case with ASD, VSD, and

single coronary artery arising from the pulmonary trunk was found to have no trisomic cells in

affected heart tissue (case 2 from Garber et al. (1994)). Conversely, a case was reported to have

11% trisomic cells by FISH in heart tissue, yet the heart was structurally normal (Johnson et al.

2000). In addition, lower birth weight (corrected for gestational age) was associated with

developmental delay. The significant association between trisomy in amniotic fluid and birth

weight suggests that the birth weight and development delay association may be confounded.

98 The postnatal prognosis of infants after prenatal diagnosis of CPM 16 is generally optimistic. Most demonstrated catch-up growth. The majority (78%) had normal development, and significantly, all 10 cases with no evidence of trisomy on amniocentesis developed normally. These findings are reassuring and will be useful for genetic counselling after CPM 16 prenatal diagnosis.

99 5.6 References

Devi AS, Velinov M, Kamath MV, Eisenfeld L, Neu R, Ciarleglio L, Greenstein R, Benn P (1993) Variable clinical expression of mosaic trisomy 16 in the newborn infant. Am J Med Genet 47:294-298

Dorfmann AD, Perszyk J, Robinson P, Black SH, Schulman JD (1992) Rare non-mosaic trisomies in chorionic villus tissue not confirmed at amniocentesis. Prenat Diagn 12:899- 902

Garber A, Carlson D, Schreck R, Fischel-Ghodsian N, Hsu WT, Oeztas S, Pepkowitz S, Graham JM, Jr. (1994) Prenatal diagnosis and dysmorphic findings in mosaic trisomy 16. Prenat Diagn 14:257-266

Greally JM, Neiswanger K, Cummins JH, Boone LY, Lenkey SG, Wenger SL, Lewis JL, Fischer D, Paul RA, Steele MW (1996) A molecular anatomical analysis of mosaic trisomy 16. Hum Genet 98:86-90

Gutbrod T, Wolke D, Soehne B, Ohrt B, Riegel K (2000) Effects of gestation and birth weight on the growth and development of very low birthweight small for gestational age infants: a matched group comparison. Arch Dis Child Fetal Neonatal Ed 82:F208-214

Hajianpour MJ (1995) Postnatally confirmed trisomy 16 mosaicism: follow-up on a previously reported patient. Prenat Diagn 15:877-879

Hsu LY, Yu MT, Neu RL, Van Dyke DL, Benn PA, Bradshaw CL, Shaffer LG, Higgins RR, Khodr GS, Morton CC, Wang H, Brothman AR, Chadwick D, Disteche CM, Jenkins LS, Kalousek DK, Pantzar TJ, Wyatt P (1997) Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations. Prenat Diagn 17:201-242

Hsu WT, Shchepin DA, Mao R, Berry-Kravis E, Garber AP, Fischel-Ghodsian N, Falk RE, Carlson DE, Roeder ER, Leeth EA, Hajianpour MJ, Wang JC, Rosenblum-Vos LS, Bhatt SD, Karson EM, Hux CH, Trunca C, Bialer MG, Linn SK, Schreck RR (1998) Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases. Am J Med Genet 80:473-480

Johnson MP, Childs MD, Robichaux AG, 3rd, Isada NB, Pryde PG, Koppitch FC, 3rd, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108

Johnson P, Duncan K, Blunt S, Bell G, Ali Z, Cox P, Moore GE (2000) Apparent confined placental mosaicism of trisomy 16 and multiple fetal anomalies: case report. Prenat Diagn 20:417-421

Lee PA, Chernausek SD, Hokken-Koelega AC, Czernichow P (2003) International Small for Gestational Age Advisory Board consensus development conference statement: management of short children born small for gestational age, April 24-October 1, 2001. Pediatrics 111:1253-1261

100 Leung AK, Kao CP (1999) Evaluation and management of the child with speech delay. Am Fam Physician 59:3121-3128, 3135

Limperopoulos C, Majnemer A, Shevell MI, Rohlicek C, Rosenblatt B, Tchervenkov C, Darwish HZ (2002) Predictors of developmental disabilities after open heart surgery in young children with congenital heart defects. J Pediatr 141:51-58

Lindor NM, Jalal SM, Thibodeau SN, Bonde D, Sauser KL, Karnes PS (1993) Mosaic trisomy 16 in a thriving infant: maternal heterodisomy for chromosome 16. Clin Genet 44:185- 189

Ogden CL, Kuczmarski RJ, Flegal KM, Mei Z, Guo S, Wei R, Grummer-Strawn LM, Curtin LR, Roche AF, Johnson CL (2002) Centers for Disease Control and Prevention 2000 growth charts for the United States: improvements to the 1977 National Center for Health Statistics version. Pediatrics 109:45-60

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong SL, Lewis S, Bruyere H, Howard- Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X- chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446

Pletcher BA, Sanz MM, Schlessel JS, Kunaporn S, McKenna C, Bialer MG, Alonso ML, Zaslav AL, Brown WT, Ray JH (1994) Postnatal confirmation of prenatally diagnosed trisomy 16 mosaicism in two phenotypically abnormal liveborns. Prenat Diagn 14:933-940

Schneider AS, Bischoff FZ, McCaskill C, Coady ML, Stopfer JE, Shaffer LG (1996) Comprehensive 4-year follow-up on a case of maternal heterodisomy for chromosome 16. Am J Med Genet 66:204-208

Simensen RJ, Colby RS, Corning KJ (2003) A prenatal counseling conundrum: mosaic trisomy 16. A case study presenting cognitive functioning and adaptive behavior. Genet Couns 14:331-336

Usher R, McLean F (1969) Intrauterine growth of live-born Caucasian infants at sea level: standards obtained from measurements in 7 dimensions of infants born between 25 and 44 weeks of gestation. J Pediatr 74:901-910

Williams J, 3rd, Wang BB, Rubin CH, Clark RD, Mohandas TK (1992) Apparent non-mosaic trisomy 16 in chorionic villi: diagnostic dilemma or clinically significant finding? Prenat Diagn 12:163-168

Woo V, Bridge PJ, Bamforth JS (1997) Maternal uniparental heterodisomy for chromosome 16: case report. Am J Med Genet 70:387-390

101 Table 5.1 CPM16 cases meeting the inclusion criteria

Case # Local case # Origin of postnatal follow-up data 1 - Hsu et al. (1998) case 9 2 92.48 Unpublished data 3 93.103 Unpublished data 4 93.73 Penaherrera et al. (2000); unpublished data 5 - Unpublished data 6 93.94 Schneider et al. (1996) 7 94.21 Penaherrera et al. (2000); unpublished data 8 98.92 Penaherrera et al. (2000); unpublished data 9 90.90 Johnson et al. (1993) 10 91.10 Unpublished data 11 92.95 Hsu et al. (1997) case XIV-11; unpublished data 12 - Hajianpour (1995); Hsu et al. (1998) case 5 13 - Unpublished data 14 - Lindor et al. (1993) 15 - Pletcher et al. (1994) case 1 16 - Unpublished data 17 - Unpublished data 18 - Williams et al. (1992); Garber et al. (1994) 19 - Woo et al. (1997); unpublished data 20 - Unpublished data 21 - Unpublished data 22 - Unpublished data 23 - Unpublished data 24 - Simensen et al. (2003) 25 - Hsu et al. (1997) case XIV-3 26 - Rubin et al. (unpublished case from Devi et al. (1993) 27 - Dorfmann et al. (1992)

Full references in Appendix B.

102 Table 5.2 Clinical data for included CPM16 cases

Case Amnio UPD Malformations GA Sex Asc % 1 10% BPD VSD, anterior anus, abnormality of 34 F B cervical vertebrae, short forearm, hypoplastic left thumb 2 0% BPD hypospadias 36 M U 3 - UPD none 37 M U 4 0% BPD none 37 F u 5 - BPD hypospadias 37 M B 6 4% UPD hypospadias, 5th finger clinodactyly 39 M U 7 0% UPD none 40 F B 8 3.7% UPD ASD, other cardiac anomalies 29 F B 9 0 UPD none 41 F U 10 0 BPD none 36 F U 11 3%a UPD hypospadias 36 M u 12 6/20 BPD microcephaly, dolicocephaly, sacral 35 F B colonies6; dimple, high arched palate, mild pectus 0% in excavatum, widely spaced, hypoplastic repeat nipples, unilateral hypoplastic labia majoria, tapering fingers, partial cutaneous syndactyly between 2nd and 3rd toes, mild asymmetry in hand and foot size 13 0% UPD suspected thyroid agenesis, inguinal hernia 35 F U 14 45% UPD dolicocephaly, fingers were minimally 35 F B tapered distally, 5th finger clinodactyly 15 39% BPD DORV, PDA, endocardial cushion defect, 40 M B brachycephaly, high narrow palate, wide spaced hypoplatic nipples, hyposapdias with cordee, right inguinal hernia, small umbilical hernia, scoliosis, deep sacral dimple, 5th finger clinodactyly, dorsiflexed toes, talipes calcaneovalgus 16 0% UPD ASD 37 F B 17 3% BPD inguinal hernia, scoliosis, assymemmtrical 38 M ? skull, unilateral cryptorchidism 18 0% BPD ASD, VSD, PDA 37 F ? 19 0% UPD unilateral talipes equinovarus, left 33 F U unilateral renal agenesis, left foot larger 20 0% BPD None 30 F B

21 mosaic BPD None 42 F B 22 25% - None 38 F B 23 21% UPD ? 34 F B 24 50% bicuspid aortic valve, hypoplastic left 32 F U thumb, small 2nd finger, left side body smaller than right 25 9% - None ? F ? 26 mosaic - None <37 ? ? 27 0% - None F u

"GA" = gestational age in weeks. "Asc" = ascertainment, where U = unbiased and B = biased. "-" = not done; '?" = not known. Malformations exclude facial dysmorphism and skin anomalies. a7%, then 1% in repeat amniocentesis. b6/20 colonies had at least 1 trisomy 16 cell, of which only 1 colony (with 3 cells) was fully trisomic.

103 Table 5.3 Follow-up data for length/height, weight and developmental outcome

Case Length/ Catch• Weight Catch• Development Normal Height up up 1 - - 2 yr = Developmental N delay. 2 Birth = 0.26 N/A Birth = -0.66 N/A 9 yr 9 mo = Reached Y 7.5 mo = 0.09 7.5 mo = 0.20 all developmental 11 mo = -0.07 11 mo = -0.15 milestones on time; 9 yr 9 mo = 0.86 9 yr 9 mo = 1.48 very advanced vocabulary for age. 3 Birth = 0.36 N/A Birth = -0.41 N/A 6 yr 9 mo = Speech N 6 mo = -1.32 6 mo = -0.89 development delayed. 1 yr = -0.50 1 yr = -0.98 3yr = 2.51 3yr= 1.50 4 Birth = -1.31 N/A Birth = -1.25 N/A Met developmental Y 3 mo = 0.87 milestones for rolling 6 mo = -1.32 6 mo = -0.45 over (4.5 mo), sitting 1 yr = -0.41 1 yr = -0.78 (6 mo), walking (1 2yr = 0.19 2 yr = -0.33 yr), first words (1 yr), 3 yr = -0.93 3 yr = -0.89 and phrases (1 yr 6 4yr = -1.53 4yr = -1.19 mo). 5 Birth = -1.45 N/A Birth = -1.46 N/A Met developmental Y 1 mo = -1.87 1 mo = -1.52 milestones for rolling 3 mo = 1.29 3 mo = 0.90 over at (3 mo), sitting 6 mo = 0.33 6 mo = 0.56 (8 mo), walking (1 yr 1 yr = 0.03 1 yr = 0.30 1 mo), and speech 2 yr = 0.05 2 yr = 0.07 development (mama, 4 yr = 0.25 4 yr = 0.18 papa) (1 yr) 6 Birth = -1.98 N/A Birth = -3.18 Y 3 yr = Average in all Y 3 mo = -2.36 3 mo= 1.69 areas of the Hawaii 6 mo = -1.21 6 mo = -1.66 Early Learning 8 mo = -0.65 8 mo = -1.02 Profile (HELP) and 10 mo = 0.00 10mo = -1.15 Receptive Expressive 1 yr = -0.35 lyr = -1.46 Emergent Language- 1 yr 6 mo = -1.57 1 yr 6 mo = -2.06 2 (REEL-2) with 2 yr = -0.76 2 yr = -2.24 strengths in fine 3 yr = -0.95 3yr = -1.68 motor skills and 4yr = -1.36 4yr = -1.60 receptive and Report: fussy Report: fussy expressive language; eater. eater. active and sociable. 7 Birth = -1.20 N/A Birth = -0.67 N/A 1 yr 6 mo = Normal Y 1 yr 6 mo = 50- 1 yr 6 mo = 75- psychomotor. 75th 90th 8 Parental anecdote: N 2 yr = Occupational, physical and speech therapy; close to achieving - - - - developmental milestones on time. Cognitively 'right on target'. History of seizures. 8 yr = Math at grade level, above average in other subjects; occupational and

104 Case Length/ Catch• Weight Catch• Development Normal Height up up speech therapy to be stopped soon. Evidence of obsessive-compulsive disorder (OCD). 9 1 yr 4 mo = Bright, Y - - - - sociable, normal developmental milestones. 10 Birth = 0.58 N/A 1 yr 8 mo = Normal Y - - 2 y r 8 mo = - development 0.08 13 yr 6 mo = 10th 11 Birth = -3.80 Y Birth = -3.03 Y 1 yr 1 mo = Normal Y 1 mo = -5.23 1 mo = -1.61 development 2 mo = -1.58 2 mo = -1.26 4 mo = -2.48 4 mo = -1.02 5 mo = -3.66 5 mo = -1.90 6.5 mo = -0.80 6.5 mo = -0.97 9 mo = -0.62 9 mo = -1.56 1 yr 1 mo = -1.35 1 yr 1 mo = -1.40 1 yr 2.5 mo = - 1.92 12 Birth = -7.73 Y Birth = -2.29 Y 2 yr 4.5 mo = Mild N 3 mo = 40,h 3 mo = 40th developmental and 2 yr 4.5 mo = 10(h 2 yr 4.5 mo = 3rd speech delay. 3 yr 9 mo = Developmental and speech delay. 13 Birth = -4.70 Y Birth = -2.08 N 4 yr 3 mo = Normal Y 3 mo = -2.91 3 mo = -2.98 developmental 11 mo = -1.50 11 mo = -2.16 milestones 2 yr 7 mo = -1.87 2 yr 7 mo = -3.45 14 11 mo = Y Development of social and communication skills appeared normal. - - - - Met developmental milestone for rolling over (6-7 mo), delayed for sitting alone (8 mo), and met milestone for walking (1 yr 2 mo). 15 Birth = -3.76 Y Delayed in rolling N 2 yr 2 mo = 10th over and sitting unsupported (9 mo), but met developmental milestone for walking (1 yr 2 mo). Delayed for putting 2 words together (2.5 yr). Febrile seizure post-

105 Case Length/ Catch• Weight Catch• Development Normal Height up up operatively at 6 mo. Early intervention services including speech therapy, with resulting good neurodevelopmental progress. 16 Birth = -1.45 N/A Birth = -2.25 N 1 yr 5 mo = Normal Y 6 mo = -2.09 6 mo = -2.62 developmental 1 yr 5 mo = -1.20 1 yr 5 mo = -4.52 milestones 17 Birth = -2.44 Y Birth = -2.99 Y 8 mo = Central motor N 5 yr = 0.30 5 yr= 1.30 disturbance. 11 mo = Mild central motor impairment and statomotoric developmental delay. 5 yr = Developmental delay, central motor impairment, speech and motor therapy. 18 Birth = -1.00 N/A 2 yr 2 mo = Normal Y 2 yr 2 mo = 40th psychomotor - - development 3 yr 6 mo = Appeared developmentally normal. 19 Birth = -1.45 N/A Birth = -1.42 N/A Met developmental Y 1 yr = -1.09 1 yr = -1.84 milestones for hand 2yr = -0.10 2 yr = -0.38 eye coordination (appropriate at 1 yr), sitting (at least 7 mo), walking (1 yr). 3 yr = Normal development; full psychomotor assessment showed normal results on the Bayley scales. 20 Birth = -3.97 Y Birth = -2.58 Y 1 yr 8 mo = Normal Y 1 yr 8 mo = -0.10 1 yr 8 mo = -0.44 development. 21 - - - - 2 yr = Normal Y development. 22 Birth = -1.50 N/A Birth = -2.32 Y Parental report: Y 1 mo = -0.64 1 mo = -0.93 1 yr 6 mo = 'all 2 mo = -0.83 2 mo = -0.51 developmental 4 mo = -0.83 4 mo = -0.76 milestones at 6 mo = -0.35 6 mo = -0.72 appropriate time' 9 mo = -0.69 9 mo = -1.66 1 yr = -1.15 1 yr = -1.55 1 yr 4 mo = -0.12 23 Birth = -2.15 Y Birth = -3.43 Y 1 yr 2 mo = 'Normal Y 6 mo = -1.36 6 mo = -1.04 baby' lyr2mo = -1.00 1 yr 2 mo = -1.25 24 Birth = -0.81 N/A Birth = 0.15 N/A 5 yr 6 mo = Normal Y 5 yr 6 mo = 0.60 5 yr 6 mo = 0.79 cognitive assessment. Early development

106 Case Length/ Catch• Weight Catch• Development Normal Height up up within normal limits. 25 - - - - lyr 5 mo = 'Normal' Y 26 - - - - 2 yr = 'Normal Y phenotype' 27 3 yr = 'Clinically Y - - - - normal with no significant complications to date'

Birth weight and length data only shown for cases with long-term follow-up. Bolded birth weight and lengths were SGA.

