MATERNAL UNIPARENTAL DISOMY OF HUMAN 7

Katariina Hannula

Department of Medical Genetics Haartman Institute and Hospital for Children and Adolescents Helsinki University Central Hospital

University of Helsinki Finland

Academic dissertation

To be publicly discussed, with the permission of the Medical Faculty of the University of Helsinki, in Biomedicum Lecture Hall 2, Haartmaninkatu 8, Helsinki, on the 15th of June 2001, at 12 noon.

Helsinki 2001 Supervised by: Juha Kere, MD, PhD Professor Director, Finnish Genome Center University of Helsinki and Department of Medical Genetics Haartman Institute University of Helsinki, Finland

Reviewed by: Helena Kääriäinen, MD, PhD Docent, Chief Physician Department of Medical Genetics The Family Federation of Finland

Raimo Voutilainen, MD, PhD Professor Department of Pediatrics University of Kuopio, Finland

Official opponent: Gudrun Moore, PhD Reader in Molecular Biology Institute of Reproductive and Developmental Biology Imperial College School of Medicine London, United Kingdom

ISBN 952-91-3473-8 ISBN 951-45-9991-8 (pdf version: http://ethesis.helsinki.fi)

Yliopistopaino Helsinki 2001 To my mother and father TABLE OF CONTENTS

LIST OF ORIGINAL PUBLICATIONS 1

ABBREVIATIONS 2

INTRODUCTION 4

REVIEW OF THE LITERATURE 6 UNIPARENTAL DISOMY 6 Mechanisms of UPD formation 8 UPD as a cause of disorders 10 UPD15 12 patUPD11p15.5 13 Detecting UPD 13 Methods 13 Strategies 14 GENOMIC IMPRINTING 15 Characteristics of imprinted 17 Parent-of-origin-specific methylation 17 Clustering 18 Tissue-, isoform-, promotor-, and time-specific expression 18 Asynchronous replication 19 Repeat sequences 19 Mouse and human homology 19 Mechanisms 19 DNA methylation 20 structure 21 RNA transcripts 22 Imprinted genes and imprinting disorders 22 Growth and development 24 Cancer 25 Methods for identifying imprinting in humans 25 Parent-of-origin-specific DNA methylation 26 Parent-of-origin-specific expression 27 Location-based approach 28 UNIPARENTAL DISOMY OF 28 MatUPD7 28 PatUPD7 32 Candidate chromosome 7 genes for SRS and growth retardation 33 Imprinted genes 33 PEG1/MEST 33 COPG2, γ2-COP 34 MEG1/GRB10 35 Genes not imprinted 36 Human chromosome 7 and mouse homology 37 SILVER-RUSSELL SYNDROME 37 Clinical manifestation 37 Growth 39 Dysmorphic features 40 Development and behavior 41 Differential diagnosis 42 Etiology 43 Familial SRS 43 Chromosome aberrations 44 GROWTH RETARDATION 45 Intrauterine growth retardation 46 Postnatal growth retardation 47

AIMS OF THE STUDY 48

MATERIALS AND METHODS 49 PATIENTS 49 Silver-Russell syndrome patients 49 Growth-retarded patients 50 PatUPD7 patient 50 GENOTYPING 51 SOUTHERN HYBRIDIZATION 52

RESULTS AND DISCUSSION 53 IDENTIFICATION OF CASES OF MATUPD7 IN SRS (I, II, III) 53 CHARACTERIZATION OF THE MATUPD7 PHENOTYPE (I) 55 DELINEATION OF THE CANDIDATE SRS REGION TO 7Q31-QTER BY SEGMENTAL MATUPD7 (II) 58 THE ROLE OF MATUPD7 IN GROWTH RETARDATION (III) 60 MatUPD7 is confined to SRS 60 MatUPD7 screening as a diagnostic tool 61 PARENT-OF-ORIGIN-SPECIFIC METHYLATION OF CHROMOSOME 7 GENES IN LYMPHOBLAST DNA AND BLOOD DNA (IV) 62

CONCLUSIONS AND FUTURE PROSPECTS 64

SUMMARY 66

ACKNOWLEDGMENTS 68

REFERENCES 70

1

LIST OF ORIGINAL PUBLICATIONS

I Hannula K, Kere J, Pirinen S, Holmberg C, and Lipsanen-Nyman M. Do patients with maternal uniparental disomy for chromosome 7 have a distinct mild Silver-Russell phenotype? Journal of Medical Genetics 2001; 38:273-278.

II Hannula K, Lipsanen-Nyman M, Kontiokari T, and Kere J. A narrow segment of maternal uniparental disomy of chromosome 7q31-qter in Silver-Russell syndrome delimits a candidate region. American Journal of Human Genetics 2001; 68:247- 253.

III Hannula K, Lipsanen-Nyman M, Kristo P, Kaitila I, Simola KOJ, Lenko HL, Tapanainen P, Holmberg C, and Kere J. Genetic screening for maternal uniparental disomy of chromosome 7 in pre- and postnatal growth retardation of unknown etiology. Submitted

IV Hannula K, Lipsanen-Nyman M, Scherer SW, Holmberg C, Höglund P, and Kere J. Maternal and paternal 7 show differential methylation of many genes in lymphoblast DNA. Genomics 2001; 73:1-9.

The original articles are referred to in the text by their Roman numerals. In addition, some unpublished data are presented. 2

ABBREVIATIONS ag androgenetic AGA appropriate for gestational age AP-PCR arbitrarily primed polymerase chain reaction AS Angelman syndrome bp BWS Beckwith-Wiedemann syndrome cDNA complementary deoxyribonucleic acid CF cystic fibrosis CLD congenital chloride diarrhea cM centiMorgan COPG2, γ2-COP gene for coatomer complex, subunit γ CPM confined placental mosaicism DMR differently methylated region DNA deoxyribonucleic acid EGFR gene for epidermal growth factor receptor EST expressed sequence tagged site GH growth hormone IC imprinting center IGFBP1 gene for insulin-like growth factor binding protein 1 IGFBP3 gene for insulin-like growth factor binding protein 3 IM imprinting mutation IQ intelligence quotient IUGR intrauterine growth retardation kb kilobase pairs (1kb=1000 bp) LB lymphoblast LOI loss of imprinting mat maternal matUPD7 maternal uniparental disomy of chromosome 7 Mb megabase pairs MEG1/GRB10 gene for maternally expressed gene 1, growth factor receptor bound protein 10 MIM# Mendelian Inheritance in Man catalog number p short arm of chromosome PAGE polyacrylamide gel electrophoresis pat paternal patUPD7 paternal uniparental disomy of chromosome 7 PAX4 gene for paired box gene 4 PCR polymerase chain reaction PEG1/MEST gene for paternally expressed gene 1/mesoderm-specific transcript pg parthenogenetic PNGR postnatal growth retardation PWS Prader-Willi syndrome q long arm of chromosome qter telomere of the long arm of chromosome RLGS restriction landmark genome scanning RNA ribonucleic acid RT-PCR reverse transcriptase polymerase chain reaction 3