107 Table 5.4 Association between trisomy in amniotic fluid and developmental delay

Development Normal Delayed Trisomy at Present (>0%) 9 5 14 amniocentesis Absent (0%) 11 0 11

Presence (>0%) and absence (0%) of trisomy in amniotic fluid as assessed by amniocentesis. Fisher Exact test, p = 0.038.

Table 5.5 Association between malformation and developmental delay

Development Normal Delayed Malformation Present 10 6 16 Absent 10 0 10

Fisher Exact test, p = 0.035.

108 Figure 5.1 Association between birth weight and developmental delay

0.00'

-1.001

BW

-2.00^

-3.00

-4.00H

Normal development Developmental delay

The mean birth weight and standard deviation in cases with normal development was -1.60±1.10 and in cases with developmental delay -2.66±1.52. The sample sizes were 18 and 5, respectively, t = 1.76, df = 21, p = 0.047.

109 6 Cytokeratin staining in villus cultures from miscarriage and CVS9

6.1 Note

I wrote this chapter/manuscript, and did the experiments, data organization and analysis, with the following clarifications and exceptions. Ascertainment of miscarriage placental samples was coordinated by Dr. D. McFadden. CVS placental sample dissection and cultures were done by the Clinical Cytogenetics laboratory at C&W. The immunochemistry was performed by myself with the protocol and equipment of Dr. C. MacCalman (Obstetrics and

Gynaecology).

6.2 Introduction

Chorionic villus cultures are routinely used for clinical cytogenetic diagnosis of spontaneous abortions, as well as of ongoing pregnancies via chorionic villus sampling (CVS) in the late first-trimester. These cultures are thought to consist of cells from the villus mesenchymal core, which ultimately derive from the inner cell mass (ICM) of the blastocyst. In contrast, the trophoblast derives from the trophectoderm, which is the outer epithelial covering of the blastocyst (Larue et al. 1994). Cytokeratin is an epithelial marker that has been used to assess trophoblast contamination of chorionic villus cultures: using antibody to cytokeratin-8 or

-18, or a pan-cytokeratin antibody, both cytokeratin-negative and -positive cells have been observed by immunochemistry of first-trimester chorionic villus cultures from CVS or terminations of pregnancy (Willers et al. 1990; Zimmer et al. 1993; Haigh et al. 1999). This suggests that trophoblast contamination is common in chorionic villus cultures. Recently, however, Blaschitz et al. (2000) conclusively demonstrated that only cytokeratin-7 (CK7) is

9 A version of this chapter will be submitted for publication. Yong PJ, McFadden DE, MacCalman CD, Robinson WP. Developmental origin of chorionic villus cultures from miscarriage and chorionic villus sampling (CVS).

110 specific for the trophoblast in histological sections, while other cytokeratins are also expressed in the mesenchymal core. I hypothesized that true trophoblast contamination (CK7-positive cells) of chorionic villus cultures would be rare, while mosaicism for mesenchymal cells expressing other cytokeratins would be present as seen in previous studies. Therefore, in this

study chorionic villus cultures from CVS, as well as from miscarriages, were assessed for both

CK7 and CK18 by immunochemistry.

6.3 Methods

6.3.1 Tissue processing and culture

CVS cultures were ascertained from the Clinical Cytogenetics laboratory at the

Children's and Women's Health Centre of British Columbia (C&W). CVS placental samples

are processed as follows at the Clinical Cytogenetics laboratory: approximately 15-20 mg of

dissected chorionic villi are washed in Hanks Balanced Salt Solution (HBSS) (with calcium and

magnesium) for 10 min at 37 degrees, followed by digestion with 1 mL of collagenase (1

mg/mL in HBSS) for 20 min at 37 degrees with vortexing at 10 min, 15 min and 20 min. After

the last vortex, the supernatant (trophoblast suspension) is removed. The remaining villus

mesenchymal cores are then washed once in HBSS, and incubated in lmL of cold diluted

collagenase (0.33 mg/mL) overnight at 4 degrees. The next day, the diluted collagenase is

removed, and the remaining villus mesenchyme resuspended with culture medium (Amniomax

+ Amniomax serum supplement +1% antibiotic/antimycotic), vigorously disrupted by pipetting

once per second for 60 sec (or until the tissue was disaggregated into a suspension), and then

placed into a vented tissue culture flask. Leftover backup cultures were sent to our laboratory

after approximately 2 weeks in culture. The cells were passaged once, then grown on coverslips

for immunochemistry (see below).

Ill Placental samples from first-trimester miscarriages were ascertained from the

Embryopathology laboratory at BC Women's Hospital. Chorionic villi were dissected from chorionic plate, and cleared of maternal decidua and blood clots. As performed for clinical cytogenetic diagnosis of miscarriages, the villi were simply minced and cultured in culture

medium in vented tissue culture flasks. The cells were passaged once, then grown on coverslips

for immunochemistry.

JEG-3 choriocarcinoma cells, provided courtesy of Dr. C. MacCalman, were grown on

coverslips as a positive control for CK18 and CK7 immunochemistry. Approval was granted by

the ethics committees of the University of British Columbia and C&W (Appendix A).

6.3.2 Immunochemistry

The coverslip-grown cells were washed three times in phosphate buffered saline (PBS)

and fixed with 4% paraformaldehyde in PBS for 15 min. The cells were then washed three

more times in PBS, permeabilized in methanol (with 2% hydrogen peroxide to inhibit

endogenous peroxidases) for 20 min, washed in water for 5 min, and then blocked twice for 5

minutes with a blocking solution consisting of 10% Automation Buffer (Biomeda M30) and 1%

bovine albumin in distilled H2O. Then, the cells were blocked again with 10% normal serum

(Vector S2000) in blocking solution for 20 min at 37 degrees. Next, the cells were incubated for

45 min at 37 degrees with mouse anti-human primary antibody to CK7 (Dako M7018) diluted

1/50 in blocking solution or primary antibody to CK18 (Dako M7010) diluted 1/25 in blocking

solution. Negative controls consisted of blocking solution alone in lieu of primary antibody.

After blocking twice for 5 min with blocking solution, the cultures were incubated for 30 min at

37 degrees with biotinylated horse anti-mouse IgG secondary antibody (Vector BA-2000)

diluted 1/200 in blocking solution. After blocking twice more for 5 min with blocking solution,

the cultures were incubated for 30 min with streptavidin-biotin-horseradish peroxidase. After

112 washing in blocking solution for 5 min twice, the cells were incubated for 5 min in the chromogen, 3,3'-diaminobenzidine (DAB) solution, 0.05% in blocking solution with 1% hydrogen peroxide substrate. The cells were then washed in running water for 5 min, then stained with hematoxylin (30 sec), decolourized in 4% acetic acid solution (20 sec), and then

'blued' in 1% lithium carbonate solution (30 sec), each step separated by washing in running water for 2 min. The coverslips were then dehydrated in ethanol (5 min, 3x) and xylene (5 min,

3x), and then mounted on slides in Permount mounting medium (Fisher SP15-100).

6.3.3 Statistical analysis

Statistical analyses were performed using SPSS 10.0 and the VassarStats Web Site for

Statistical Computation (http://facultv.vassar.edu/lowry/VassarStats.htinl).

6.4 Results

Chorionic villus cultures from CVS and miscarriages showed little or no CK7 staining, while the JEG-3 cells did stain positive for CK7 (Figures 6.1 and 6.2; Table 6.1). In contrast, the CVS cultures had variable numbers of CK18-negative and -positive cells, while cultures from miscarriages showed little or no CK18 staining (Table 6.1). The proportion of CK18- positive cells was significantly higher than the proportion of CK7-positive cells in the CVS cultures (paired-sample t = 2.59, df = 4, p = 0.031, 1-tailed). The difference between CVS and miscarriage cultures in CK18-positivity was also statistically significant (Mann-Whitney test, p

< 0.02, 2-tailed).

6.5 Discussion

This study confirms the hypothesis that trophoblast contamination in chorionic villus cultures from first-trimester spontaneous abortions and CVS is minimal or non-existent.

113 Therefore, clinical cytogenetic diagnoses from these cultures do reflect the villus mesenchymal core, and ultimately, the inner cell mass (ICM) of the blastocyst. As seen in previous studies

(Willers et al. 1990; Zimmer et al. 1993), variable CK18 expression was seen in cultures from

CVS. Unexpectedly, little or no CK18 staining was evident in the cultures from miscarriages.

This may reflect the fact that the CVS cultures were 2 weeks old before transfer to our laboratory, since culture conditions can induce non-CK7 cytokeratin expression in villus cultures (von Koskull and Virtanen 1987). Alternatively, there may be a true difference in composition of mesenchymal cells in chorionic villus cultures from CVS and miscarriages, with

CK18 expressing mesenchymal cells absent in the latter. This difference may relate to time of sampling: CVS samples placentas from viable ongoing pregnancies, while placentas from miscarriages are affected by tissue degeneration during the period of retention between intrauterine demise and diagnosis. Future studies should include further characterization of these cultures to determine which components of the mesenchymal core are growing in vitro, including fibroblasts, myofibroblasts, endothelial cells, macrophages, and/or smooth muscle cells (Benirschke and Kaufmann 1995). It is possible that chromosome abnormalities diagnosed in different mesenchymal core cell-types may differ in functional and clinical significance.

114 6.6 References

Benirschke K, Kaufmann P (1995) Pathology of the Human Placenta. Springer-Verlag, New York

Haigh T, Chen C, Jones CJ, AplinJD (1999) Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage. Placenta 20:615-625

Larue L, Ohsugi M, Hirchenhain J, Kemler R (1994) E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci U S A 91:8263-8267 von Koskull H, Virtanen I (1987) Induction of cytokeratin expression in human mesenchymal cells. J Cell Physiol 133:321-329

Willers I, Blankenfeld J, Goedde HW (1990) Characterization of long-term cell cultures of human chorion villi and fibroblasts using antibodies to cytoskeletal proteins. Arch Gynecol Obstet 248:87-92

Zimmer N, Gottert E, Kraus J, Zang KD, Henn W (1993) Immunophenotyping of mitotic cells from long-term cultures of chorionic villi. Hum Genet 91:317-320

115 Table 6.1 CK7 and CK18 staining

Sample CK7 staining CK18 staining

CVS1 0% 31% CVS2 -1% 18% CVS3 -1% -1% CVS4 -1% 5% CVS5 0% 22% SAB1 0 0 SAB2 0 0 SAB3 0 0 SAB4 0 0 SAB5 -1% -1%

116 Figure 6.1 CK7 and CK18 staining in CVS cultures

lOOx. a) No CK7 staining in SAB and CVS cultures; b) CK18 staining in CVS cultures. See Table 1.

117 Figure 6.2 CK7 staining in JEG-3 cells (positive control)

200x.

118 7 EVT outgrowth in trisomic miscarriage

7.1. Note

I wrote this chapter/manuscript, and did the experiments, data collection and analysis, with the following clarifications and exceptions. Placental samples from miscarriages were ascertained from the Embryopathology laboratory, coordinated by Dr. D. McFadden.

Microsatellite PCR to confirm trisomy in the trophoblast was performed by R. Jiang (technician,

Robinson laboratory). I performed the immunochemistry using the protocol and equipment of

Dr. C. MacCalman.

7.2 Introduction

Extravillus trophoblast (EVT) are key to normal placentation. They arise from the villus cytotrophoblast and proliferate to form columns and a shell that adhere the placenta to the uterine tissue (Figure 7.1). From the columns/shell, an invasive subpopulation of EVT migrate further into the decidualized endometrium and myometrium (interstitial EVT) and invade the spiral arteries (endovascular EVT), resulting in spiral artery remodeling to produce a low- resistance high-flow circuit (Figure 7.1). Poor invasion and spiral artery remodeling is classically associated with preeclampsia (Chapter 4), but has also been found in miscarriage

(Khong et al. 1987; Hustin et al. 1990; Michel et al. 1990). In early pregnancy, endovascular

EVT are also thought to form plugs that prevent full maternal blood flow from entering the intervillus space (IVS) until the end of the first-trimester; premature high-velocity flow of oxygenated maternal blood into the IVS in the first-trimester may damage the placenta contributing to the pathogenesis of miscarriage (Jauniaux et al. 2003a; Jauniaux et al. 2003b;

Jauniaux et al. 2003c). About half of spontaneous abortions have been shown by histology to be

10 A version of this chapter will be submitted for publication. Yong PJ, McFadden DE, MacCalman CD, Robinson WP. Extravillus trophoblast outgrowth in trisomic miscarriage.

119 associated with an absence or reduction of the EVT columns, shell, interstitial and endovascular subpopulations, and spiral artery remodelling in the basal plate, compared to no such changes in placentas from elective terminations (Hustin et al. 1990). This abnormal placentation in miscarriages may be influenced by the karyotype.

There have been several studies of EVT growth and function in chromosomally abnormal miscarriages. Hustin et al. (1990) found that the frequency of poor EVT column and shell formation and decidual spiral artery remodelling in miscarriage was increased among cases with histopathological findings associated with chromosomal abnormalities; however, actual karyotypes were not available. Other groups did not observe a significant association between poor placentation on histology and the karyotype of the miscarriage (Khong et al. 1987; Khong and Ford 1997; Sebire et al. 2002). In a study of uterine artery resistance (reflecting, in part, poor spiral artery remodelling) at the time of chorionic villus sampling (CVS), Bindra et al.

(2001) found no significant difference between euploid pregnancies and individual groups of chromosomally abnormal pregnancies (trisomy 21, trisomy 18, trisomy 13, monosomy X and

'other'). Also, Greenwold et al. (2003) showed no difference between euploid and chromosomally abnormal missed abortions in the distribution of intervillus maternal blood flow using Doppler, which depends in part on adequate spiral artery invasion. Together, these findings suggest that EVT are not likely to be significantly altered in all chromosomally abnormal pregnancies relative to euploid pregnancies. However, it is possible that only certain chromosomal abnormalities are associated with abnormal EVT. Autosomal trisomies are more likely to affect EVT growth and function. Trisomy 16, in particular, is the most common trisomy in miscarriage, and when confined to the placenta, is associated with preeclampsia

(Chapter 4): these clinical outcomes could be caused by abnormally functioning trisomy 16

EVT. Thus, I hypothesized that consistent with previous studies, there would be no difference on average between euploid and chromosomally abnormal miscarriages in EVT outgrowth in

120 vitro, although chromosome-specific effects may be present. In this Chapter, using a different methodology from the previous studies, EVT outgrowth in vitro was assessed by culturing chorionic villi from miscarriages on Matrigel, with outgrowth compared between different karyotypes.

7.3 Methods

7.3.1 Miscarriage cases

Placental samples were obtained from 52 cases of first-trimester spontaneous abortion examined in the Embryopathology laboratory at the BC Women's Hospital. These include cases with spontaneous expulsion, and cases requiring uterine evacuation following diagnosis of missed or incomplete abortion. Karyotyping was performed by the Clinical Cytogenetics laboratory at the Children's and Women's Health Centre of British Columbia (C&W), which usually involved G-banding of metaphase spreads of cultures of minced chorion or chorionic

villi. In cases of culture failure, comparative genomic hybridization (CGH) of chorion or chorionic villi was used. The cases had the following karyotypes: euploid (n = 17); trisomy 15

(n = 8); trisomy 16 (n = 4); trisomy 22 (n = 4); trisomy 20 (n = 2); trisomy 21 (n = 2), 47,-15,

+i(15) (n = 2) (equivalent in dosage to free trisomy 15); triploidy (n = 2); trisomy 4 (n = 1); trisomy 9 (n = 1); trisomy 10 (n = 1); trisomy 13 (n = 1); trisomy 17 (n = 1); trisomy 18 (n = 1); monosomy X (n = 1); mosaic monosomy X (n = 1); monosomy X and trisomy 22 (n = 1); trisomy 8 and trisomy 20 (n = 1); and an unbalanced translocation (n = 1). Excluding the monosomy X and triploid cases, there were 30 females and 18 males. The average gestational

age of the cases (at spontaneous expulsion or uterine evacuation) was 10.2 weeks ±1.6 weeks (±

standard deviation), with a range of 6-14 weeks. The experiments were done blind to karyotype.

Because there had been no a priori hypothesis regarding gestational age and EVT outgrowth in culture (Aplin 2000), the experimenter (PJY) was aware of gestational age when placental

121 samples were received from Embryopathology. The study was approved by the ethics committees of the University of British Columbia and C&W (Appendix A).

7.3.2 Tissue culture and processing

Extravillus trophoblast (EVT) outgrowths were propagated from first-trimester chorionic

villus explants using a modified protocol based on the methods first described by Yagel et al.

(1989) and Genbacev et al. (1992). Chorionic villi were dissected, washed in Dulbeco's

Minimal Essential Medium (DMEM), and then minced into 2-5mm pieces. Following further

washings in DMEM, the minced villi (explants) were placed in 6-well plates coated with a thin-

layer of Matrigel (0.5mm thick) (BD Biosciences 354603) (compared to the regular 1.0mm

thick Matrigel-coated 6-well plates (BD Biosciences 354432)). The number of explants varied

between cases due to different amounts of placental tissue available. The average number of

explants was 182 ± 161 explants per case (± standard deviation), with a range of 24 to 742

explants. The explants were allowed to attach in a minimal amount of DMEM for 1-1.5 hours.

Then, 1.5mL of culture medium was placed into the wells and changed every 2 days. The

culture medium consisted of DMEM (with high glucose and without L-glutamine and sodium

pyruvate) + 10% fetal bovine serum (FBS) + 1% L-glutamine +1% antibiotic-antimycotic.

7.3.3 Immunochemistry

After 10 days, the cultures were washed three times in phosphate buffered saline (PBS)

and fixed directly on the Matrigel with 4% paraformaldehyde in PBS for 15 min. The cultures

were then washed three more times in PBS before standard immunocytochemistry was

performed in situ. The cultures were permeabilized in methanol for 20 min, washed in water for

5 min, then blocked twice for 5 min with a blocking solution consisting of 10% Automation

Buffer (Biomeda M30) and 1% bovine albumin in distilled FLO. Then, they were blocked again

122 with 10% horse normal serum in blocking solution for 20 min at 37 degrees. The cultures were incubated for 45 min at 37 degrees with primary antibody: monoclonal mouse anti-human antibody against cytokeratin-7 (CK-7) (DAKO M7018), diluted 1/50 in blocking solution; or monoclonal mouse antibody against vimentin (DAKO M0725), a mesenchymal marker

(Blaschitz et al. 2000), diluted 1/50 in blocking solution. After blocking twice for 5 minutes with blocking solution, the cultures were incubated for 30 min at 37 degrees with secondary antibody: fluoroscein-conjugated (Vector FI-2000) or Texas Red-conjugated (Vector TI-2000) horse anti-mouse antibody against IgG, diluted 1/200 in blocking solution. Following incubation in secondary antibody and washing twice for 5 min with blocking solution, the cultures were incubated for 30 min in 5|ig/mL 4',6-diamidino-2-phenylindole (DAPI) in PBS.

After washing, the cultures were examined under a fluorescent microscope, and the presence and number of explants with CK7-positive trophoblast outgrowths and vimentin positive villus mesenchymal outgrowths were counted for each case.

7.3.4 Karyotype confirmation

Griffin et al. (1997) found 6.1% (4/65) of miscarriages had different karyotypes in different placental lineages after cytogenetic analysis of metaphases from cultured mesenchymal cells and direct metaphases from spontaneous divisions of the villus cytotrophoblast. To check for such karyotypic discrepancy, chromosome-specific multiplex microsatellite PCR was performed on trophoblast DNA for 6 cases with a trisomy 15 karyotype found in the chorion or villus mesenchyme by conventional cytogenetics or CGH. Trophoblast was isolated by digesting chorionic villi for 20 min at 37 degrees with lmg/mL collagenase IA diluted in Hank's

Balanced Salt Solution, vortexing every 5 minutes. After 20 min, the villi were vigorously vortexed, and the resulting trophoblast suspension removed. This technique has been confirmed histologically (I. Barrett, personal communication), and the resulting trophoblast suspension

123 contains both villus and extravillus trophoblast (D. McFadden, personal communication).

Chromosome 15 multiplex PCR was performed using fluorescent-labeled primers for the following microsatellite loci: D15S541, GABRB3, and D15S11. The alleles for each locus were visualized using the ABI Prism 310; an approximate 1:1:1 or 2:1 allelic ratio for each locus was taken as evidence of trisomy in the trophoblast.

7.3.5 Statistical analysis

All statistical analysis was done using SPSS 10.0 or the VassarStats Web Site for

Statistical Computation (http://faculty.vassar.edu/lowrv/Vassai-Stats.html). All p-values are 1- tailed due to a priori evidence or rational mechanisms, unless otherwise noted.

7.4 Results

Cell outgrowths from the explants appeared after ~4 days and exhibited 2 common morphologies under the light microscope: column-like (Figure 7.2) and fibroblast-like (Figure

7.3). The column-like outgrowths resembled the EVT column outgrowths on collagen described by (Aplin et al. 1999). The column-like outgrowths stained positive for CK-7 (Figure 7.4) and negative for vimentin, confirming that they are EVT. The fibroblast-like outgrowths were negative for CK-7 and positive for vimentin (data not shown), suggesting a villus mesenchymal origin. The CK-7 positive EVT column outgrowths were present in 48% (25/52) of cases.

The relationship between EVT outgrowths and karyotype may be confounded by the number of explants, sex of the embryo, or gestational age. There was no association between the number of explants per case and the presence of EVT outgrowths (t = 0.41, df = 50, p =

0.35). Neither was there an association between sex of the embryo and the presence of EVT outgrowths (Fisher Exact test, n = 48, p = 1.00, 2-tailed). However, there was an association between gestational age and EVT outgrowths (Figure 7.5). Figure 7.5 illustrates that there

124 appears to be a threshold at 10 weeks gestation, with outgrowths more likely to occur in cases

<10 weeks compared to cases >10 weeks. Analyzed statistically, miscarriages <10 weeks were significantly more likely to have the presence of EVT outgrowths (Fisher Exact test, n = 49, p =

0.019, 2-tailed; RR = 1.88; Table 7.1).

To eliminate the confounding effect of gestational age, only the cases <10 weeks gestation (n = 28) were considered in the following analyses. Among these cases, there was a sufficient number for comparison only for euploids (n = 9), trisomy 15 (n = 6), and trisomy 16

(n = 3). The distribution for the presence or absence of EVT outgrowths was similar between the euploid and trisomy 15 cases (Fisher Exact test, n = 15, p = 0.54; Table 7.2). When the proportion of explants with EVT outgrowths was compared between the euploid and trisomy 15 cases, the distribution was again similar between the 2 groups (t = 0.12, df = 13, p = 0.45;

Figure 7.6). Trophoblast suspensions were available for 4/6 trisomy 15 cases <10 weeks: chromosome 15-specific multiplex microsatellite PCR confirmed trisomy 15 in the suspensions for each case. In contrast, no outgrowths were observed among the trisomy 16 cases, which compared to the euploid cases was statistically significant (Fisher Exact test, n = 12, p = 0.045;

RR = 0; Table 7.3). For the remaining cases <10 weeks, the cases with outgrowths (n = 6) had the following karyotypes: trisomy 10; trisomy 13; trisomy 21; trisomy 8 and trisomy 20; 45,X and trisomy 22; and 45,X mosaicism. The cases that did not have any outgrowths (n = 3) had the following karyotypes: trisomy 17, trisomy 22, and triploidy. When EVT outgrowth was compared between the euploid cases and the chromosomally abnormal cases as a total group (n

= 19), there was no significant difference in the presence of outgrowths (Fisher Exact test, n =

28, p = 0.20; Table 7.4) or in the proportion of explants with outgrowths (t = 0.12, df = 26, p =

0.45; Figure 7.7).

The number of explants was not significantly associated with the presence or absence of

EVT outgrowths among cases <10 weeks, with the trend in the opposite direction (cases with

125 EVT outgrowths had fewer explants on average) (Welch's approximate t = 1.62, df = 12.8, p =

0.07). Similarly, the number of explants was not significantly different between the euploid and trisomy 16 cases <10 weeks, again with the trend in the opposite direction (trisomy 16 cases had more explants on average) (t = 1.02, df = 10, p = 0.17). In addition, the sex of the embryo was not significantly associated with the presence of EVT outgrowths among cases <10 weeks

(Fisher Exact test, n = 25, p = 0.43, 2-tailed).

7.5 Discussion

There were no differences on average between euploid and chromosomally abnormal

miscarriages in EVT in vitro outgrowth, which was the hypothesized finding based on previous

studies. However there was some evidence of a chromosome-specific effect; specifically,

trisomy 16 EVT may have poor outgrowth ability compared to euploid EVT. It should be noted,

however, that only cases <10 weeks gestation could be analyzed, as older miscarriages

demonstrated few or no EVT outgrowths. Considering all chromosomally abnormal cases, EVT

outgrowth was quite variable. This supports the observations of previous authors that EVT

structure and spiral artery remodeling of chromosomally abnormal pregnancies in vivo are quite

variable and not significantly different as a total group from that of euploid pregnancies (Khong

et al. 1986; Khong et al. 1987; Hustin et al. 1990; Khong and Ford 1997; Bindra et al. 2001;

Sebire et al. 2002; Greenwold et al. 2003). Only Khong and Ford (1997) studied trisomy 16

specifically; they found that 40% (2/5) of trisomy 16 miscarriages showed poor placentation at

histology, which was higher compared to euploid miscarriages (21%; 4/19), although not

significantly so (Fisher Exact test, n = 24, p = 0.37).

Considering all cases, a reduction in EVT outgrowth was observed after 10 weeks

gestation. Previous studies using tissue from terminations have reported EVT cultures in

specimens up to 12 weeks (Irving et al. 1995) and 13 weeks (Aplin et al. 1999) gestation, with

126 no mention of altered growth at later gestational ages. Thus, Aplin (2000) concluded that late first-trimester chorionic villi continue to be able to form new anchoring sites. The reduction in outgrowth in this study is likely due to the period of intrauterine retention and tissue degeneration in miscarriages before expulsion or diagnosis. Cases retained for longer periods

(and hence with later gestational ages at expulsion or diagnosis) would therefore have undergone more tissue degeneration, which presumably affects tissue culture. The difficulty with culturing human miscarriage material due to tissue retention and degeneration also limited sample size in this study, as did the relatively small probability of ascertaining any one trisomy.

The EVT morphology on a thin-layer of Matrigel resembled the anchoring columns seen on collagen (Aplin et al. 1999), rather than the invasive outgrowths previously reported on

Matrigel (Caniggia et al. 1997; Aplin et al. 1999). The reasons for this are not clear, although it could be related to the tissue source (miscarriage vs. termination) or to the fact that wells pre- coated with a thin-layer of Matrigel was used in this study. Also, the onset of the EVT outgrowths (~4 days) represented a significant delay compared to previous studies using

Matrigel or collagen (~1 day) (e.g. Caniggia et al., 1997; Aplin et al., 1999). In previous studies using collagen, fibroblasts have been previously shown to grow only after weeks of culture; therefore, the fibroblast-like outgrowths observed at ~4 days in this study represent a significant acceleration in growth. These differences are likely also due to the tissue degeneration in miscarriages, which may affect trophoblast more than the mesenchyme.

The abnormal trisomy 16 EVT outgrowth may explain in part the common occurrence of trisomy 16 in miscarriages, and the high rate of pregnancy complication (IUGR and preeclampsia) seen in ongoing pregnancies with trisomy 16 confined to the placenta (Chapters

2-5). The altered EVT outgrowth may be related to the possible over-expression of the chromosome 16-encoded E-cadherin (E-cad). E-cadherin is well expressed in villus cytotrophoblast, but is progressively downregulated proximal to distal from the villus tip to the

127 invasive EVT (Zhou et al. 1997; Floridon et al. 2000; Shih le et al. 2002). Therefore, overexpression of E-cadherin in trisomy 16 (which has not yet been investigated) may inhibit

EVT growth and differentiation in trisomy 16 pregnancies. There is also evidence for a different distribution of maternal immune cells in the decidua between euploid and chromosomally abnormal (in particular, trisomy 16) spontaneous abortions (Yamamoto et al.

1999; Quack et al. 2001). Therefore, trisomy 16 miscarriages may have unique pathogenic mechanisms compared to euploid miscarriages.

The similar distribution of EVT outgrowth between trisomy 15 and euploid cases suggests that the aspects of EVT growth assessed in vitro with this methodology do not differ between trisomy 15 and euploid EVT. Trisomy 15 is common in miscarriages, present in -5% of spontaneous abortions at BC Women's Hospital (D. McFadden and W. Robinson, unpublished data). Thus mechanisms other than poor EVT outgrowth may be involved in trisomy 15 miscarriage. It is also notable that the one trisomy 21 case (<10 weeks gestation) did exhibit EVT outgrowths. Khong and Ford (1997) found normal placentation at histology in a single case of trisomy 21 miscarriage. However in a recent study of 4 second-trimester terminations of Down syndrome pregnancies, there was a clear defect in EVT outgrowth on

Matrigel (Wright et al. 2004). Additional studies with larger sample size are needed to clarify these apparently contradictory findings in trisomy 21.

In conclusion, EVT outgrowth in vitro was similar to previous in vivo histological findings: chromosomally abnormal miscarriages show variable degrees of placentation, with no significant difference, on average, compared to euploid miscarriages. Trisomy 16 EVT may be abnormal, while trisomy 15 EVT, at least for initial outgrowth on Matrigel, was similar to EVT from euploid miscarriages. Further investigations should include assessment of E-cadherin expression in the trisomy 16 placenta.

128 7.6 References

Aplin J (2000) Maternal influences on placental development. Semin Cell Dev Biol 11:115-125

Aplin JD, Haigh T, Jones CJ, Church HJ, Vicovac L (1999) Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin alpha5betal. Biol Reprod 60:828-838

Bindra R, Curcio P, Cicero S, Martin A, Nicolaides KH (2001) Uterine artery Doppler at 11-14 weeks of gestation in chromosomally abnormal fetuses. Ultrasound Obstet Gynecol 18:587-589

Blaschitz A, Weiss U, Dohr G, Desoye G (2000) Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta 21:733- 741

Caniggia I, Taylor CV, Ritchie JW, Lye SJ, Letarte M (1997) Endoglin regulates trophoblast differentiation along the invasive pathway in human placental villous explants. Endocrinology 138:4977-4988

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130 Table 7.1 Asssociation between gestational age and the presence of EVT outgrowths

EVT outgrowths Absent Present Total Gestational <10 weeks 11 17 38 age >10 weeks 16 5 21

EVT outgrowths were less frequent in miscarriages <10 weeks (Fisher Exact test, p = 0.011).

Table 7.2 EVT outgrowth in euploid and trisomy 15 cases <10 weeks gestation

EVT outgrowths Absent Present Total Karyotype Trisomy 15 2 4 6 Euploid 2 7 9

Fisher Exact test, p = 0.54.

Table 7.3 EVT outgrowth in euploid and trisomy 16 cases <10 weeks gestation

EVT outgrowths Absent Present Total Karyotype Trisomy 16 3 0 3 Euploid 2 7 9

Fisher Exact test, p = 0.045.