SD standard deviation SDS standard deviation score SGA small for gestational age SRS Silver-Russell syndrome UPD uniparental disomy UTR untranslated region VNTR variable number of tandem repeats wt wild type

Human genes are in italics in uppercase letters. Murine genes are in italics in lowercase letters. 4

INTRODUCTION

A child normally inherits one chromosome of a chromosome pair from the mother and the other from the father. In uniparental disomy (UPD), both chromosomes are exceptionally inherited from only the mother (maternal UPD, matUPD) or the father (paternal UPD, patUPD). The most common mechanisms leading to UPD include chromosomal nondisjunction in the first or second meiotic division, but errors in mitosis and somatic recombination may also lead to UPD. UPD can comprise the entire chromosome or be confined to only a small segment of it. In isodisomic UPD, two identical copies of a chromosome are present, and in heterodisomic UPD, the chromosomes are not identical but are inherited from only one parent.

In humans, UPD has been reported for virtually all chromosomes, including the XX and XY pairs, with the exceptions of chromosomes 3, 12, 18, and 19. UPD may lead to disorders because maternal and paternal genomes are not equivalent on all chromosomes. This is caused by genomic imprinting, the differential expression of genes according to their parental origin. The most likely mechanism of imprinting is DNA methylation, which generally suppresses . Genomic imprints are acquired differently during oogenesis and spermatogenesis, and thus, some genes are expressed only from the maternal and others only from the paternal chromosome. Over 40 imprinted genes have been identified in the mouse and human, and many of these have significant roles during embryonic development. Both maternal and paternal genomes are required for normal growth and development, and therefore, UPD of many chromosomes leads to abnormal growth, development, and behavior.

Maternal UPD of chromosome 7 (matUPD7) is associated with pre- and postnatal growth retardation and dysmorphic features typical of Silver-Russell syndrome (SRS). Over 30 cases of matUPD7 have been reported, all with short stature and at least 25 with SRS. Paternal UPD of chromosome 7 (patUPD7), in contrast, appears to have no effect on growth and development. The different phenotypes observed in maternal and paternal UPD7 suggest that one or more imprinted genes, which regulate growth and development, reside on human chromosome 7.

The molecular etiology of SRS is unclear and diagnosis is based on the clinical phenotype. Manifestation of typical SRS characteristics varies enormously among individuals, which 5 leads to great heterogeneity among patients who have been diagnosed with SRS and also complicates diagnosis. Furthermore, no diagnostic criteria have been set for SRS. MatUPD7 is observed in approximately 10% of SRS patients, but the roles of the three known imprinted genes on human chromosome 7 at 7q32 (PEG1/MEST, COPG2) and 7p11.2-p12 (GRB10) remain unsettled.

The aims of this thesis were to evaluate the role of matUPD7 on the SRS phenotype and in growth retardation in general as well as to further characterize candidate gene regions for imprinted genes underlying SRS. 6

REVIEW OF THE LITERATURE

UNIPARENTAL DISOMY

Uniparental disomy (UPD) is the rare inheritance of both homologous chromosomes exceptionally from only one parent [Engel, 1980]. UPD is referred to as isodisomy when it comprises two identical copies of one chromosome and heterodisomy when the entire chromosome pair is inherited from only one parent [Engel, 1980]. Segments of isodisomy and heterodisomy can also occur alternately, forming a mixed iso/heterodisomy [Spence et al., 1988; Engel, 1993]. UPD can comprise the whole chromosome or be confined to only a small segment [Spence et al., 1988; Engel, 1993].

UPD has been reported for virtually all human chromosomes, including the XX and XY pairs, with the exceptions of chromosomes 3, 12, 18, and 19 (Table 1). The frequency of UPD has been estimated for some chromosomes by taking into account the frequency of occurrence of a syndrome and the proportion of UPD cases in a syndrome [Engel, 1998]. Maternal and paternal UPD15 in Prader-Willi (PWS) and Angelman syndromes (AS) is estimated to occur in 1/80 000 live births [Robinson et al., 1996], patUPD11p (Beckwith- Wiedemann syndrome, BWS) in 1/75 000, and patUPD6 (transient neonatal diabetes mellitus) in 1/1 250 000 [Engel, 1998]. The frequency of UPD in the general population is uncertain, but an estimate can be obtained from cases of UPD that have emerged from large genotyping projects or from routine HLA-screening, such as UPD1 which was found in 1/203, UPD6 in 1/700, and UPD8 in 1/2300 of subjects studied [van den Berg-Loonen et al., 1996; Engel, 1998; Field et al., 1998; Karanjawala et al., 2000]. Engel (1980) estimated the frequency of UPD, by the number of aneuploid gametes and their chance reunion, to be as high as 2.8/10 000 conceptions.

UPD is unequally observed for different chromosomes, and maternal UPD is approximately three times more frequent than paternal UPD [Kotzot, 1999]. The higher incidence of maternal UPDs can be explained by a higher frequency of aneuploidy in oocytes (18-19%) than sperm (3-4%) [Martin et al., 1991]. Trisomy is chiefly maternal in origin, and trisomy rescue with loss of one chromosome (paternal) appears to be the most common mechanism for UPD formation [Kotzot, 1999]. Increasing maternal age is well known to be associated 7

Table 1. Observed maternal and paternal UPDs and associated abnormal growth and phenotype. Modified from Hurst and McVean (1997) and Kotzot, (1999). Additional references are listed.*= case report of autosomal recessive disorder (Table 2), §=other than BWS or PWS cases.