Table 7.4 EVT outgrowth in euploid and chromosomally abnormal cases <10 weeks gestation

EVT outgrowths Absent Present Total Karyotype Abnormal 9 10 19 Euploid 2 7 9

Fisher Exact test, p = 0.20.

131 Figure 7.1. Extravillus trophoblast (EVT) columns deriving from the chorionic villus

Chorionic villus

The columns join to form a shell, while invasive EVT migrate to form endovascular plugs in maternal arteries (BV) in early pregnancy, and then remodel the arteries to create a low- resistance circuit.

132 Figure 7.2 EVT outgrowths

lOOx.

Figure 7.3 Fibroblast-Iike outgrowths

lOOx.

133 Figure 7.4 Cytokeratin-7 staining of EVT outgrowths

lOOx.

134 Figure 7.5 Association between gestational age and EVT outgrowth

i i i i 6-9 10 11 12-14

Gestational age (weeks)

The black bars represent the number of cases where EVT outgrowths were observed; the white bars represent the number of cases where no EVT outgrowths were observed. The proportion of cases with EVT outgrowths is as follows: 6-9 weeks = 67% (10/15), 10 weeks = 54% (7/13), 11 weeks = 33% (3/9), 12-14 weeks = 17% (2/12).

135 Figure 7.6 Proportion of explants with EVT outgrowths for euploid and trisomy 15 cases

0.40

0.30 H

0.20 H

0.10i

o.oo H

Euploid Trisomy 15

For cases with gestational age <10 weeks. Means and standard deviations for the euploid and trisomy 15 cases were 0.07±0.11 and 0.08±0.08, respectively. The sample sizes were 9 and 6, respectively, t = 0.12, df = 13, p = 0.45.

136 Figure 7.7 Proportion of explants with EVT outgrowths for euploid and abnormal cases

0.60 H

0.40 H

0.20'

0.00- 1 Euploid Abnormal

For cases with gestational age <10 weeks. Means and standard deviations for euploid and all chromosomally abnormal cases were 0.07±0.11 and 0.08±0.15, respectively. The sample sizes were 9 and 19, respectively, t = 0.12, df = 26, p = 0.45.

137 8 Protein kinase profiling in trisomic miscarriage11

8.1 Note

I wrote this chapter/manuscript, and did the experiments, data collection and analysis, with the following clarifications and exceptions. Placental samples from miscarriages were ascertained from the Embryopathology laboratory, coordinated by Dr. D. McFadden.

Microsatellite PCR to check for maternal contamination and to confirm trisomy was done by myself and R. Jiang. I performed the immunochemistry using the protocol and equipment of Dr.

C. MacCalman. RNA extractions were done by myself and Dr. C. Anderson (post-doc,

Robinson lab); RT-PCR to check for DNA contamination was done by Dr. C. Anderson.

Protein expression profiling was performed by staff at Kinexus Bioinformatics Corporation by commercial agreement. RNA expression profiling was performed by A. Haegert at the Prostate

Centre Array Facility by commercial agreement.

8.2 Introduction

The phenotype of trisomy is ultimately due to developmental perturbation from altered protein expression secondary to the extra chromosome; however, the pathways are not well elucidated. Two hypotheses have been proposed for the pathogenesis of trisomy: the gene dosage hypothesis and the amplified instability hypothesis (Reeves et al. 2001). In the gene dosage hypothesis, the trisomic phenotype is considered primarily due to over-expression of specific genes on the particular trisomic chromosome and their corresponding functional effects at the protein level (Epstein 1990). This would account for the specific phenotypic features of the different trisomies. In the amplified developmental instability hypothesis, the trisomic phenotype is thought to result because the expression of hundreds of trisomic genes disrupts

11 A version of this chapter will be submitted for publication. Yong PJ, McFadden DE, MacCalman CD, Robinson WP. Protein kinase profiling in trisomic miscarriage.

138 cellular homeostasis non-specifically (i.e. regardless of chromosome) and causes the cell (and organism) to have a non-specific amplified sensitivity to environmental effects and genomic mutations and polymorphisms during development (Shapiro 1983; Shapiro 1989). This hypothesis is supported by the observation that Down syndrome phenotypic traits (e.g. malformations) are also present at a low rate in the general population (Pritchard and Kola

1999), and that certain bilateral traits (e.g. dermatoglyphics) show increased fluctuating asymmetry (FA) in Down syndrome (Katznelson et al. 1999). If environmental and genetic factors are randomly distributed in the population regardless of karyotype, then the hypothesis can account for the many overlapping features of different trisomies. A corollary is that environmental effects and genome-wide mutations and polymorphisms can act as 'modifiers' of the phenotype of an individual with a given trisomy. Reeves et al. (2001) contend that the two hypotheses are not mutually exclusive and could be integrated at the molecular level.

Furthermore, Shapiro (2001) argues that when comparing gene expression between a particular trisomy and a normal karyotype, another trisomy must be used as a control to demonstrate that the differences in gene expression are either specific to the particular trisomy (gene dosage hypothesis) or common to both trisomies (amplified instability hypothesis).

No studies have assessed gene dosage effects (using another trisomy as a control) or amplified instability using protein expression profiling in trisomic individuals. Protein kinases were chosen for profiling in the present study as they act as fundamental effectors of signal transduction by catalyzing reversible protein phosphorylation (Manning et al. 2002), and so can be sensitive indicators of functional perturbation in cell function. Further, both protein and

RNA levels were examined, as they do not necessarily correlate (Greenbaum et al. 2003). I hypothesized that both amplified instability and gene dosage effects would operate simultaneously at the molecular level in trisomic cells. In this study, the expression of seventy- five protein kinases were assessed by 2D Western blots and oligonucleotide microarrays in chorionic villus fibroblast cultures from trisomy 16 miscarriages, and compared to euploid miscarriages using trisomy 15 miscarriages as control. Expression data were analyzed to determine any differences in level of expression (gene dosage hypothesis) or differences in variability in expression (amplified instability hypothesis) between the karyotypes.

8.3 Methods

8.3.1 Miscarriage cases

Chorionic villi, chorion or chorionic plate, and decidua were obtained from a series of miscarriages (n = 73) from the Embryopathology laboratory at BC Women's Hospital. Routine karyotype information for each case was provided by the Clinical Cytogenetics laboratory at the

Children's and Women's Health Centre of British Columbia (C&W) after the experiments were performed. The study was approved by ethics committees of the University of British Columbia and C&W (Appendix A)

8.3.2 Tissue processing and culture

As karyotype was not known ahead of time, all samples were initially processed in the same manner. The tissues were dissected and cleaned of blood, and samples of the maternal decidua, placental chorionic villi, and chorion (or chorionic plate) were taken. The chorionic villi were cultured, while maternal decidua, and leftover chorionic villi and/or chorion, were frozen for future DNA extraction.

The chorionic villus cultures were set up as described for CVS samples in Chapter 6.

Culture medium was changed every 2 days. Appearance of outgrowths from attached explants

(and/or single cells) was highly variable, sometimes evident after 2 days, but in other cases taking about 1 week. At the second passage, cells were transferred to another culture flask and also to coverslips for immunochemistry. In addition, some cells at the second passage were

140 frozen for future DNA extraction. At the third passage, the cells from the flask were transferred to 5 other flasks, from which the cells were harvested for protein and RNA extraction at -75% confluence.

8.3.3 Immunochemistry and PCR

Immunochemistry was carried out to characterize the cultures from a subset of the cases: the cultures appeared to consist primarily of fibroblasts (Table 8.1). Immunochemistry was carried out as described in Chapter 7, with a human skin fibroblast cell line (provided by Dr. D.

Speert, UBC Department of Paediatrics) used as a positive control. Primary antibodies to the following antigens were used: cytokeratin-7 (CK-7) (Dako M7018), vimentin (mouse monoclonal IgG) (Dako M0725), CD45/Leucocyte Common Antigen (LCA) (mouse monoclonal IgG) (Dako M0701), CD31/platelet endothelial cell adhesion molecule-1 (PECAM-

1) (mouse monoclonal IgG) (Dako M0823), desmin (rabbit polyclonal IgG) (Dako A0611), and fibroblast surface protein (FSP) (mouse monoclonal IgM) (Sigma F4771). Vimentin is specific for the chorionic villus mesenchymal core (Blaschitz et al. 2000), while the other antibodies specify components of the mesenchymal core: LCA for hematopoietic cells (Blaschitz et al.

2000); PECAM-1 for endothelium (Blaschitz et al. 2000) and megakaryocytes, platelets, myeloid cells, natural killer cells, some T-cell subsets, and B-cell precursors (Dako); desmin for muscle (Blaschitz et al. 2000); and FSP for fibroblasts (Blaschitz et al. 2000) and tissue macrophages and 95% of peripheral blood monocytes (Sigma). The normal serum utilized was normal horse serum (Vector S2000), except for desmin and FSP, where normal goat serum

(Vector S-1000) was used. The following dilutions in blocking solution were used for the primary antibodies: CK-7 (1/50), vimentin (1/50), PECAM-1 (1/20), LCA (1/50), desmin

(1/20), and FSP (1/500). The secondary antibody was fluoroscein-conjugated (Vector FI-2000) or Texas Red-conjugated (Vector TI-2000) horse anti-mouse IgG antibody, with the exception

141 of FITC-conjugated goat anti-rabbit IgG antibody (Vector FI-1000) for desmin and Texas Red- conjugated goat anti-mouse IgM antibody (Vector TI-2020) for FSP. In addition to immunochemistry, genotyping of microsatellites by PCR of the maternal decidua, chorionic villi and/or chorion, and chorionic villus cultures was utilized to rule out maternal contamination and trisomic/euploid mosaicism in the cultures used for profiling experiments.

8.3.4 Protein kinase protein profiling

Protein kinases were profiled at the protein level in fibroblast cultures for each of the following cases (Table 8.1): Eu-4, Eu-5, Eu-6, Eu-7 (n = 4, euploid); T16-4, T16-5, T16-6 (n =

3, trisomy 16); and T15-1, T15-2, T15-3 (n = 3, trisomy 15). Profiling was done using the

Kinetworks KPKS-1.2 screen for 75 protein kinases developed and performed by Kinexus

Bioinformatics (Appendix C). Details for protein extraction are provided on the Kinexus website (http://www.kinexus.ca). Briefly, cultured cells at the third passage were harvested using a brief treatment of warm 0.2% trypsin-EDTA, and the cell pellet sonicated in lysis buffer

(containing protease and phosphatase inhibitors and 0.5% Nonidet P-40 detergent in a 20 mM

MOPS buffer at pH 7.2). The homogenate was centrifuged at 1000,000 g for 30 min, the supernatant was removed, and protein concentration determined by a Bradford assay (Bio-Rad) using bovine serum albumin as a concentration standard. Then, 750 p:L of protein was diluted to 1 p:g/uL in SDS sample buffer, and boiled for 4 min before freezing at -80 degrees.

The Kinetworks KPKS-1.2 screen involves SDS-PAGE, Western transfer, and immunoblotting using a 20-lane multiblotter apparatus from Bio-Rad (allowing a different cocktail of antibodies in each well/lane). Further details are available (Pelech et al. 2003).

Isoforms for a particular kinase are detectable by shifts in molecular weight. Protein expression levels were determined by using Quantity One software (Bio-Rad) to quantify band signal

142 intensity, arising from chemoluminescence detected with a FluorS Max Multi-Imager (Bio-

Rad). The raw band signal intensity for each kinase was used as the 'expression level' without any statistical normalization.

Appendix AH, which lists the protein kinases in this study, differs from the corresponding table on the Kinexus website in two ways. First, JNK2 is not included because it is very close to another band, and its accuracy has been called into question. Second, glycogen synthase kinase 3-a and 3-(3 were considered separate kinases because the isoforms are encoded on different genes. Third, the expression levels of the 3 isoforms for CK2 are added together as a composite measure, because although they are encoded on different genes, they function as a tetramer and are therefore non-independent due to stochiometric relationships. In addition,

KPKS-1.2 has one lane with an antibody specific to ERK2, and a second lane with an antibody to both ERK1 and ERK2. The ERK2 (37) isoform was taken to be the average from the corresponding bands in the two lanes. In the second lane, the ERK2 (39) isoform is difficult to distinguish from the ERK1 (40) isoform (due to overlapping of the bands), and in our analyses, only the ERK1 (40) band was called. Thus, the ERK2 (39) isoform was taken to be its band in the first lane, while the 'true' ERK1 (40) isoform was calculated by subtracting the band intensity of the ERK2 (39) isoform in the first lane from the band intensity of the ERK1 (40) isoform in the second lane. Finally, the ERK1 (41) isoform was taken to be its band in the second lane.

8.3.5 Protein kinase RNA profiling

Protein kinases were profiled at the RNA level in fibroblast cultures for each of the following cases (Table 8.1): Eu-6, Eu-7, Eu-8, Eu-9 (n = 4); T16-5, T16-6 (n = 2); and T15-2,

T15-3, T15-4 (n = 3). A human 14,000 oligonucleotide microarray developed and performed by the Gene Array Facility of the Prostate Centre at the Vancouver Hospital and Health Sciences

143 Centre (VHHSC) (http://www.prostatecentrexonVresearch/genearray.html) was utilized for

RNA profiling. All KPKS-1.2 kinase genes were present on the microarray except for calcium/calmodulin-dependent kinase kinase a.

Cultured cells at the third passage were harvested using a brief treatment of warm 0.2% trypsin-EDTA. RNA was extracted immediately (Eu-6, T16-5, T15-3), or the cell pellet was first placed in RNAlater (Ambion) using the provided protocol (Eu-7, Eu-8, Eu-9, T16-6, T15-1,

T15-2). RNA was extracted using a GenElute Mammalian Total RNA Miniprep kit (Sigma) according to the provided protocol. Isolated RNA was precipitated with absolute ethanol overnight at 4 degrees. RT-PCR using primers upstream and downstream of the transcribed region of the XIST gene was used to exclude DNA contamination of the RNA. Next, the RNA was run on an Agilent 2100 Bioanalyzer, and its quality assessed in two ways (Auer et al. 2003):

1) degradation peak signals between the small RNAs and the 18S peak (evidence of degradation); and 2) decreased 28S/18S peak ratio and 28S breakdown peaks above and below the 18S peak (evidence of apoptosis). Using Degradometer software (Auer et al. 2003; http://www.dnaarrays.org), the cases showed minimal degradation (degradation factor less than

1.5%), but the average 28S/18S ratio was decreased in the samples stored in RNAlater compared to the samples extracted immediately (mean peak area ratio: 1.07±0.08 (n = 6) vs.

1.31±0.16 (n = 3), respectively; t = 2.50, df = 7, p = 0.041). However, RNAlater can cause a specific reduction in the 28S-specific peak by changing 28S secondary structure (Ambion) and none of the cases demonstrated 28S breakdown peaks. Hence, apoptosis was not considered significant.

The Prostate Centre Gene Array Facility uses the 3DNA Array 350 kit (Genisphere) for

RNA profiling. The detailed protocol is available at http://www.genisphere.com. Briefly, case

(test) RNA was matched to an equal amount of universal reference human (control) RNA: either

144 1.5 jag (Eu-7, T16-6, T15-2) or 5 [ig (Eu-6, Eu-8, Eu-9, T16-5, T15-1, T15-3) was used. The test RNA was reverse transcribed with poly(T) primers with a specific capture sequence extension, while the control RNA was reverse transcribed with poly(T) primers with a different specific capture sequence extension. The test and control cDNA were then each amplified by

PCR to produce probes. The test and control probes were co-hybridized onto the microarray chip overnight. After washing, the microarray chips were co-hybridized with Cy3-labelled

3DNA capture reagent (-375 Cy3 per molecule) that binds the test specific capture sequence and Cy5-labelled 3DNA capture reagent that binds the control specific capture sequence. The microarray chips were then washed, imaged, and Cy3 and Cy5 signals quantified using Imagene

5.6 software. After quantification, the Cy3 and Cy5 signal data were normalized with

GeneSpring software. A standard intensity-dependent (non-linear or Lowess) normalization was utilized to account for dye-related artefacts, which works by adjustment of control Cy5 signals. The natural logarithm of the ratio between the test Cy3 signal and adjusted control Cy5 signal at each microarray spot was used as the 'expression level' for that spot (i.e. oligonucleotide for a particular gene).

8.3.6 Profiling data analysis

8.3.6.1 Confounding

The euploid and trisomic cases were not perfectly matched for sex of the conceptus or gestational age because of the difficulty in ascertaining any one particular trisomy, and the prevalence of culture failure and maternal contamination in cultures from miscarriage. To check for confounding, associations between the protein expression level of each of the kinases, and sex or gestational age, were carried out using the two-sample t-test and Pearson correlation coefficient, respectively. Five kinases were significantly associated with sex (CAMK1) or

145 gestational age (RAFB, PKC-cc, PKC-(i, ROK-a) and were therefore excluded from the study, leaving seventy kinases for the rest of the analyses. Twenty-three kinases were undetectable at the protein level in all cases, and also removed from the study. RNA expression was detected for all kinases. However, inspection of RNA expression data showed two outliers with extreme levels (both > 8 standard deviations above the global mean) that were considered methodology- related (poor control probe hybridization) and so were excluded.

8.3.6.2 Protein kinase expression at the protein/RNA levels

Pair-wise comparisons of protein expression levels of each kinase were made between karyotypes (euploid vs. trisomy 16; and euploid vs. trisomy 15). Because of the difference in variance between the groups (see text), the non-parametric 2-sample t-test of ranked data (Zar

1996) was utilized for the pair-wise comparisons. For kinases encoded by genes on the trisomic chromosome, a one-tailed test was used. A correction for multiple comparisons was not made because the small number of samples limited statistical power. For each of the kinases with statistically significant differences when compared between karyotypes at the protein level, the corresponding analysis was performed at the RNA level also using a 2-sample t-test rank test because of differences in variance.

8.3.6.3 Inter-individual variation in expression

Inter-individual variation in protein and RNA expression was assessed within each karyotype. For the cases within a given karyotype, the mean and standard deviation for protein or RNA level was calculated for each kinase. Then the standard deviation was divided by the mean to give the coefficient of variation (CV) in protein or RNA level for each kinase within each karyotype. The coefficients were then compared between karyotypes using the non- parametric Wilcoxon paired-sample test. Because CV is a dimensionless parameter,

146 comparisons can be made between samples with difference measurement scales (Zar 1996).

Hence, the CV for protein and CV for RNA were compared within each karyotype using the

Wilcoxon test.

8.3.7 Statistical analysis

Statistical analyses were performed using SPSS 10.0 and the VassarStats Web Site for

Statistical Computation (http://facultv.vassar.edu/lowry/VassarStats.html). In the text, X ± Y represents mean ± standard deviation unless otherwise noted.

8.4 Results

8.4.1 Protein kinase expression at the protein level

Seven kinases showed significantly different expression in trisomy 16 compared to the euploid group (CDK1, CDK7, PKC-E, PKG1, ERK1, S6K p70, IKK-a) (Table 8.2), two of which were also significantly different in trisomy 15 compared to the euploid group (S6K p70,

IKK-a). Seven other kinases were significantly altered only in trisomy 15 (CKl-e, SRC,

CDK9, DNAPK, MEK2, PKC-z, PKC-(3) (Table 8.2). For those kinases with genes on chromosome 16 (CK2 [isoform-oc], PKC-(3, ERK1), only ERK1 was significantly overexpressed in trisomy 16. The larger of the two ERK1 isoforms (with apparent MW 41kDa) was 2.8x more expressed in trisomy 16 (mean = 3675 ± 1938) than in euploids (mean = 1297 ± 389) (p =

0.034). In a separate experiment, Kinexus phospho-antibody to T202/Y204 on ERK1 was used for 4 of the cases, which showed a 2.9x increase in trisomy 16 (2075 and 6949; mean = 4512) compared to euploids (1738 and 1370; mean = 1554). Hence the activated phosphorylated form of ERK1 was also increased in trisomy 16. The upstream ERK1 activators, MEK1 and MEK2

147 (Roux and Blenis 2004), were not differentially expressed in trisomy 16 (data not shown), which supports a specific gene dosage-related increase in ERK1 expression.

Interestingly, there were 13 significant decreases and 3 significant increases in kinase expression in the trisomic groups (combined) compared to euploid; if one assumes a null hypothesis that the direction of (significant) expression change should be random with respect to whether it is increased or decreased in the trisomic groups, then the trend of decreased expression in trisomy was statistically significant (Sign test, p = 0.021).

8.4.2 Protein kinase expression at the RNA level

RNA levels were compared between the trisomic groups and the euploid group for the kinases whose comparisons were statistically significant at the protein level (Table 8.3). Two kinases, ERK1 (16pl2-pl 1.2) and CDK1 (10q21.1), had significantly altered expression in the same direction at both the RNA and protein levels: ERK1 (higher) and CDK1 (lower) in trisomy

16 compared to euploid (Tables 8.2 and 8.3). Figure 8.1 illustrates how the chromosome 16- encoded ERK1 is significantly increased at both the RNA and protein levels in trisomy 16 compared to the euploid group. In contrast, PKG1 and DNAPK showed inverse relationships between RNA and protein expression (Tables 8.2 and 8.3).

8.4.3 Inter-individual variation in expression

The coefficients of variation (CV) for RNA and protein expression for each karyotype are illustrated in Figure 8.2 and compared in Table 8.4. The coefficient (CV) was used as a measure of inter-individual variability in expression. At the protein level, the coefficient of variation was significantly higher in both trisomic groups compared to the euploid group. At the

RNA level, the coefficient of variation was significantly lower in both trisomic groups compared to the euploid group. Figure 8.2 and Table 8.4 illustrate that for the euploid group,

148 the coefficients at the RNA and protein levels were equal, while for both trisomic groups, the coefficient was significantly lower at the RNA level compared to the protein level.

8.5 Discussion

Consistent with the hypothesis, this study demonstrated the simultaneous operation of both gene dosage effects and amplified instability on protein kinase expression in trisomy 16 placental fibroblasts in vitro. The mostly unique pattern of differentially expressed protein kinases in trisomy 16 versus euploid, using trisomy 15 as a control, supports the gene dosage hypothesis. The gene dosage hypothesis is also supported by the ERK1 over-expression at both the RNA and protein levels in trisomy 16. The two other trisomy 16 genes, however, did not exhibit protein over-expression, which is consistent with the finding of no change in protein expression for most chromosome 21 genes in Down syndrome fetal cerebral cortex (Cheon et al.

2003a; Cheon et al. 2003b; Cheon et al. 2003c; Cheon et al. 2003d; Ferrando-Miguel et al.

2004). These protein results are in contrast to the increased RNA expression on average of genes on the trisomic chromosome (Chrast et al. 2000; FitzPatrick et al. 2002; Gross et al. 2002;

Mao et al. 2003; Saran et al. 2003; Amano et al. 2004; Giannone et al. 2004; Kahlem et al. 2004;

Lyle et al. 2004; Tang et al. 2004; Dauphinot et al. 2005).

The trisomic groups combined showed a significant trend toward decreased protein kinase expression, an observation also seen in studies of signalling proteins in Down syndrome

(Engidawork and Lubec 2003). This trend may be related to the undetectable levels of S6K p70 in both trisomic groups (Table 8.2), since S6K p70 promotes translation of ribosomal proteins and translational factors (Shah et al. 2000). Similarly, of 9 translation factors that were examined in Down syndrome fetal cerebral cortex, 3 were down-regulated and 5 were unchanged (Freidl et al. 2001; Engidawork and Lubec 2003). Thus, a translational 'deficiency' could account for decreased expression of protein kinases in trisomy.

149 Increased inter-individual variation (quantified by the coefficient of variation) in protein kinase expression at the protein level provides support for the amplified instability hypothesis in both trisomy 16 and trisomy 15. The finding of decreased inter-individual variation at the RNA level, however, is contradictory to previous studies. Saran et al. (2003) observed increased

RNA expression variation between trisomic mice on inspection of graphs produced by principal components analysis (PCA), while Mao et al. (2003) observed no difference for human fetal

Down syndrome brain on inspection of graphs of the coefficient of variation. The difference between previous studies and this study may be related to differences in methodology: Saran et

al. (2003) and Mao et al. (2003) used genome-wide Affymetrix microarrays, as opposed to only

75 protein kinases in this study; and the biological interpretations of PCA and the coefficient of

variation are not identical. Also, the coefficients of variation were statistically compared only in

this study. Regardless, the increased inter-individual variation in trisomy observed at only the

protein level in this study suggests amplified instability manifests specifically on protein

expression for these protein kinases. This may be due to amplified sensitivity to environmental

differences (e.g. slight variation between cell cultures) or, more likely, to genome-wide genetic

variation between trisomic conceptuses. Since variability at the RNA level was not increased,

trisomic protein expression may be particularly sensitive to genetic variation specific to the

translational machinery (e.g. regulatory factors binding cis elements on RNA transcripts) or to

protein degradation (e.g. proteases).

A Western blot approach was utilized in this study instead of 2-D gel electrophoresis

techniques because the 2-D gel positions of fewer than two-dozen kinases have been elucidated

(Pelech et al. 2003). Protein kinases are difficult to identify on 2-D gels because their

intracellular concentrations are much lower (100-1000x) than those of proteins involved in

metabolism or cell structure (Pelech et al. 2003). Our study was limited by small sample size,

in part because of the difficulty with culturing of human miscarriage material and relatively

150 small probability of obtaining any one trisomy; this limited the power of the analyses, and as a consequence, a correction was not made for multiple comparisons of kinase expression levels between karyotypes. Furthermore, the conclusions may be only applicable to this set of protein kinases in trisomy 16. It is possible that different patterns of gene expression may be present with other protein kinases or other types of signalling proteins and in other trisomies. Also, cell cultures are always artificial models of complex in vivo systems, where gene expression is affected by complex cell-cell and cell-matrix interactions within a finely tuned hormonal and growth factor milieu. The chorionic villus mesenchymal fibroblasts are also only one cell type in the placenta, and different expression patterns may be present in the functionally important trophoblast subpopulations.

Despite the limitations of this study, the findings add some insight into the pathogenesis

of trisomy 16 in miscarriages, as well as in ongoing pregnancies complicated by confined

placental mosaicism (CPM). It is interesting that ERK1, a major intracellular regulator of cell

proliferation (Roux and Blenis 2004), showed gene dosage-related over-expression in trisomy

16. It remains to be seen whether a small increase (~2x) in ERK1 expression can alter signal

transduction pathways enough to perturb trisomy 16 embryological development. The presence

of amplified instability in vitro raises the possibility that genetic variation (e.g. single gene

mutations) or environmental variation (e.g. maternal effects during pregnancy) may play a role

in the trisomy 16 pregnancies in vivo. The presence of both dosage effects and amplified

instability supports chromosome 16-specific, as well as genome-wide and multifactorial factors,

in the pathogenesis of trisomy 16.

151 8.6 References

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Auer H, Lyianarachchi S, Newsom D, Klisovic MI, Marcucci G, Kornacker K (2003) Chipping away at the chip bias: RNA degradation in microarray analysis. Nat Genet 35:292-293

Blaschitz A, Weiss U, Dohr G, Desoye G (2000) Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta 21:733- 741

Cheon MS, Bajo M, Kim SH, Claudio JO, Stewart AK, Patterson D, Kruger WD, Kondoh H, Lubec G (2003a) Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part II). Amino Acids 24:119-125

Cheon MS, Kim SH, Ovod V, Kopitar Jerala N, Morgan JI, Hatefi Y, Ijuin T, Takenawa T, Lubec G (2003b) Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part III). Amino Acids 24:127-134

Cheon MS, Kim SH, Yaspo ML, Blasi F, Aoki Y, Melen K, Lubec G (2003c) Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I). Amino Acids 24:111-117

Cheon MS, Shim KS, Kim SH, Hara A, Lubec G (2003d) Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis (Part IV). Amino Acids 25:41-47

Chrast R, Scott HS, Papasavvas MP, Rossier C, Antonarakis ES, Barras C, Davisson MT, Schmidt C, Estivill X, Dierssen M, Pritchard M, Antonarakis SE (2000) The mouse brain transcriptome by SAGE: differences in gene expression between P30 brains of the partial trisomy 16 mouse model of Down syndrome (Ts65Dn) and normals. Genome Res 10:2006-2021

Dauphinot L, Lyle R, Rivals I, Dang MT, Moldrich RX, Golfier G, Ettwiller L, Toyama K, Rossier J, Personnaz L, Antonarakis SE, Epstein CJ, Sinet PM, Potier MC (2005) The cerebellar transcriptome during postnatal development of the TslCje mouse, a segmental trisomy model for Down syndrome. Hum Mol Genet 14:373-384

Engidawork E, Gulesserian T, Fountoulakis M, Lubec G (2003) Aberrant protein expression in cerebral cortex of fetus with Down syndrome. Neuroscience 122:145-154

Engidawork E, Lubec G (2003) Molecular changes in fetal Down syndrome brain. J Neurochem 84:895-904

152 Epstein CJ (1990) The consequences of chromosome imbalance. Am J Med Genet Suppl 7:31- 37

Ferrando-Miguel R, Cheon MS, Lubec G (2004) Protein levels of genes encoded on chromosome 21 in fetal Down Syndrome brain (Part V): overexpression of phosphatidyl-inositol-glycan class P protein (DSCR5). Amino Acids 26:255-261

FitzPatrick DR, Ramsay J, McGill NI, Shade M, Carothers AD, Hastie ND (2002) Transcriptome analysis of human autosomal trisomy. Hum Mol Genet 11:3249-3256

Freidl M, Gulesserian T, Lubec G, Fountoulakis M, Lubec B (2001) Deterioration of the transcriptional, splicing and elongation machinery in brain of fetal Down syndrome. J Neural Transm Suppl:47-57

Giannone S, Strippoli P, Vitale L, Casadei R, Canaider S, Lenzi L, D'Addabbo P, Frabetti F, Facchin F, Farina A, Carinci P, Zannotti M (2004) Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome. Ann Hum Genet 68:546-554

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153 Pritchard MA, Kola I (1999) The "gene dosage effect" hypothesis versus the "amplified developmental instability" hypothesis in Down syndrome. J Neural Transm Suppl 57:293-303

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Saran NG, Pletcher MT, Natale JE, Cheng Y, Reeves RH (2003) Global disruption of the cerebellar transcriptome in a Down syndrome mouse model. Hum Mol Genet 12:2013- 2019

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Shapiro BL (2001) Developmental instability of the cerebellum and its relevance to Down syndrome. J Neural Transm Suppl: 11-34

Tang Y, Schapiro MB, Franz DN, Patterson BJ, Hickey FJ, Schorry EK, Hopkin RJ, Wylie M, Narayan T, Glauser TA, Gilbert DL, Hershey AD, Sharp FR (2004) Blood expression profiles for tuberous sclerosis complex 2, neurofibromatosis type 1, and Down's syndrome. Ann Neurol 56:808-814

Zar JH (1996) Biostatistical Analysis. Prentice Hall, Upper Saddle River, New Jersey

154 Table 8.1 Immunochemistry, and protein/RNA profiling of mesenchymal core cultures

Case Karyotype GA CK-7 Vimen LCA PECAM Desmin FSP P R

Eu-1 46,XY 12 - - - - + Eu-2 47,XY 10 - - - Eu-3 47,XY 10 + - - - + Eu-4 46.XX 1" * Eu-5 46,XY 13 * Eu-6 46,XY 6 * * Eu-7 46,XX 1st - * * Eu-8 46,XY 10 * Eu-9 46,XX 12 * T16-1 47,XX,+ 16 8 - + - - - + T16-3 47,XY,+16 10 - + - - - + T16-4 47,XY,+ 16 12 - + - - ± * T16-5 47,XX,+16 8 - * * T16-6 47,XX+16 10 - + - - ± * * T15-1 47,XY,+15 10 - + - - + * T15-2 Male,+15' 11 - - * * T15-3 47,XX,+15 10 * * T15-4 47,XY,+15 8 *

"GA" = gestational age; "Is" = first-trimester, but exact gestational age unknown. "Vimen" = Vimentin. "-" = <1% cells stained positive. "±" = most cells were FSP positive, but relatively less (both in proportion of cells and intensity) compared to vimentin staining. "P" = protein profiling was done. "R" = RNA profiling was done. 'CGH failed for this sample; the sex and extra chromosome 15 was determined by microsatellite PCR.