Number UPD Growth Phenotype Reference of reports mat1 * 3 pat1 * 2 Chen et al., 1999 mat2 IUGR, oligohydramnios, hypospadia, undescended testes, 6 Heide et al., 2000 PNGR hypothyroidism, hyaline membrane disease mat4 * 2 pat5 * 1 mat6 IUGR, sarcoidosis, chronic tubulointerstitial nephritis, * 2 PNGR pat6 IUGR, transient neonatal diabetes mellitus, * 10 Hermann et al., 2000 PNGR mat7 IUGR, Silver-Russell syndrome,* >14 Table4 PNGR pat7 * 2 mat8 early-onset ileal tumor 3 Karanjawala et al., 2000 pat8 * 1 mat9 PNGR mental retardation, indistinct speech, minor 3 anomalies (Trisomy 9 mosaicism),* mat10 1 pat10 1 pat11 PNGR Beckwith-Wiedemann syndrome,* 1§ mat13 2 pat13 4 Soler et al., 2000 mat14 IUGR, Precocious puberty, small hands and feet, hypotonia, >20 Sanlaville et al., PNGR motor developmental delay, * 2000 pat14 PNGR Mental retardation, blepharophimosis, short neck, 4 small thorax, skeletal dysplasia mat15 PNGR Prader-Willi syndrome,* 1§ pat15 Angelman syndrome mat16 IUGR, +/- minor anomalies, congenital heart and digestive >20 Chan et al., 2000 PNGR tract anomalies (CPM), body stalk anomaly, * pat16 IUGR Bilateral pes calcaneus, rudimentary mandibular 2 Kohlhase et al., 2000 dental arch * mat17 none 1 Genuardi et al., 1999 mat20 IUGR, hyperactive, minor facial dysmorphology 1 PNGR pat20 microcephaly, absence of external ears 1 mat21 none early embryonic failure 4 Rogan et al., 1999 pat21 none 3 mat22 none 6 pat22 none 1 matX none association with Turner's syndrome, * 5 patX none association with Turner's syndrome, * 4

with a higher frequency of chromosomal abnormalities and meiotic nondisjunction, and thus, possibly also with an increased risk for UPD [Robinson et al., 1993; Robinson, 2000]. However, advanced paternal age has also been suggested to increase the occurrence of aneuploidy [Robinson et al., 1993]. This is in accordance with the requirement for the fusion of two aneuploid gametes in one mechanism of UPD formation (Figure 1A) [Kotzot, 1999]. 8

The higher amount of reported UPD cases for some chromosomes is probably biased by the greater interest focused on specific phenotypes associated with UPD (e.g. pat6 and transient neonatal diabetes mellitus, mat7 and Silver-Russell syndrome (SRS), pat11p15.1 and BWS, mat14 and the “UPD14 phenotype”, mat15 and PWS, pat15 and AS, and mat16 and the “matUPD16 phenotype”) [Kotzot, 1999]. A higher risk for UPD has been recognized in individuals with structural chromosome abnormalities, i.e. Robertsonian translocations, isochromosomes, and marker chromosomes [James et al., 1995; Berend et al., 2000].

Mechanisms of UPD formation Mechanisms leading to UPD include (1) gamete complementation, (2) trisomy rescue, (3) monosomy duplication, and (4) postfertilization errors including mitotic recombination and gene conversion [Spence et al., 1988; Engel, 1993; Robinson, 2000] (Figure 1). The first three result from fertilization between one or two aneuploid gametes, originating from segregation errors in either meiosis I or II, and subsequent return to a normal diploid chromosome pair [Robinson, 2000]. Nondisjunction at meiosis I will lead to heterodisomy and at meiosis II to isodisomy at the pericentromeric region [Spence et al., 1988; Engel, 1993].

A) Gamete B) Trisomy rescue C) Monosomy to D) Postfertilization complementation isodisomy error

gametes

zygote

somatic tissues heterodisomy heterodisomy isodisomy isodisomy segmental meiosis I error meiosis I error isodisomy

(isodisomy if (isodisomy if meiosis II error) meiosis II error)

Figure 1. Mechanisms leading to UPD. Adapted from Spence et al. (1988) and Robinson (2000). 9

In gamete complementation, fertilization occurs between two aneuploid gametes, one nullisomic and the other disomic [Spence et al., 1988] (Figure 1A). The disomic gamete contains a chromosome pair resulting from nondisjunction in meiosis I or II [Spence et al., 1988]. In trisomy rescue, a normal monosomic gamete and a diploid gamete unite, thus leading to temporary trisomy [Spence et al., 1988] (Figure 1B). The trisomy is then reduced to diploidy by extraction of one chromosome, which in one-third of the cases is the only chromosome provided by the monosomic gamete and the two remaining chromosomes are both from the diploid gamete, thus uniparental in origin [Engel, 1993]. Trisomy rescue is presumed to be the most frequent mechanism leading to UPD [Kotzot, 1999]. Gamete complementation and trisomy rescue may lead to heterodisomy or isodisomy of the entire chromosome, but intervening segments of isodisomy or heterodisomy, respectively, are quite frequently observed, thus forming the mixed iso/heterodisomy UPDs [Spence et al., 1988; Robinson, 2000]. The intervening segments arise from meiotic recombinations and the number of segments correlates with the number of recombinations [Spence et al., 1988]. In monosomy duplication, a monosomic and nullisomic gamete unite, resulting in a monosomic zygote [Spence et al., 1988] (Figure 1C). Duplication of the only chromosome present leads to isodisomy of the whole chromosome [Spence et al., 1988; Robinson, 2000].

Postfertilization errors at early embryonic stages may also cause UPD, leading to UPD of the entire or only part of the chromosome [Spence et al., 1988] (Figure 1D). Mitotic nondisjunction and subsequent duplication of the remaining chromosome leads to complete isodisomy [Spence et al., 1988; Robinson, 2000]. UPD for only part of a chromosome may arise from somatic recombination or gene conversion, leading to a minute UPD segment [Spence et al., 1988]. A recombination in an early mitotic division followed by loss of the other daughter cell line will produce a segmental UPD without mosaicism [Robinson, 2000]. If, however, both daughter cells persist, mosaicism will be observed [Robinson, 2000]. Mosaicism is expected to be high in postfertilization errors [Spence et al., 1988], as in BWS, where mosaicism is extremely frequent for patUPD11p15.5 originating from postzygotic recombination [Slatter et al., 1994; Bischoff et al., 1995]. However, the presence of a normal disomic or compensatory UPD cell line might mask the molecular and phenotypic observation of a postfertilization error, which could explain the few reported cases [Robinson, 2000]. Segmental UPD has been reported only for chromosomes 2p16 [Stratakis et al., 2001], 4q21-q35 [Yang et al., 1999], 6q24-qter [Das et al., 2000], 11p15 [Henry et al., 1993], and 14q23-q24.2 [Martin et al., 1999]. Somatic recombination has also been suggested 10 on chromosome 15 for PWS [Gregory et al., 1991]. Chromosome replacement is an alternative, yet rare, mechanism where an abnormal chromosome is lost postzygotically and replaced by a duplicate of the initial normal chromosome [Petersen et al., 1992; Engel, 1993; Robinson, 2000]. It has been reported in at least two individuals with a partial chromosome 21 deletion [Petersen et al., 1992].