155 Table 8.2 Protein levels of kinases with significant differences in protein expression

Kinase Chr Euploid Trisomy 16 Trisomy 15 Euploid Euploid Mean Mean Mean vs. vs. expression expression expression Trisomy 16 Trisomy 15 CDK1 10q21.1 520 ± 202 101 ± 174 362 ±319 p = 0.010 - CDK7 5ql2.1 887 ±114 569 ±100 729 ± 73 p = 0.012 - PKC-e 2p21 2708 ± 301 1271 ±621 1408±966 p = 0.012 - PKG1 10qll.2 3401 ±743 5642 ±419 3970± 1874 p = 0.012 - ERK1 16pl2- 6115 ±1120 12148 ±4326 6985± 1559 p = 0.006 pl 1.2 S6K 17q23.2 180 ± 80 0+-0 0±0 p = 0.012 p = 0.012 p70 IKK-a 14ql3 1484 ± 254 743 ± 35 1019±185 p = 0.012 p = 0.012 CKl-e 22ql3.1 192 ± 99 36 ±62 0±0 - p = 0.012 SRC 20ql2- 59 ± 17 14 ±24 0±0 - p = 0.012 ql3 CDK9 9q34.1 5234 ± 805 4728 + 419 3401 ±785 - p = 0.012

DNAPK 8qll 755 ± 270 577 + 401 0±0 - p = 0.012 MEK2 19pl3.3 1754 ± 220 1340±703 1015 ±441 - p = 0.012 PKC-z lp36.33- 15778 ±1968 13839± 1817 9968 ± 3248 - p = 0.012 p36.2 PKC-P 16pll.2 5994 ± 583 5294 ± 1267 9882 ±2371 - p = 0.012

Bolded mean expression levels are the higher of each statistically significant pair-wise comparison (2-sample rank t-test; see text). Italicized mean expression levels are of the other trisomy not involved in the statistically significant pair-wise comparison.

156 Table 8.3 RNA levels of kinases with significant differences in protein expression

Kinase Chr Euploid Trisomy 16 Trisomy 15 Euploid vs. Euploid vs. Mean Mean Mean Trisomy 16 Trisomy expression expression expression 15 CDK1 10q21.1 1.03 ± 0.25 0.47 ± 0.36 - p = 0.042 - CDK7 5ql2.1 1.15 + 0.46 1.54 ±0.14 - p = 0.41 - PKC-e 2p21 1.23 ±0.16 0.98 ±0.12 - p = 0.31 - PKG1 10qll.2 1.37 ± 0.44 1.00 ±0.008 - p = 0.042 - ERK1 16pl2- 1.12 ± 0.12 1.54 ± 0.07 - p = 0.021 pll.2 S6K p70 17q23.2 0.88 ± 0.23 0.92 +- 0.009 0.88 ± 0.25 p = 0.41 p = 0.76 IKK-oc 14ql3 0.78 ± 0.22 0.74 ± 0.004 0.79 ±0.10 p= 1.00 p = 0.53 CKl-e 22ql3.1 0.49 ± 0.07 - 0.45 ±0.21 - p = 0.53 SRC 20ql2-ql3 1.07 ±0.53 - 0.89 ± 0.26 - p = 0.76 CDK9 9q34.1 0.98 ±0.18 - 1.04 ±0.15 - p = 0.76 DNAPK 8ql 1 0.68 ± 0.04 - 0.87 ±0.18 - p = 0.012 MEK2 19pl3.3 1.20 ±0.16 - 1.15 ±0.24 - p= 1.00 PKC-z lp36.33- 0.41 ±0.07 - 0.52 ±0.11 - p = 0.33 p36.2 PKC-p 16pll.2 1.27 ±0.36 - 1.10 ±0.01 - p= 1.00

Bolded mean expression levels are the higher of a statistically significant pair-wise comparison (2-sample rank t-test; see text).

157 Table 8.4 Coefficients of variation (CV) at the RNA and protein levels

Karyotype Mean Karyotype Mean n Comparison [Level] [Level] Euploid [RNA] 0.32 + 0.19 Euploid [Protein] 0.31 ±0.24 47 p = 0.50 Trisomy 16 0.19 ± 0.18 Trisomy 16 0.49 ± 0.50 44 p < 0.001 [RNA] [Protein] Trisomy 15 0.24 ±0.18 Trisomy 15 0.52 ± 0.42 42 p < 0.001 [RNA] [Protein] Euploid [RNA] 0.32 ±0.19 Trisomy 16 0.19±0.18 47 p < 0.001 [RNA] Euploid [RNA] 0.32 ±0.19 Trisomy 15 0.25 ±0.17 47 p = 0.02 [RNA] Euploid 0.27 ±0.14 Trisomy 16 0.49 ± 0.50 44 p < 0.01 [Protein] [Protein] Euploid 0.29 ± 0.24 Trisomy 15 0.52 ± 0.42 42 p < 0.001 [Protein] [Protein]

Bolded mean expression levels are the higher of each statistically significant pair-wise comparison (Wilcoxon test; see text). There are different numbers of kinases (n) in these paired-sample comparisons because for the trisomic karyotypes, some kinases were not expressed at the protein level such that the coefficient of variation was undefined.

158 Figure 8.1 ERK1 RNA and protein expression

1.50 H 15000

RNA Protein

1.00 J U IOOOO Eu T16 T15

0.50 J - 5000

0 0 RNA Protein

Mean ± standard error. ERK1 RNA and protein expression in trisomy 16 (T16), but not in trisomy 15 (T15), are significantly increased compared to euploid (Eu) expression (see Tables 8.2 and 8.3, and Figure 8-1.

159 Figure 8.2 Coefficient of variation (CV) of RNA and protein expression

0.60'

0.501

CV 0.40-

0.30 •

Eu T16 T15 0.201 I Eu T16 T15 0.10-L

RNA Protein

Mean ± standard error. For statistical comparisons, see Table 8.4 and the text. 9 Conclusion

This thesis research has focused on cytogenetic, biological, and clinical aspects of trisomy in the placenta. Trisomy CPM pregnancies have reduced placental weight and birth weight, and birth weight was determined in part by the level of trisomic trophoblast, through an alteration in placental function independent of placental weight (Chapter 2). Pregnancies with trisomy 16 CPM (CPM 16) were at particularly high-risk for abnormal outcomes such as IUGR and malformation, and the risk was modulated by factors such as the presence of trisomy 16 cells in amniotic fluid, full trisomy on chorionic villus sampling (CVS), sex of the fetus, mode of ascertainment, and uniparental disomy for chromosome 16 (upd(16)mat) in the fetus (Chapter

3). CPM 16 pregnancies were also at increased risk for preeclampsia (Chapter 4). However, infants from such pregnancies had a good postnatal prognosis in terms of catch-up growth and developmental progress (Chapter 5). Predictors of developmental delay included trisomy in amniotic fluid, presence of malformation, and low birth weight for gestational age, which may all be markers of low-level trisomy mosaicism in tissues of the infant.

The higher risk of abnormal outcome in CPM 16 pregnancies and the high incidence of trisomy 16 in miscarriages may be related in part to the observed poorer outgrowth of trisomy

16 extravillus trophoblast (EVT) in vitro (Chapter 7). Other than trisomy 16, EVT outgrowth in vitro was found to be variable among other chromosomally abnormal miscarriages as a group; trisomy 15 in particular showed a similar distribution in EVT outgrowth compared to euploid miscarriages. Although gene expression in trisomy 16 trophoblast could not be examined (due to no outgrowths), it is apparent that there is an altered distribution of protein kinase expression in trisomy 16 placental fibroblasts compared to euploid placental fibroblasts, and in a manner that differs from trisomy 15 placental fibroblasts (Chapter 8). A dose-related increase in ERK1 protein expression was present in trisomy 16. There was also an increase in the variance in

161 protein kinase expression in the trisomic groups, which interestingly, manifested only at the protein level. These observations indicate that both dosage-effects and amplified instability due to the effects of genomic and environmental variation, can operate simultaneously in trisomy.

There have been a number of studies involving post-partum cytogenetic analysis of the placenta in pregnancies with small-for-gestational age (SGA) newborns, with widely divergent results in the frequency of CPM (Kalousek and Dill 1983; Verp and Unger 1990; Kennerknecht et al. 1993; Wolstenholme et al. 1994; Artan et al. 1995; Krishnamoorthy et al. 1995; Wilkins-

Haug et al. 1995; Stipoljev et al. 2001; Masuzaki et al. 2004; Grati et al. 2005). The variable results are likely related to the heterogeneity in methodology, including the type of cytogenetic analysis (conventional vs. FISH vs. molecular/PCR), the number of placental sites sampled, and the tissue-types analyzed (e.g. trophoblast, mesenchymal core, and/or chorionic plate). No systematic study of post-partum placental sampling has been published for pre-eclampsia, though such a study is in-progress in the laboratory of my supervisor (W. Robinson). For idiopathic IUGR or preeclamptic pregnancies, it is possible that identification of more 'high- risk' cases will yield higher rates of CPM at post-partum cytogenetic investigation. Examples include severe SGA newborns (<3rd percentile), with or without malformation, from pregnancies with pre-term labour, PROM and/or other complications such as oligohydramnios. In addition, most of the studies on SGA pregnancies depended on tissue culture for cytogenetic analysis. It is reasonable to expect that chromosomally abnormal cells may be less likely to divide in vitro and produce adequate metaphases, resulting in some cases of CPM being missed with such methods. As an alternative, methods that not depend on cell divisions can be used, such as comparative genomic hybridization (CGH). For example, Amiel et al. (2002) used CGH to look for CPM in IUGR pregnancies with other features 'suspicious' of chromosome abnormality as determined by a perinatal pathologist. The majority (61%; 14/24) of the IUGR cases showed chromosome abnormalities, compared to 0/6 controls (Fisher Exact test, p = 0.013); however, in

162 only a few cases was there any attempt to confirm the findings by FISH. It is possible that

selected cases of complicated pregnancy may harbour undiagnosed chromosome abnormalities in the placenta.

The mostly normal developmental outcome of CPM 16 suggests that trisomy 16 completely confined to the placenta has minimal postnatal consequences. However, there is

evidence from mouse models that an abnormality confined to the placenta can contribute to

neurologic affects in the fetus. In two fascinating studies, de Bruin et al. (2003) and Wu et al.

(2003) showed that retinoblastoma (Rb) knockout mice have extensive placental abnormalities,

and that when Rb knockout embryos are 'rescued' by aggregating with a tetraploid embryo to

produce a tetraploid placenta (James and West 1994), much of the Rb knockout fetal phenotype,

including some of the neurologic abnormalities, were corrected. This suggests that an abnormal

placenta, in this case lacking the Rb gene, can contribute to neurologic outcome. There are

well-developed mouse models of Down Syndrome involving partial trisomy for the region of

mouse chromosome 16 (orthologous to human chromosome 21) carried on a marker

chromosome, originally produced as a translocation from chromosome 16 (Dierssen et al. 2001).

To further address the role of the placenta in neurodevelopment, trisomic embryos that are

produced from a balanced carrier parent could be rescued with a tetraploid placenta to determine

whether some aspect of the mental retardation could be rectified.

In conclusion, this thesis research has further clarified the clinical implications of

trisomy confined to the placenta, in particular trisomy 16 CPM, as well as cytogenetic and

biological aspects of the pathogenesis of placental mosaicism. It is likely that with properly

conducted post-partum studies of the placenta, confined placental trisomy will be more widely

recognized as an etiological factor in idiopathic pregnancy complications. In the future, clinical

features that make a postnatal diagnosis of CPM more likely should be identified, and in such

pregnancies, CPM listed in the differential diagnosis. 9.1 References

Amiel A, Bouaron N, Kidron D, Sharony R, Gaber E, Fejgin MD (2002) CGH in the detection of confined placental mosaicism (CPM) in placentas of abnormal pregnancies. Prenat Diagn 22:752-758

Artan S, Basaran N, Hassa H, Ozalp S, Sener T, Sayli BS, Cengiz C, Ozdemir M, Durak T, Dolen I, et al. (1995) Confined placental mosaicism in term placentae: analysis of 125 cases. Prenat Diagn 15:1135-1142

de Bruin A, Wu L, Saavedra HI, Wilson P, Yang Y, Rosol TJ, Weinstein M, Robinson ML, Leone G (2003) Rb function in extraembryonic lineages suppresses apoptosis in the CNS of Rb-deficient mice. Proc Natl Acad Sci U S A 100:6546-6551

Dierssen M, Fillat C, Crnic L, Arbones M, Florez J, Estivill X (2001) Murine models for Down syndrome. Physiol Behav 73:859-871

Galdzicki Z, Siarey R, Pearce R, Stoll J, Rapoport SI (2001) On the cause of mental retardation in Down syndrome: extrapolation from full and segmental trisomy 16 mouse models. Brain Res Brain Res Rev 35:115-145

Grati FR, Miozzo M, Cassani B, Rossella F, Antonazzo P, Gentilin B, Sirchia SM, Mori L, Rigano S, Bulfamante G, Cetin I, Simoni G (2005) Fetal and placental chromosomal mosaicism revealed by QF-PCR in severe IUGR pregnancies. Placenta 26:10-18

Heller JH, Spiridigliozzi GA, Doraiswamy PM, Sullivan JA, Crissman BG, Kishnani PS (2004) Donepezil effects on language in children with Down syndrome: results of the first 22- week pilot clinical trial. Am J Med Genet A 130:325-326

Heller JH, Spiridigliozzi GA, Sullivan JA, Doraiswamy PM, Krishnan RR, Kishnani PS (2003) Donepezil for the treatment of language deficits in adults with Down syndrome: a preliminary 24-week open trial. Am J Med Genet A 116:111-116

Hunter P, Smith N, Fernandez J, Tawhai M (2005) Integration from proteins to organs: the IUPS Physiome Project. Mech Ageing Dev 126:187-192

Hunter PJ, Borg TK (2003) Integration from proteins to organs: the Physiome Project. Nat Rev Mol Cell Biol 4:237-243

James RM, West JD (1994) A chimaeric animal model for confined placental mosaicism. Hum Genet 93:603-604

Johnson N, Fahey C, Chicoine B, Chong G, Gitelman D (2003) Effects of donepezil on cognitive functioning in Down syndrome. Am J Ment Retard 108:367-372

Kalousek DK, Dill FJ (1983) Chromosomal mosaicism confined to the placenta in human conceptions. Science 221:665-667

164 Kennerknecht I, Kramer S, Grab D, Terinde R, Vogel W (1993) A prospective cytogenetic study of third-trimester placentae in small-for-date but otherwise normal newborns. Prenat Diagn 13:257-269

Kishnani PS, Sullivan JA, Walter BK, Spiridigliozzi GA, Doraiswamy PM, Krishnan KR (1999) Cholinergic therapy for Down's syndrome. Lancet 353:1064-1065

Kondoh T, Amamoto N, Doi T, Hamada H, Ogawa Y, Nakashima M, Sasaki H, Aikawa K, Tanaka T, Aoki M, Harada J, Moriuchi H (2005) Dramatic improvement in Down syndrome-associated cognitive impairment with donepezil. Ann Pharmacother 39:563- 566

Krishnamoorthy A, Gowen LC, Boll KE, Knuppel RA, Sciorra LJ (1995) Chromosome and interphase analysis of placental mosaicism in intrauterine growth retardation. J Perinatol 15:47-50

Lott IT, Osann K, Doran E, Nelson L (2002) Down syndrome and Alzheimer disease: response to donepezil. Arch Neurol 59:1133-1136

Masuzaki H, Miura K, Yoshiura KI, Yoshimura S, Niikawa N, Ishimaru T (2004) Detection of cell free placental DNA in maternal plasma: direct evidence from three cases of confined placental mosaicism. J Med Genet 41:289-292

Prasher VP (2004) Review of donepezil, rivastigmine, galantamine and memantine for the treatment of dementia in Alzheimer's disease in adults with Down syndrome: implications for the intellectual disability population. Int J Geriatr Psychiatry 19:509-515

Prasher VP, Adams C, Holder R (2003) Long term safety and efficacy of donepezil in the treatment of dementia in Alzheimer's disease in adults with Down syndrome: open label study. Int J Geriatr Psychiatry 18:549-551

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Stipoljev F, Latin V, Kos M, Miskovic B, Kurjak A (2001) Correlation of confined placental mosaicism with fetal intrauterine growth retardation. A case control study of placentas at delivery. Fetal Diagn Ther 16:4-9

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Wilkins-Haug L, Roberts DJ, Morton CC (1995) Confined placental mosaicism and intrauterine growth retardation: a case-control analysis of placentas at delivery. Am J Obstet Gynecol 172:44-50

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165 Wu L, de Bruin A, Saavedra HI, Starovic M, Trimboli A, Yang Y, Opavska J, Wilson P, Thompson JC, Ostrowski MC, Rosol TJ, Woollett LA, Weinstein M, Cross JC, Robinson ML, Leone G (2003) Extra-embryonic function of Rb is essential for embryonic development and viability. Nature 421:942-947 Appendix B Extra references

References for CPM cases in Table 2.1 in Chapter 2

Johnson MP, Childs MD, Robichaux III AG, Isada NB, Pryde PG, Koppitch III FC, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108.