UPD is occasionally found in the diploid cells of a fetus with confined placental mosaicism (CPM) [Cassidy et al., 1992; Hurst and McVean, 1997]. CPM presents as an abnormal cell line (usually trisomic) in the placenta but a normal diploid cell line in the fetus [Robinson et al., 1997]. CPM is found in approximately 2% of viable pregnancies [Wang et al., 1993]. In UPD cases associated with CPM, most likely a meiotic error leads to trisomy, but subsequent trisomy rescue results in a disomic UPD cell line in the fetus, with trisomy being sustained in the placenta [Robinson et al., 1997; Robinson, 2000]. UPD and CPM are both associated with intrauterine growth retardation (IUGR) and prenatal abnormalities [Kalousek et al., 1991; Hurst and McVean, 1997; Robinson et al., 1997]. Therefore, in cases of UPD originating from CPM, it is difficult to ascertain whether IUGR is caused by imprinting effects brought on by the UPD in the fetus, the UPD cell line in the placenta, or the trisomic cell line in the placenta [Hurst and McVean, 1997; Robinson et al., 1997]. In true chromosomal mosaicism, both the abnormal and normal cell lines are present in the placenta and fetus. UPD is occasionally also found in the diploid cell line of mosaic chromosomal aberrations such as trisomy mosaicism [Willatt et al., 1992; Shaffer et al., 1996; Robinson, 2000].

UPD as a cause of disorders UPD may lead to disorders by disrupting the balance of imprinted genes or by reduction to homozygosity for a recessive disorder [Engel, 1980; Engel, 1998]. UPD may, however, have no effect on an individual when the maternal and paternal genomes are equivalent in terms of imprinting and when no recessive disease alleles are transmitted [Field et al., 1998]. In the most extreme case, UPD may be lethal [Kotzot, 1999]. Both of these scenarios, a normal phenotype or inviability, will most likely go undetected [Engel, 1998; Kotzot, 1999]. However, ongoing large-scale genomewide genotyping projects will probably detect additional UPD cases in healthy individuals with no apparent abnormal phenotype [Engel, 1998; Field et al., 1998]. 11

For many human chromosomes, UPD is associated with growth retardation with varying degrees of dysmorphic features and developmental delay [Hurst and McVean, 1997; Kotzot, 1999] (Table 1). This is consistent with knowledge that imprinted genes affect growth, development, and behavior [Tilghman, 1999]. The initial effects of UPD on the phenotype were shown by experiments producing mice with UPDs of whole chromosomes or small segments [Cattanach and Kirk, 1985]. The UPD mice showed abnormal growth, behavior, and survival associated with a specific chromosomal region of either maternal or paternal origin [Cattanach and Kirk, 1985].

UPD is an unusual cause of recessive disorders, with only one parent a carrier of the disease allele [Engel, 1980] (Table 2). The first case of UPD was in fact revealed by monoparental inheritance of an autosomal recessive disorder [Spence et al., 1988]. In addition to autosomal recessive disorders, X-linked disorders have been transmitted from father to son and observed in females due to UPD [Vidaud et al., 1989; Quan et al., 1997].

Table 2. Case reports of recessive disorders resulting from maternal or paternal UPD. Modified from Engel (1998) and Kotzot (1999).

UPD Autosomal recessive disorder Reference mat1 Herlix junctional epidermolysis bullosa Pulkkinen et al., 1997 Chediak-Higashi syndrome Dufourcq-Lagelouse et al. 1999 pat1 Pycnodysostosis Gelb et al., 1998 Congenital insensitivity to pain with anhidrosis Miura et al., 2000 mat4q21-q35 Abetalipoproteinemia Yang, 1999 pat5 Spinal muscular atrophy Brzustowicz et al., 1994 mat6 Congenital adrenal hyperplasia Spiro et al., 1999 pat6 Complement C4A+C4B deficiency Welch et al., 1990 Methylmalonic acidemia Abramowicz et al., 1994 mat7 Cystic fibrosis Spence et al., 1988; Voss et al., 1989; Hehr et al., 2000 pat7 Congenital chloride diarrhea Höglund et al., 1994 Kartagener syndrome, Cystic fibrosis Pan et al., 1998 pat8 Lipoprotein lipase deficiency Benlian et al., 1996 mat9 Cartilage-hair hypoplasia Sulisalo et al., 1997 pat11 Beta-thalassemia major Beldjord et al., 1992 mat14 Rod monochromacy Pentao et al., 1992 mat15 Bloom syndrome Woodage et al., 1994 mat16 Familial mediterranean fever Korenstein et al., 1994 pat16 Hydrops fetalis alpha talassemia Ngo et al., 1993 matXX Duchenne muscular dystrophy Quan et al., 1997 patXY Hemophilia A Vidaud et al., 1989 12

UPD15 Maternal and paternal UPD of chromosome 15 are associated with the Prader-Willi syndrome (PWS, MIM 176270) and the Angelman syndrome (AS, MIM105830), respectively [Nicholls et al., 1989; Malcolm et al., 1991]. PWS and AS are clinically distinct syndromes, that are caused by defects of imprinted genes in the 15q11-q13 region [Nicholls et al., 1998; Mann and Bartolomei, 1999]. A cluster of imprinted genes, with a common regulatory imprinting center (IC), is located at 15q11-q13 [Buiting et al., 1995; Nicholls et al., 1998]. To date, this cluster contains twelve imprinted genes, the majority of which are paternally expressed (Table 3).

PWS patients are characterized by delayed onset of and reduced fetal activity, hypotonia and failure to thrive in infancy, and hyperphagia and obesity from early childhood onward [Cassidy, 1997]. In addition, mild to moderate mental retardation, hypogonadism with diminished puberty, short stature, small hands and feet, and facial anomalies are observed [Cassidy, 1997]. AS, previously referred to as the “happy puppet syndrome”, manifests as severe motor and mental retardation, inappropriate paroxysmal laughter, absence of speech, epilepsy, ataxic gait, mild hypotonia, a protruding tongue, and a large mandible [Bower and Jeavons, 1967; Clayton-Smith and Pembrey, 1992]. The incidence of PWS is ~1 in 10 000 to 15 000, and AS 1 in 20 000 live births [Clayton-Smith and Pembrey, 1992; Cassidy, 1997].

MatUPD15 is found in 25% of PWS and patUPD15 in 2% of AS patients [Nicholls et al., 1998]. Large paternal and maternal deletions are found in 70% of PWS and AS patients, respectively [Nicholls et al., 1998]. Gene mutations are identified in 20% of AS patients, but none have been found in PWS patients so far [Nicholls et al., 1998; Mann and Bartolomei, 1999]. Imprinting mutations account for less than 5% and balanced translocations for less than 0.1% of cases in both syndromes [Nicholls et al., 1998]. Defective function of the maternally expressed gene UBE3A is consistent with AS [Kishino et al., 1997; Matsuura et al., 1997]. SNRPN and the associated SNURF mRNA transcript are likely candidates for PWS, but a single gene has not yet been identified [Mann and Bartolomei, 1999]. Multiple paternally expressed genes have been suggested to be involved in PWS [Mann and Bartolomei, 1999]. 13 patUPD11p15.5 Beckwith-Wiedemann syndrome (BWS, MIM 130650) is associated with patUPD11p15.5 in approximately 20% of sporadic BWS cases [Henry et al., 1993; Slatter et al., 1994]. The 11p15.5 region encompasses the second cluster of imprinted genes in humans and epigenetic alterations as well as mutations of the imprinted genes are observed in BWS patients [Reik and Maher, 1997; Engel et al., 2000; Maher and Reik, 2000]. The clinical features of BWS include pre- and postnatal overgrowth, macroglossia, visceromegaly, hemihypertrophy, genitourinary abnormalities, increased risk for specific tumors, and mental retardation [Engström et al., 1988; Elliott and Maher, 1994]. The estimated incidence of BWS is 1/13 700 [Engström et al., 1988; Elliott and Maher, 1994]. The genetics of BWS is complex; ~15% of cases are familial, ~2% have cytogenetic abnormalities in the 11p15.5 region, and the majority are sporadic [Reik and Maher, 1997; Maher and Reik, 2000]. In over 40% of familial cases, mutations in the CDKN1C gene are found, whereas in sporadic cases patUPD11p15.5 or loss of imprinting (LOI) of IGF2 or other imprinted genes are predominantly observed [Maher and Reik, 2000]. Among sporadic cases, one subgroup of BWS patients has a defect in an imprinting control element, which leads to LOI of IGF2 and silencing of H19 [Reik et al., 1995; Engel et al., 2000]. Another subgroup has loss of methylation at a differently methylated region (KvDMR) within the KCNQ1 gene [Smilinich et al., 1999; Engel et al., 2000]. These different epigenotypes appear to be associated with different BWS phenotypes [Engel et al., 2000].

Detecting UPD Methods UPD can be detected by analysis of the parental origin of alleles on a specific chromosome [Spence et al., 1988; Nicholls et al., 1989; Malcolm et al., 1991]. Lack of alleles from either the mother or the father indicates paternal UPD or maternal UPD, respectively [Spence et al., 1988; Nicholls et al., 1989; Malcolm et al., 1991; Höglund et al., 1994]. Genotyping with highly polymorphic microsatellite markers is fast and feasible with PCR (polymerase chain reaction) and PAGE (polyacrylamide gel electrophoresis) [Kotzot et al., 1995; Ledbetter and Engel, 1995; Eggermann et al., 1997]. Automatization, by use of fluorescently labeled primers and computer-controlled detection systems, enables genotyping of multiple samples expeditiously [Ledbetter and Engel, 1995]. VNTR (variable number of tandem repeats) analysis by Southern hybridization is an alternative approach, although more tedious than PCR [Spence et al., 1988; Nicholls et al., 1989; Malcolm et al., 1991]. Both PCR and 14

Southern hybridization-based techniques require at least one parental sample, preferably both, to ascertain parental origin of alleles. However, calculating the probability of a child consistently sharing maternal or paternal alleles can be used to detect UPD when only one parental sample is available [Spence et al., 1988; Manzoni et al., 2000]. Isodisomy can be ascertained by calculating the probability of a child being homozygous for numerous markers. UPD can also be verified without parental samples by parent-of-origin-specific methylation analysis for a known differently methylated region (DMR) [Kosaki et al., 2000b] (see page 26).

Strategies UPD is probable for human chromosomes carrying imprinted genes or with homology to mouse imprinted regions [Ledbetter and Engel, 1995]. Novel UPD cases and imprinted genes might also be observed in disorders, in which etiology remains obscure [Engel and DeLozier- Blanchet, 1991]. Patients of a well-defined clinical entity, who lack the common cause of the entity, such as a deletion or chromosomal rearrangement, might turn out to have UPD for the aberrant region as in PWS (matUPD15q11-q13), AS (patUPD15q11-q13), and BWS (patUPD11p15.1) [Engel and DeLozier-Blanchet, 1991; Slatter et al., 1994; Nicholls et al., 1998]. Especially growth disorders of unknown etiology with a well-defined phenotype, such as Silver-Russell, Brachmann-deLange, Rubinstein-Taby, and Sotos syndromes, are regarded as good candidates for an imprinting etiology [Engel and DeLozier-Blanchet, 1991]. SRS is already associated with matUPD7 and an imprinted gene(s) is very likely in its etiology [Moore et al., 1999]. Sotos syndrome, on the other hand, does not appear to be associated with UPD [Smith et al., 1997]. Disorders where a deletion or mutation is preferentially inherited on either the paternal or maternal chromosome or when the severity of the disorder is associated with parental origin of inheritance may also have imprinted genes in their etiology and reveal cases of UPD [Engel and DeLozier-Blanchet, 1991].

UPD might be the cause of divergent clinical findings in patients with an inherited balanced translocation but an additional abnormal phenotype or in patients with a recessive disorder who also present with unexpected growth patterns, anomalies, or developmental problems [Engel and DeLozier-Blanchet, 1991; Schinzel, 1991]. Moreover, the association of two or more diseases not caused by cytogenetic aberrations may be caused by UPD by reduction to homozygosity and imprinting imbalance or by the occurrence of two recessive traits in an isodisomic segment [Nicholls, 1991]. 15

GENOMIC IMPRINTING

Mendelian inheritance presumes that the maternal and paternal copies of a gene are equal in function. This is, however, not the case in genomic imprinting, where the expression of a gene depends on its parental origin [Bartolomei and Tilghman, 1997; Reik and Surani, 1997]. Over 40 imprinted genes have been found to date in the mouse and human (Table 3), and only a small proportion of genes, approximately 100-200, are estimated to be expressed in a parent-of-origin-specific manner [Tilghman, 1999]. An imprinted gene is active on only the maternal or paternal chromosome while the homologous allele is silent [Bartolomei and Tilghman, 1997]. The silenced allele is considered the imprinted allele, that is, a maternally imprinted gene is silent on the maternal chromosome and expressed from the paternal chromosome [Bartolomei and Tilghman, 1997]. The imprints are established differently during oogenesis and spermatogenesis, most likely by DNA methylation [Surani et al., 1984; Surani, 1998]. Differential marking of maternal and paternal alleles thus distinguishes the maternally and paternally derived genomes after fertilization [Bartolomei and Tilghman, 1997]. Imprints are maintained throughout development and in adult somatic tissues, but they are erased in the germline during gametogenesis and then re-established according to the sex of the individual [Barlow, 1997].