Kalousek DK, Langlois S, Barrett IJ, Yam I, Wilson DR, Howard-Peebles PN, Johnson MP, Giorgiutti E (1993) Uniparental disomy for chromosome 16 in humans. Am J Hum Genet 52:8-16.

Kalousek DK, Langlois S, Robinson WP, Telenius A, Bernard L, Barrett IJ, Howard- Peebles PN, Wilson RD (1996) Trisomy 7 CVS mosaicism: pregnancy outcome, placental and DNA analysis in 14 cases. Am J Med Genet 65:348-352.

Kuchinka BD, Barrett IJ, Moya G, Sanchez JM, Langlois S, Yong SL, Kalousek DK, Robinson WP (2001) Two cases of confined placental mosaicism for chromosome 4, including one with maternal uniparental disomy. Prenat Diagn 21:36-39

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong S-L, Lewis S, Bruyere H, Howard-Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446.

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard-Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927.

Shaffer LG, Langlois S, McCaskill C, Main DM, Robinson WP, Barrett IJ, Kalousek DK (1996) Analysis of nine pregnancies with confined placental mosaicism for trisomy 2. Prenat Diagn 16:899-905

References for CPM16 cases in Methods in Chapter 3

Excluded CPM16 cases of paternal origin of the trisomy

Kohlhase J, Janssen B, Weidenauer K, Harms K, Bartels I (2000) First confirmed case with paternal uniparental disomy of chromosome 16. Am J Med Genet 91:190-191.

Paulyson KJ, Sherer DM, Christian SL, Lewis KM, Ledbetter DM, Salafia CM, Meek JM (1996) Prenatal diagnosis of an infant with mosaic trisomy 16 of paternal origin. Prenat Diagn 16:1021-1026.

168 Excluded CPM16 cases of partial trisomy

Devi AS, Egan JFX, Campbell W, Ingardia C, Rosengren S, Tezcan K, Weiser J, Benn PA (1997) Poor pregnancy outcome and the presence of trisomy 16 cells in amniotic fluid. Am J Hum Genet 61:A151.

Hsu WT, Shchepin DA, Mao R, Berry-Kravis E, Garber AP, Fischel-Ghodsian N, Falk RE, Carlson DE, Roeder ER, Leeth EA, Hajianpour MJ, Wang J-C C, Rosenblum-Vos LS, Bhatt SD, Karson EM, Hux CH, Trunca C, Bialer MG, Linn SK, Schreck RR (1998) Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases. Am J Med Genet 80:473-480.

Schinzel A, Kotzot D, Brecevic L, Robinson WP, Dutly F, Dauwerse H, Binkert F, Baumer A, Ausserer B (1997) Trisomy first, translocation second, uniparental disomy and partial trisomy third: a new mechanism for complex chromosomal aneuploidy. Eur J Hum Genet 5:308-314.

Excluded CPM16 case of concomitant aneuploidy

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard-Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927.

CPM16 cases from UBC study with some data previously published

Kalousek DK, Howard-Peebles PN, Olson SB, Barrett IJ, Dorfmann A, Black SH, Schulman JD, Wilson RD (1991) Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism. Prenat Diagn 11:743-450.

Kalousek DK, Langlois S, Barrett IJ, Yam I, Wilson DR, Howard-Peebles PN, Johnson MP, Giorgiutti E (1993) Uniparental disomy for chromosome 16 in humans. Am J Hum Genet 52:8-16.

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard-Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927.

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong S-L, Lewis S, Bruyere H, Howard-Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446.

Stavropoulos DJ, Bick D, Kalousek DK (1998) Molecular cytogenetic detection of

169 confined gonadal mosaicism in a conceptus with trisomy 16 placental mosaicism. Am J Hum Genet 63:1912-1914.

CPM16 cases from UBC study overlapping with papers by other research groups

Chan Y, Silverman N, Jackson L, Wapner R, Wallerstein R (2000) Maternal uniparental disomy of chromosome 16 and body stalk anomaly. Am J Med Genet 94:284-286.

Dworniczak B, Koppers B, Kurlemann G, Holzgreve W, Horst J, Miny P (1992) Uniparental disomy with normal phenotype. Lancet 340:1285.

Holzgreve R, Exeler R, Holzgreve W, Wittwer B, Miny P (1992) Non-viable trisomies confined to the placenta leading to poor pregnancy outcome. Prenat Diagn 12 (Suppl):S95.

Hsu LYF, Yu M-T, Neu RL, Van Dyke DL, Benn PA, Bradshaw CL, Shaffer LG, Higgins RR, Khodr GS, Morton CC, Wang H, Brothman AR, Chadwick D, Disteche CM, Jenkins LS, Kalousek DK, Pantzer TJ, Wyatt P (1997) Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations. Prenat Diagn 17:201-242.

Hsu WT, Shchepin DA, Mao R, Berry-Kravis E, Garber AP, Fischel-Ghodsian N, Falk RE, Carlson DE, Roeder ER, Leeth EA, Hajianpour MJ, Wang J-C C, Rosenblum-Vos LS, Bhatt SD, Karson EM, Hux CH, Trunca C, Bialer MG, Linn SK, Schreck RR (1998) Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases. Am J Med Genet 80:473-480.

Johnson MP, Childs MD, Robichaux Ul AG, Isada NB, Pryde PG, Koppitch III FC, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108.

Kennerknecht I, Terinde R (1990) Intrauterine growth retardation associated with chromosomal aneuploidy confined to the placenta. Three observations: triple trisomy 6, 21, 22; trisomy 16; and trisomy 18. Prenat Diagn 10:539-544.

Schneider AS, Bischoff FZ, McCaskill C, Coady ML, Stopfer JE, Shaffer LG (1996) Comprehensive 4-year follow-up on a case of maternal heterodisomy for chromosome 16. Am J Med Genet 66:204-208.

Schwinger E, Seidl E, Klink F, Rehdar H (1989) Chromosome mosaicism of the placenta: a cause of developmental failure of the fetus? Prenat Diagn 9:639-647.

Verp MS, Rosinsky B, Sheikh Z, Amarose AP (1989) Non-mosaic trisomy 16 confined to villi. Lancet 2:915-916.

Wolstenholme J (1995) An audit of trisomy 16 in man. Prenat Diagn 15:109-121.

Woo V, Bridge PJ, Bamforth JS (1997) Maternal uniparental disomy for chromosome 16: case report. Am J Med Genet 70:387-390.

170 Zimmerman R, Lauper U, Streichier A, Huch R, Hugh A (1995) Elevated alpha- fetoprotein and human chorionic gonadotropin as a marker for placental trisomy 16 in the second trimester? Prenat Diagn 15:1121-1124.

CPM16 cases from other published reports to date

Abu-Amero SN, Ali Z, Abu-Amero KK, Stanier P, Moore GE (1999) An analysis of common isodisomic regions in five mUPD 16 probands. J Med Genet 36:204-207.

Association of Clinical Cytogenetics Working Party on Chorionic Villi in Prenatal Diagnosis (1994) Cytogenetic analysis of chorionic villi for prenatal diagnosis: An ACC collaborative study of U.K. data. Prenat Diagn 14:363-379.

Astner A, Schwinger E, Caliebe A, Jonat W, Gembruch U (1998) Sonographically detected fetal and placental abnormalities associated with trisomy 16 confined to the placenta. A case report and review of the literature. Prenat Diagn 18:1308-1315.

Benn P (1998) Trisomy 16 and trisomy 16 mosaicism: A review. Am J Med Genet 79:121-133.

Benn P, Craffey A, Home D, Cusick W, Smeltzer J (1995) An association between trisomy 16 (and other fetal aneuploidy) in women with grossly elevated second trimester maternal serum human chorionic gonadotropin (MSHCG). Am J Hum Genet 57:A275.

Bennett P, Vaughan J, Henderson D, Loughna S, Moore G (1992) Association between confined placental trisomy, fetal uniparental disomy, and early intrauterine growth retardation. Lancet 340:1284-1285.

Brandenburg H, Los FJ, In't Veld P (1996) Clinical significance of placenta-confined nonmosaic trisomy 16. Am J Obstet Gynecol 174(5): 1663-1664.

Callen DF, Korban G, Dawson G, Gugasyan L, Krumins EJM, Eichbaum S, Petrass J, Purvis-Smith S, Smith A, Dendulk G, Martin N (1988) Extraembryonic/fetal karyotypic discordance during diagnostic chorionic villus sampling. Prenat Diagn 8:453-460.

Caspari D, Bartels I, Rauskolb R, Prange G, Osmers R, Eiben B (1994) Discrepant karyotypes after second- and third-trimester combined placentesis/amniocentesis. Prenat Diagn 14:569-576.

Chen CP, Shih JC, Chern SR, Lee CC, Wang W (2004) Prenatal diagnosis of mosaic trisomy 16 associated with congenital diaphragmatic hernia and elevated maternal serum alpha-fetoprotein and human chorionic gonadotrophin. Prenat Diagn Jan;24(l):63-6.

Davies GAL, Gad IK, Diamond T, Papenhausen P (1995) Discordant maternal serum and amniotic fluid alpha-fetoprotein results in mosaic trisomy 16 pregnancies. Am J Hum Genet 57:A278.

Devi AS, Egan JFX, Campbell W, Ingardia C, Rosengren S, Tezcan K, Weiser J, Benn

171 PA (1997) Poor pregnancy outcome and the presence of trisomy 16 cells in amniotic fluid. Am J Hum Genet 61:A151.

Devi AS, Kamath MV, Eisenfield L, Neu R, Ciarleglio L, Greenstein R, Benn P (1992) Mosaic trisomy 16 in the newborn: A recognizable syndrome. Am J Hum Genet 51:A1193.

Devi AS, Velinov M, Kamath MV, Eisenfield L, Neu R, Ciarleglio L, Greenstein R, Benn P (1993) Variable clinical expression of mosaic trisomy 16 in the newborn infant. Am J Med Genet 47:294-298.

Dorfmann AD, Perszyk J, Robinson P, Black SH, Schuman JD (1992) Rare non-mosaic trisomies in chorionic villus tissue not confirmed at amniocentesis. Prenat Diagn 12:899-902.

Farra C, Giudicelli B, Pellissier MC, Philip N, Piquet C (2000) Fetoplacental chromosomal discrepancy. Prenat Diagn 20:190-193

Fryburg JS, Dimaio MS, Mahoney MJ (1992) Postnatal placental confirmation of trisomy 2 and trisomy 16 detected at chorionic villus sampling: a possible association with intrauterine growth retardation and elevated maternal serum alpha-fetoprotein. Prenat Diagn 12:157-162

Fryburg JS, Dimaio MS, Yang-Feng TL, Mahoney MJ (1993) Follow-up of pregnancies complicated by placental mosaicism diagnosed by chorionic villus sampling. Prenat Diagn 13:481-494

Garber A, Carlson D, Schreck R, Fischel-Ghodsian N, Hsu WT, Oeztas S, Pepkowitz S, Graham JM, Jr. (1994) Prenatal diagnosis and dysmorphic findings in mosaic trisomy 16. Prenat Diagn 14:257-266

Gollop TR, Piere P De C, Naccache NF, Bittencourt EA (1990) Brazilian chorionic villus sampling (CVS): Experience with 900 cases. Am J Hum Genet 47:A1087.

Groli C, Cerri V, Tarantini M, Bellotti D, Jacobello C, Gianello R, Zanini R, Lancetti S, Zaglio S (1996) Maternal serum screening and trisomy 16 confined to the placenta. Prenat Diagn 16:685-689.

Hajianpour MJ (1995) Postnatally confirmed trisomy 16 mosaicism: follow-up on a previously reported patient. Prenat Diagn 15:877-879.

Hajianpour MJ, Randolph LM, Parvizpour D, Habibian R (1992) Trisomy 16 mosaicism in amniotic fluid cells and poor pregnancy outcome associated with unexplained elevated maternal serum alpha-fetoprotein. Am J Hum Genet 51:A409.

Hashish AF, Monk NA, Lovell-Smith MPF, Bardwell LM, Fiddes TM, Gardner RJM (1989) Trisomy 16 detected at chorion villus sampling. Prenat Diagn 9:427-432.

Hogge WA, Schonberg SA, Golbus MS (1986) Chorionic villus sampling: Experience of

172 the first 1,000 cases. Am J Obstet Gynecol 154:1249-1252.

Holzgreve R, Exeler R, Holzgreve W, Wittwer B, Miny P (1992) Non-viable trisomies confined to the placenta leading to poor pregnancy outcome. Prenat Diagn 12 (Suppl):S95.

Hsu LYF, Yu M-T, Neu RL, Van Dyke DL, Benn PA, Bradshaw CL, Shaffer LG, Higgins RR, Khodr GS, Morton CC, Wang H, Brothman AR, Chadwick D, Disteche CM, Jenkins LS, Kalousek DK, Pantzer TJ, Wyatt P (1997) Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations. Prenat Diagn 17:201-242.

Hsu WT, Shchepin DA, Mao R, Berry-Kravis E, Garber AP, Fischel-Ghodsian N, Falk RE, Carlson DE, Roeder ER, Leeth EA, Hajianpour MJ, Wang J-C C, Rosenblum-Vos LS, Bhatt SD, Karson EM, Hux CH, Trunca C, Bialer MG, Linn SK, Schreck RR (1998) Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases. Am J Med Genet 80:473-480.

Huff DS, Watkins C, Davis G, Wallerstein D, Lee M, Dyer K, McMorrow LE (1991) Mosaic trisomy 16 detected by mid-trimester amniocentesis. Am J Hum Genet Suppl 49:174.

Jalal S, Lindor NM, Bonde D, Karnes P (1992) Trisomy 16 mosaicism in a six month old infant. Am J Hum Genet 51: A290.

Johnson A, Wapner RJ, Davis GH, Jackson LG (1990) Mosaicism in chorionic villus sampling: an association with poor perinatal outcome. Obstet Gynecol 75:573-577.

Johnson MP, Childs MD, Robichaux HI AG, Isada NB, Pryde PG, Koppitch III FC, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108.

Johnson P, Duncan K, Blunt S, Bell G, Ali Z, Cox P, Moore GE (2000) Apparent confined placental mosaicm of trisomy 16 and multiple fetal anomalies: case report. Prenat Diagn 20:417-421.

Kalousek DK, Howard-Peebles PN, Olson SB, Barrett U, Dorfmann A, Black SH, Schulman JD, Wilson RD (1991) Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism. Prenat Diagn 11:743-450.

Leschot NJ, Wolf H (1991) Is placental mosaicism associated with poor perinatal outcome? Prenat Diagn 11:403-404.

Leschot NJ, Wolf H, Verjaal M, Van Prooijen-Knegt LC, De Boer EG, Kanhai HHH, Christiaens GCML (1987) Chorionic villi sampling: cytogenetic findings in 500 pregnancies. BMJ 295:407-410.

Lindor NM, Jalal SM, Thibodeau SN, Bonde D, Sauser KL, Karnes PS (1993) Mosaic

173 trisomy 16 in a thriving infant; maternal heterodisomy for chromsome 16. Clin Genet 44:185-189.

Moore, GE, Ali Z, Khan RU, Blunt S, Bennett PR, Vaughan JI (1997) The incidence of uniparental disomy associated with intrauterine growth retardation in a cohort of thirty- five severely affected babies. Am J Obstet Gynecol 176(2):294-299.

Morssink LP, Sikkema-Raddatz B, Beekhuis JR, Dewolf BTHM, Mantingh A (1996) Placental mosaicism is associated with unexplained second trimester elevation of MShCG levels but not with elevation of MS AFP levels. Prenat Diagn 16:845-851.