Because of genomic imprinting, mammalian maternal and paternal genomes are not functionally equivalent and both are required for normal embryonic development [Barton et al., 1984; McGrath and Solter, 1984; Surani et al., 1984; Cattanach and Kirk, 1985]. Evidence for imprinting was obtained from early mouse studies by the production of androgenetic (completely paternal genome) and gynogenetic (completely maternal genome) mouse embryos in nuclear transfer experiments [McGrath and Solter, 1984; Surani et al., 1984]. Androgenotes exhibit poor embryonic development but relatively good development of extraembryonic tissues, whereas gynogenotes have a well-developed small embryo but poor extraembryonic tissue development [Barton et al., 1984; Surani et al., 1984]. Neither is viable to term [Barton et al., 1984; Surani et al., 1984]. Further evidence for the crucial role of imprinted genes in mammalian development was shown by Cattanach and Kirk [1985], who produced mice with UPD for specific chromosomes or subchromosomal regions. Aberrant phenotypes, including growth retardation and overgrowth, abnormal behavior, lethality in early embryonic development, and mid-fetal to early neonatal death were observed for some regions, indicating imprinted genes within them [Cattanach and Kirk, 16

1985; Cattanach and Beechey, 1990; Cattanach and Beechey, 1997]. To date, imprinted genes have been identified on mouse chromosomes 2, 6, 7, 9, 10, 11, 12, 14, 17, 18, 19, and X [Beechey et al., 2000] and human chromosomes 1, 6, 7, 11, 13, 14, 15, 19, 20, and X (Table 3).

Table 3. Human and mouse imprinted genes. Modified from Morison et al. (2001) and the Imprinted Gene Catalog (http://www.otago.ac.nz/IGC). m= maternal, p= paternal, ni= not imprinted.

Human HUMAN GENE Expressed Mouse Mouse Expressed Human homology Chromosome allele in Chr. Gene allele in human (cM) mouse 1p36 p73 m 2(104) Gnas m/p 20q13 1p31 ARHI p 2(88) Nnat p 20q11.2-q12 6q24.1-q24.3 HYMAI p6(1)Sgce p 7q32 6q24-q25 PLAGL1 p6(7)Peg1/Mest p 7q32 6q25.3 IGF2R conflicting 6(7) Copg2 m 7q32 6q25.3-q26 MAS1 disputed 6(7) Mit1 p 7q32 7p11-p13 GRB10 m/p 7(6) Peg3 p 19q13.4 7q32 PEG1/MEST p7(6)Usp29 p 19q13.4 7q32 COPG2 p disputed 7(6) Zim1 m 7q32 CIP m 7(27) Snrpn p 15q11-q13 11p13 WT1 m/p 7(28) Ndn p 15q11-q13 11p15.4 ZNF215 m 7(28) Magel2 p 15q11-q13 11p15.5 H19 m 7(28) MBII-13 p 15q11-q13 11p15.5 IGF2 p 7(28) MBII-85 p 15q11-q13 11p15.5 ASCL2 m 7(28) Ipw p 15q11-q13 11p15.5 MTR1 p 7(28) MBII-52 p 15q11-q13 11p15.5 KCNQ1 m 7(28) Ube3a m 15q11-q13 11p15.5 KvLQT1 m 7(29) Zfp127 p 15q11-q13 11p15.5 LIT p 7(69) H19 m 11p15.5 11p15.5 CDKN1C m 7(69) Igf2 p 11p15.5 11p15.5 SLC22A1L m 7(69) Mash2 m 11p15.5; ASCL2 11p15.5 TSSC3 m 7(69) Kvlqt1 m 11p15.5 13q14 HTR2A m 7(69) Cdnk1c m 11p15.5 14q32 GTL2 m 7(69) Orctl2 m 11p15.5; SLC22A1L 14q32 DLK1 p 7(69) Tssc3 m 11p15.5 15q11-q13 MKRN3 p 7(69) Tssc4 m 11p15.5; ni 15q11-q13 NDN p 7(69) Ins2 p 11p15.5; ni 15q11-q13 MAGEL2 p 7(69) Obph1 m11 15q11-q13 SNRPN, SNURF p 7(69) Nap2 m 11p15.5 15q11-q13 PAR-SN p 7(69) Msuit m 15q11-q13 HBII-13 p 9(50) Rasgrf1 p 15q24 15q11-q13 PAR5 p 10(15) Zac1 p 6q24-q25: PLAGL1 15q11-q13 HBII-85 p 11(8) Grb10 m 7p11-p13 15q11-q13 IPW p 11(12) U2af1-rs1 p 5q; U2AFBL ni 15q11-q13 PAR1 p 12(52) Dlk p 14q32 15q11-q13 HBII-52 p 12(54) Gtl2 m 14q32 15q11-q13 UBE3A m 14(41) Htr2a m 13q14 19q13.4 PEG3 p 17(7) Igf2r m 6q25.3 20q13 GNAS1 m/p 17(7) Mas p disputed 6q25.3-q26 Xq13.2 XIST p18Impact p 18q11.2-q12.1; ni 19(49) Ins1 p possible X(57) Esx1 m X(42) Xist p Xq13.2 17

It is puzzling why evolution has chosen the reduction from diploidy to haploidy for some genes [Tilghman, 1999]. While no single sound hypothesis exists for the evolution of genomic imprinting, the parent-offspring conflict theory is considered the most likely [Moore and Haig, 1991; Hurst, 1997]. This theory is based on the parasite-like relationship of the mammalian fetus to its mother and its need for maternally provided nutrients [Tilghman, 1999]. It is in the father’s interest to ensure the fitness of his offspring, whereas the mother’s interest lies in ensuring viability of all her offspring and herself [Moore and Haig, 1991]. Therefore, in this “parental battle”, paternal genes are suggested to enhance growth of progeny and extraembryonic tissues, and maternal genes to act as growth suppressors [Moore and Haig, 1991; Hurst, 1997]. The known growth effects of many imprinted genes support this hypothesis [Hurst and McVean, 1997]. Other theories include imprinting as a host defence against foreign DNA invasion or an endogenous surveillance and correction mechanism against aneuploidy or even cancer, where development would arrest due to imprinting imbalance [Thomas, 1995; Hurst, 1997].

Characteristics of imprinted genes Imprinted genes share many common characteristics which can facilitate distinguishing new imprinted genes and may also aid in unraveling imprinting mechanisms and further understanding of why imprinting has evolved [Bartolomei and Tilghman, 1997]. Although many common characteristics have been identified, their functions are not yet thoroughly understood [Constancia et al., 1998].

Parent-of-origin-specific methylation Parent-of-origin-specific methylation has been observed within or in the vicinity of all imprinted genes [Constancia et al., 1998; Reik and Walter, 1998]. These differently methylated regions (DMRs) are most commonly found at the 5’ ends of genes, but DMRs have also been found at the promotors, introns, and exons in the body of a gene, and regions distal to the unit [Bartolomei and Tilghman, 1997]. The size of DMRs ranges from over 8 kb for H19 to small localized patches situated on other genes [Barlow, 1997; Bartolomei and Tilghman, 1997]. The methylated allele is generally the silenced or repressed allele, however, methylation is seen on the active allele for some imprinted genes, e.g. Igf2r [Stoger et al., 1993] and Igf2 [Feil et al., 1994]. In addition, repression is not complete for all genes, and up to 5% expression has been observed from the “silent” allele [Barlow, 1997]. 18

Clustering Imprinted genes occur in clusters in both the mouse and human, possibly due to common regulatory mechanisms [Bartolomei and Tilghman, 1997]. The two best characterized clusters of imprinted genes in humans are the AS/PWS cluster on 15q11-q13 and the BWS cluster on 11p15.5 [Reik and Maher, 1997; Nicholls et al., 1998]. Imprinted gene clusters may extend over several megabases and can contain both maternally and paternally imprinted genes as well as nonimprinted genes [Nicholls et al., 1998; Maher and Reik, 2000]. An imprinting center (IC) has been identified at the PWS/AS cluster, and at least two imprinting control elements appear to be located in the BWS cluster [Sutcliffe et al., 1994; Buiting et al., 1995; Joyce et al., 1997; Maher and Reik, 2000].

Furthermore, H19 and Igf2, in the BWS cluster, share a common regulatory element by way of enhancer competition [Leighton et al., 1995b], and Igf2 imprinting is influenced by the DMR adjacent to H19 [Leighton et al., 1995a]. On the maternal chromosome, the two common enhancers induce transcription of the unmethylated H19, while Igf2 is silent [Leighton et al., 1995b]. On the paternal chromosome, where H19 is methylated and silent, Igf2 is expressed by the effect of the same enhancers. A deletion on the maternal chromosome encompassing the H19 DMR leads to activation of Igf2 on both maternal and paternal chromosomes [Leighton et al., 1995a].

Tissue-, isoform-, promotor-, and time-specific expression

The imprinting status of imprinted genes can be tissue-specific (e.g. KVLQT1, UBE3A)orcan depend on developmental stage (e.g. IGF2) [Bartolomei and Tilghman, 1997]. However, some imprinted genes, such as the mouse H19 and Snrpn genes, retain their imprinting at all stages of development [Bartolomei and Tilghman, 1997]. Imprinting can also be governed in an isoform-specific way (e.g. PEG1/MEST, GRB10) introducing a wide variety of expression patterns together with tissue-specific expression [Blagitko et al., 2000; Kosaki et al., 2000a]. A promotor-specific expression pattern has been observed for the IGF2 and GNAS1 genes in humans [Vu and Hoffman, 1994; Ekström et al., 1995]. IGF2 is paternally expressed during embryonic development, but its imprinting is later relaxed in the liver by the effect of an additional promotor, leading to biallellic expression in the adult [Vu and Hoffman, 1994]. GNAS1 has two reciprocally imprinted promotors located 11 kb apart [Hayward et al., 1998]. The 5’ promotor encodes a maternally expressed protein NESP55 and the promotor located 19 towards the 3’ end encodes a paternally expressed protein XLαs [Hayward et al., 1998]. In addition, GNAS1 codes a biparentally expressed protein Gsαs [Hayward et al., 1998].

Asynchronous replication Asynchronous replication appears to be a common feature for imprinted genes and was first observed for Igf2r, Igf2, H19,andSnrpn [Kitsberg et al., 1993]. Generally, homologous chromosome loci replicate synchronously, with active alleles replicating early while silenced alleles replicating late [Kitsberg et al., 1993; Bartolomei and Tilghman, 1997]. Imprinted genes, however, show predominantly early paternal replication in both maternally and paternally expressed genes [Kitsberg et al., 1993; Knoll et al., 1994; Bartolomei and Tilghman, 1997].

Repeat sequences Simple repeat elements are observed in many imprinted genes [Neumann et al., 1995]. Nonetheless, no homology exists between the repeats for different genes, which vary in size, sequence, and number of repeats [Bartolomei and Tilghman, 1997]. The exact role of the repeats is not known, but they may help in distinguishing imprinted genes from nonimprinted genes in gametes during establishment of imprinting marks, or they might produce secondary structures that facilitate silencing of the gene [Neumann et al., 1995; Bartolomei and Tilghman, 1997].

Mouse and human homology Imprinting patterns are usually concordant between mice and humans, but all are not faithfully conserved [Bartolomei and Tilghman, 1997]. U2AFBPL (U2af1-rs1)andIGF2R are both imprinted in the mouse but not in the human [Kalscheuer et al., 1993; Pearsall et al., 1996]. Opposite imprinting in the mouse and human was recently noted for GRB10,whichis maternally expressed in the mouse but paternally in humans [Blagitko et al., 2000; Hitchins et al., 2001].

Mechanisms The mechanisms for setting and maintaining imprinting are not completely understood [Constancia et al., 1998; Brannan and Bartolomei, 1999]. Criteria for the imprint include that it must be set differently in the maternal and paternal genomes, preferably during gametogenesis, it must sustain throughout mitotic cell divisions, and it must be able to be 20 erased and reset appropriately in male and female gametogenesis [Bartolomei and Tilghman, 1997]. So far, methylation of 5’-cytosine residues is the most consistent epigenetic mark for imprinting [Li et al., 1993; Jaenisch, 1997].

DNA methylation Approximately 60% to 90% of mammalian 5’-cytosines in CpG dinucleotides are methylated [Bird, 1986]. DNA methylation represses gene expression, which is suggested to be caused by either the direct inhibition of transcription factors binding to methylated CpG sites [Tate and Bird, 1993] or by methyl-CpG binding (e.g. MeCP1 and MeCP2) that compete with transcription factors for binding sites on methylated DNA or remodel DNA into tight chromatin structures unable to be transcribed [Boyes and Bird, 1992]. CpG islands are DNA regions with an over 50% C+G content that range over 200 bases and are often associated with genes [Gardiner-Garden and Frommer, 1987]. They are generally unmethylated and located at the 5’ ends of widely expressed genes but more variably positioned in genes with tissue-specific expression [Bird, 1986; Larsen et al., 1992]. Approximately 30 000 CpG islands are estimated to be present in the [International Human Genome Sequencing consortium, 2001].