O'Riordan S, Greenough A, Moore GE, Bennett P, Nicolaides KH (1996) Case report: uniparental disomy 16 in association with congenital heart disease. Prenat Diagn 16:963-965.

Pletcher BA, Sanz MM, Schlessel JS, Kunaporn S, Mckenna C, Bialer MG, Alonso ML, Zaslav A-L, Brown WT, Ray JH (1994) Postnatal confirmation of prenatally diagnosed trisomy 16 mosaicism in two phenotypically abnormal liveborns. Prenat Diagn 14:933- 940.

Post JG, Nijhuis JG (1992) Trisomy 16 confined to the placenta. Prenat Diagn 12:1001- 1007.

Roeder E, Immken L, Bansal V, Favou A, Hersney D, Tabors E, Curry CJR (1994) New clinical findings in mosaic trisomy 16. Am J Med Genet 52:383.

Roland B, Lynch L, Berkowitz G, Zinberg R (1994) Confined placental mosaicism in CVS and pregnancy outcome. Prenat Diagn 14:589-593.

Rosenblum-Vos LS, Roberson AE, Meyers CM, Cohen MM (1993) Trisomy 16 mosaicism identified in mid-trimester amniocentesis and confirmed in fetal tissues. Am JHum Genet 53:1808.

Sanchez JM, Lopez De Diaz S, Panal MJ, Moya G, Kenny A, Iglesias D, Wolstenholme J (1997) Severe fetal malformations associated with trisomy 16 confined to the placenta. Prenat Diagn 17:777-779.

Simoni G, Brambati B, Maggi F, Jackson L (1992) Trisomy 16 confined to chorionic villi and unfavourable outcome of pregnancy. Ann Genet 35:110-112.

Simoni G, Gimelli G, Cuoco C, Romitti L, Terzoli G, Guerneri S, Rossella F, Pescetto L, Pezzolo A, Porta S, Brambat B, Porro E, Fraccaro M (1986) First trimester fetal karyotyping: one thousand diagnoses. Hum Genet 72:203-209.

Simoni G, Gimelli G, Cuoco C, Terzoli GL, Rossella F, Romitti L, Dalpra L, Nocera G, Tibiletti MG, Tenti P, Fraccaro M (1985) Discordance between prenatal cytogenetic diagnosis after chorionic villi sampling and chromosomal constitution of the fetus. In: Fraccaro M, Simoni G, Brambati B (eds) First Trimester Fetal Diagnosis. Berlin: Springer-Verlag 137-143.

174 Sirchia SM, Garagiola I, Colucii G, Guerneri S, Lalatta F, Grimoldi MG, Simoni G (1998) Trisomic zygote rescue revealed by DNA polymorphism analysis in confined placental mosaicism. Prenat Diagn 18:201-206.

Smith R, Zackai EH, Donnenfeld AE (1997) Prenatally diagnosed trisomy 16 mosaicism which escapes postnatal detection in an infant with congenital anomalies. Am J Hum Genet 61 :A140.

Sundberg K, Smidt-Jensen S (1991) Non-mosaic trisomy 16 on chorionic villus sampling but normal placenta and fetus after termination. Lancet 337:1233-1234.

Sutcliffe MJ, Mueller OT, Gallardo LA, Papenhausen PR, Tedesco TA (1993) Maternal isodisomy in a normal 46,XX following trisomic conception. Am J Hum Genet 53:A1464.

Tantravahi U, Matsumoto C, Delach J, Craffey A, Smeltzer J, Benn P (1996) Trisomy 16 mosaicism in amniotic fluid cell cultures. Prenat Diagn 16:749-754.

Tharapel AT, Elias S, Shulman LP, Seely L, Emerson DS, Simpson JL (1989) Resorbed co-twin as an explanation for discrepant chorionic villus results: non-mosaic 47,XX,+16 in villi (direct and culture) with normal (46,XX) amniotic fluid and neonatal blood. Prenat Diagn 9:467-472.

Van Opstal D, van den Berg C, Galjaard RJH, Los FJ (2001) Follow-up investigations in uncultured amniotic fluid cells after uncertain cytogenetic results. Prenat Diagn 21:75- 80.

Van Opstal D, van den Berg C, Deelen WH, Brandenburg H, Cohen-Overbeek TE, Halley DJJ, Van Den Ouweland AMW, In't Veld PA, Los FJ (1998) Prospective prenatal investigations on potential uniparental disomy in cases of confined placental mosaicism. Prenat Diagn 18:35-44.

Vaughan J, Ali Z, Bower S, Bennett P, Chard T, Moore G (1994) Human maternal uniparental disomy for chromosome 16 and fetal development. Prenat Diagn 14:751- 756.

Wang BT, Peng W, Cheng K-T, Chiu S-F, Ho W, Khan Y, Wittman M, Williams J (1994) Chorionic villus sampling: laboratory experience with 4,000 consecutive cases. Am J Med Genet 53:307-316.

Wang J-C, Mamunes P, Kou S-Y, Mao R, Schmidt J, Habibian R, Hsu WT (1997) Centromeric DNA break in a 10; 16 whole arm translocation associated with trisomy 16 confined placental mosaicism and maternal uniparental disomy for chromosome 16. Am JHum Genet 61 :A142.

Wang J-C C, Mamunes P, Kou S-Y, Schmidt J, Mao R, Hsu W-T (1998) Centromeric

175 DNA break in a 10; 16 reciprocal translocation associated with trisomy 16 confined placental mosaicsm and maternal uniparental disomy for chromosome 16. Am J Med Genet 80:418-422.

Watson JD, Ward BE, Peakman D, Henry G (1988) Trisomy 16 and 12 confined chorionic mosaicism in liveborn infants with multiple anomalies. Am J Hum Genet 43:A252.

Whiteford ML, Coutts J, Al-Roomi L, Mather A, Lowther G, Cooke A, Vaughan JI, Moore GE, Tolmie JL (1995) Uniparental disomy for chromosome 16 in a growth- retarded infant with congenital heart disease. Prenat Diagn 15:579-584.

Williams III J, Wang BBT, Rubin CH, Clark RD, Mohandas TK (1992) Apparent non- mosaic trisomy 16 in chorionic villi: diagnostic dilemma or clinically significant finding? Prenat Diagn 12:163-168.

Wolstenholme J, Rooney DE, Davison EV (1994) Confined placntal mosaicism, IUGR, and adverse pregnancy outcome: a controlled retrospective U.K. collaborative study. Prenat Diagn 14:345-361.

Zimmerman R, Lauper U, Streichier A, Huch R, Hugh A (1995) Elevated alpha- fetoprotein and human chorionic gonadotropin as a marker for placental trisomy 16 in the second trimester? Prenat Diagn 15:1121-1124.

References for CPM16 cases in Table 3.1 from Chapter 3

Gallentine ML, Morey AF, Thompson, IM, Jr (2001) Hypospadias: A contemporary epidemiologic assessment. Urology 57:788-790.

Hoffman JI, Kaplan S (2002) The incidence of congenital heart disease. J Am Coll Cardiol 39(12): 1890-900.

References for CPM16 cases in Table 4.1 in Chapter 4

Hsu LYF, Yu M-T, Neu RL, Van Dyke DL, Benn PA, Bradshaw CL, Shaffer LG, Higgins RR, Khodr GS, Morton CC, Wang H, Brothman AR, Chadwick D, Disteche CM, Jenkins LS, Kalousek DK, Pantzer TJ, Wyatt P (1997) Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations. Prenat Diagn 17:201-242.

Johnson MP, Childs MD, Robichaux Ul AG, Isada NB, Pryde PG, Koppitch III FC, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108.

Kalousek DK, Langlois S, Barrett IJ, Yam I, Wilson DR, Howard-Peebles PN, Johnson MP, Giorgiutti E (1993) Uniparental disomy for chromosome 16 in humans. Am J Hum Genet 52:8-16.

176 Kennerknecht I, Terinde R (1990) Intrauterine growth retardation associated with chromosomal aneuploidy confined to the placenta. Three observations: triple trisomy 6, 21, 22; trisomy 16; and trisomy 18. Prenat Diagn 10:539-544.

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong S-L, Lewis S, Bruyere H, Howard-Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446.

Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard-Peebles PN, Langlois S, Kalousek DK (1997) Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet 60:917-927.

Schneider AS, Bischoff FZ, McCaskill C, Coady ML, Stopfer JE, Shaffer LG (1996) Comprehensive 4-year follow-up on a case of maternal heterodisomy for chromosome 16. Am J Med Genet 66:204-208.

Schwinger E, Seidl E, Klink F, Rehdar H (1989) Chromosome mosaicism of the placenta: a cause of developmental failure of the fetus? Prenat Diagn 9:639-647.

Verp MS, Rosinsky B, Sheikh Z, Amarose AP (1989) Non-mosaic trisomy 16 confined to villi. Lancet 2:915-916.

Wolstenholme J (1995) An audit of trisomy 16 in man. Prenat Diagn 15:109-121.

Woo V, Bridge PJ, Bamforth JS (1997) Maternal uniparental disomy for chromosome 16: case report. Am J Med Genet 70:387-390.

References for CPM16 cases in Table 5.1 in Chapter 5

Devi AS, Velinov M, Kamath MV, Eisenfield L, Neu R, Ciarleglio L, Greenstein R, Benn P (1993) Variable clinical expression of mosaic trisomy 16 in the newborn infant. Am J Med Genet 47:294-298.

Dorfmann AD, Perszyk J, Robinson P, Black SH, Schuman JD (1992) Rare non-mosaic trisomies in chorionic villus tissue not confirmed at amniocentesis. Prenat Diagn 12:899-902.

Johnson MP, Childs MD, Robichaux III AG, Isada NB, Pryde PG, Koppitch III FC, Evans MI (1993) Viable pregnancies after diagnosis of trisomy 16 by CVS: lethal aneuploidy compartmentalized to the trophoblast. Fetal Diagn Ther 8:102-108.

Garber A, Carlson D, Schreck R, Fischel-Ghodsian N, Hsu WT, Oeztas S, Pepkowitz S, Graham JM, Jr. (1994) Prenatal diagnosis and dysmorphic findings in mosaic trisomy 16. Prenat Diagn 14:257-266

177 Hajianpour MJ (1995) Postnatally confirmed trisomy 16 mosaicism: follow-up on a previously reported patient. Prenat Diagn 15:877-879.

Hsu LYF, Yu M-T, Neu RL, Van Dyke DL, Benn PA, Bradshaw CL, Shaffer LG, Higgins RR, Khodr GS, Morton CC, Wang H, Brothman AR, Chadwick D, Disteche CM, Jenkins LS, Kalousek DK, Pantzer TJ, Wyatt P (1997) Rare trisomy mosaicism diagnosed in amniocytes, involving an autosome other than chromosomes 13, 18, 20, and 21: karyotype/phenotype correlations. Prenat Diagn 17:201-242.

Hsu WT, Shchepin DA, Mao R, Berry-Kravis E, Garber AP, Fischel-Ghodsian N, Falk RE, Carlson DE, Roeder ER, Leeth EA, Hajianpour MJ, Wang J-C C, Rosenblum-Vos LS, Bhatt SD, Karson EM, Hux CH, Trunca C, Bialer MG, Linn SK, Schreck RR (1998) Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases. Am J Med Genet 80:473-480.

Lindor NM, Jalal SM, Thibodeau SN, Bonde D, Sauser KL, Karnes PS (1993) Mosaic trisomy 16 in a thriving infant; maternal heterodisomy for chromsome 16. Clin Genet 44:185-189.

Penaherrera MS, Barrett IJ, Brown CJ, Langlois S, Yong S-L, Lewis S, Bruyere H, Howard-Peebles PN, Kalousek DK, Robinson WP (2000) An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta. Clin Genet 58:436-446.

Pletcher BA, Sanz MM, Schlessel JS, Kunaporn S, Mckenna C, Bialer MG, Alonso ML, Zaslav A-L, Brown WT, Ray JH (1994) Postnatal confirmation of prenatally diagnosed trisomy 16 mosaicism in two phenotypically abnormal liveborns. Prenat Diagn 14:933- 940.

Schneider AS, Bischoff FZ, McCaskill C, Coady ML, Stopfer JE, Shaffer LG (1996) Comprehensive 4-year follow-up on a case of maternal heterodisomy for chromosome 16. Am J Med Genet 66:204-208.

Simensen RJ, Colby RS, Corning KJ (2003) A prenatal counseling conundrum: mosaic trisomy 16. A case study presenting cognitive functioning and adaptive behavior. Genet Couns 14:331-336.

Williams Ul J, Wang BBT, Rubin CH, Clark RD, Mohandas TK (1992) Apparent non- mosaic trisomy 16 in chorionic villi: diagnostic dilemma or clinically significant finding? Prenat Diagn 12:163-168.

Woo V, Bridge PJ, Bamforth JS (1997) Maternal uniparental disomy for chromosome 16: case report. Am J Med Genet 70:387-390.

178 Appendix C Protein kinases profiled in Chapter 8

Kinase Kinase 3-phosphoinositide dependent protein kinase 1 (PKB kinase) PDK1 Aurora2 Aurora2 Cancer Osaka thyroid oncogene (Tpl2) COT Casein kinase 2 CK2 Cyclin-dependent kinase 1 (cdc2) CDK1 Cyclin-dependent kinase 2 CDK2 Cyclin-dependent kinase 4 CDK4 Cyclin-dependent kinase 5 CDK5 Cyclin-dependent kinase 6 CDK6 Cyclin-dependent kinase 7 CDK7 Cyclin-dependent kinase 9 CDK9 DNA-activated protein kinase DNAPK dsRNA dependent kinase PKR eEF2k eEF2k Extracellular regulated kinase 1 ERK1 Extracellular regulated kinase 2 ERK2 Extracellular regulated kinase 3 ERK3 Glycogen synthase kinase 3 alpha GSK3a Glycogen synthase kinase 3 beta GSK3b IKKbeta IKKbeta MAP Kinase Kinase 1 (MKK1) MEK1 MAP kinase kinase 2 (MKK2) MEK2 MAP kinase kinase 4 (MEK4) MEK4 MAP Kinase Kinase 6 (MEK6) MEK6 p21 activated kinase 1 (PAK alpha) PAK1 p21 activated Kinase 3 (PAK beta) PAK3 p38 Hog MAP kinase p38 MAPK Protein kinase B alpha PKBa Protein kinase C alpha PKCa Protein kinase C Beta] PKCb Protein kinase C delta PKCd Protein kinase C epsilon PKCe Protein kinase C gamma PKCg Protein kinase C lambda PKC1 Protein kinase C mu PKCm Protein kinase C theta PKCt Protein kinase C zeta PKCz RhoA kinase ROKa Ribosomal S6 kinase 1 RSK1 Ribosomal S6 kinase 2 RSK2 S6 Kinase p70 S6K p70 v-mos Moloney murine sarcoma viral oncogene homolog 1 MOS1 v-raf murine sarcoma viral oncogene homolog B1 RAFB Bone marrow X kinase BMX (Etk) Bruton agammaglobulinemia tyrosine kinase BTK Calmodulin-dependent kinase 1 CaMKl Calmodulin-dependent kinase 4 CaMK4 Calmodulin-dependent kinase kinase CaMKK Casein kinase 1 delta CKld Casein kinase 1 epsilon CKle c-SRC tyrosine kinase CSK Death associated protein kinase 1 DAPK Focal adhesion kinase FAK Fyn oncogene related to SRC FYN G protein-coupled receptor kinase 2 (BARK2) GRK2 Germinal centre kinase GCK Hematopoietic progenitor kinase 1 HPK1 Inhibitor NF kB kinase alpha/beta IKKa Janus kinase 1 JAK1 Janus kinase 2 JAK2 Kinase suppressor of Ras 1 KSR1 Lymphocyte-specific protein tyrosine kinase LCK Mammalian sterile 20-like 1 MST1 MAP kinase interacting kinase 2 MNK2 Oncogene Lyn LYN Oncogene Raf 1 RAF1 Oncogene SRC SRC Protein kinase A (cAMP-dependent protein kinase) PKA Protein kinase Gl (cGMP-dependent protein kinase) PKG1 Protein tyrosine kinase 2 PYK2 Spleen tyrosine kinase SYK Yamaguchi sarcoma viral oncogene homolog 1 YES1 Zeta-chain (TCR) associated protein kinase ZAP70 ZIP kinase (death associated protein kinase 3) ZIP

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