Substantial evidence exists for the role of DNA methylation as the principal mechanism underlying imprinting [Li et al., 1993; Jaenisch, 1997]. DMRs have been observed for all imprinted genes, and DNA methylation is essential for mammalian development [Li et al., 1992; Reik and Walter, 1998]. Some DMRs are established early in gametogenesis and are sometimes regarded as true imprinting signals, while DMRs arising later in development are presumed to have roles in maintaining imprinting or controlling the epigenotypes in clusters of imprinted genes [Constancia et al., 1998]. DNA methylation fulfills the prerequisites for the main imprinting mark; it silences gene activity selectively on one parental allele, is maintained in mitosis throughout embryonic development and in adult tissues, and can specifically be erased and reset in gametogenesis [Wigler et al., 1981; Chaillet et al., 1991; Razin and Cedar, 1994]. The recent identification of specific methyltransferases, namely DNMT1, DNMT3A, and DNMT3B, clarifies the dynamic processes of DNA methylation, but the existence of specific demethylases is still controversial [Okano et al., 1998; Robertson et al., 1999; Bestor, 2000; Hsieh, 2000]. DNMT1 functions primarily as a maintenance methyltransferase, with hemimethylated DNA as its substrate, but has also de novo methylation capability [Robertson et al., 1999; Bestor, 2000]. In contrast, DNMT3A and 21

DNMT3B function mainly as de novo methyltransferases by methylating previously unmethylated DNA [Okano et al., 1998; Robertson et al., 1999]. DNMT1, DNMT3A, and DNMT3B are all crucial for normal mammalian development [Bestor, 2000]. Mouse embryos with a mutated Dnmt fail to develop properly and die in the initial phases of pregnancy [Li et al., 1993]. Disruption of the function of Dnmt results in hypomethylation of imprinted genes Igf2, Igf2r,andH19 and loss of their normal imprinted expression; the normally silent paternal allele of H19 is activated and the normally active paternal and maternal alleles of Igf2 and Igf2r, respectively, are silenced [Li et al., 1993].

DNA methylation has a significant role not only in mammalian embryonic development and genomic imprinting but also in X-inactivation and suppression of foreign DNA sequences [Razin and Shemer, 1995; Robertson and Jones, 2000]. Furthermore, 5’-cytosine methylation has a role in cancer epigenetics by causing an increased mutation susceptibility the result of differential repair efficiency, rate of spontaneous deamination, and rate of cell division of methylated DNA and also repression of tumor suppressor genes [Jones and Laird, 1999; Robertson and Jones, 2000]. Additional potential roles for methylation are suggested in DNA repair and genomic instability in general [Robertson and Jones, 2000].

De novo methylation as well as hypomethylation of CpG islands has been reported in cell lines [Wilson and Jones, 1983; Antequera et al., 1990]. These alterations may be caused by adjustment to the in vitro environment by silencing genes unnecessary for culture [Antequera et al., 1990]. Alterations in methylation usually become evident after multiple cell divisions in culture and continuously increase in conjunction with the number of divisions and time spent in culture [Shmookler Reis and Goldstein, 1982]. The methylation changes may be due to the transformation procedures used to create immortal cell lines [Jones et al., 1990] or the composition of reagent media used in culture [Doherty et al., 2000].

Chromatin structure Allele-specific chromatin structure might also account for epigenetic effects. Histone deacetylation allows a tighter chromatin structure, which is associated with hypermethylated regions and reduced gene expression [Jeppesen and Turner, 1993]. Tightly packed heterochromatin is also seen in late-replicating chromosomal regions, and discrepancy in replication timing has been observed between parental alleles of imprinted genes [Kitsberg et al., 1993]. 22

RNA transcripts Imprinted RNA or antisense transcripts have been found for H19, IPW, Igf2r, UBE3A, Igf2, SNRPN,andXIST [Bartolomei and Tilghman, 1997; Constancia et al., 1998]. Possibly, the RNA transcripts have a role in the imprinting process itself [Constancia et al., 1998]. Imprinted antisense transcripts have been reported to overlap Igf2r and UBE3A, thus suppressing expression [Wutz et al., 1997; Rougeulle et al., 1998]. XIST controls X- inactivation in cis by the accumulation of transcripts along the length of the X-chromosome. This is presumed to be required for the spread of heterochromatin from the X-inactivation center [Panning et al., 1997].

Imprinted genes and imprinting disorders Disruption of the normal balance of imprinted genes results in abnormal growth, dysmorphic features, developmental delay, and abnormal behavior [Falls et al., 1999; Tilghman, 1999; Preece and Moore, 2000]. Aberrant phenotypes observed for numerous chromosomes in UPD patients are likely caused by loss of normal imprinting (Table 1). Imprinted genes or parent- of-origin-dependent effects have also been associated with cancers, especially the Wilms’ tumor, neurobehavioral disorders such as AS and PWS, and also autism, bipolar affective disorder, and schizophrenia [Rainier et al., 1993; Isles and Wilkinson, 2000; Nicholls, 2000]. Loss of normal imprinting may be induced by UPD or other mechanisms, including mutations, deletions, or translocations, leading to loss of function of the sole active allele [Hall, 1990; Cassidy, 1995; Falls et al., 1999] (Figure 2A and B). In these cases, a familial mode of inheritance can be seen, which is, distinct from the classical autosomal dominant, recessive, and X-linked modes (Figure 2). Imprinting mutations (IMs) change the epigenotype, i.e. alter the allele-specific methylation and expression patterns [Buiting et al., 1995; Reik and Walter, 1998]. IMs may arise from mutations on the DNA as well as from epimutations which leave the DNA intact but alter the epigenotype [Nicholls et al., 1998; Reik and Walter, 1998]. IMs disable the imprinting mechanisms to switch the opposite sex parental imprint to the imprint according to the sex of the individual during gametogenesis [Reik and Walter, 1998] (Figure 2C and D). In other words, an IM inherited on the paternal chromosome is erroneously transmitted as a paternal imprint, instead of being changed to a maternal imprint, from the mother to her children [Reik and Walter, 1998]. These children then have a 50% risk of inheriting a paternal imprint from both the mother and father. Approximately 3% of AS and PWS cases have an IM, the majority due to inherited submicroscopic deletions, but some sporadic cases do exist [Nicholls et al., 1998]. 23

A)Loss-of-function of the paternal allele of a B) Loss-of-function of the maternal allele of a paternally expressed gene. maternally expressed gene.

C)Imprinting mutation on the paternal allele and D) Imprinting mutation on the maternal allele and failure to reset the paternal imprint in failure to reset the maternal imprint in oogenesis. spermatogenesis.

E)Autosomal dominant F) Autosomal recessive

G)X-linked dominant H) X-linked recessive

I) Y-linked

Figure 2. Pedigrees of loss-of-function of the expressed allele or imprinting mutations disturbing the balance of imprinted genes. A disorder is manifested when the defective gene is transmitted from either the mother (a maternally expressed gene) or the father (a paternally expressed gene). Autosomal dominant and recessive, X-linked dominant and recessive, and Y-linked modes of inheritance are shown for comparison. Modified from Mannens and Alders (1999) and Buiting et al. (1995). Pedigrees are hypothetical. 24

Growth and development Imprinted genes have crucial roles in mammalian embryonic development [Surani et al., 1984; Cattanach and Kirk, 1985; Fundele et al., 1997; Tilghman, 1999]. The effects of aberrant imprinting are most readily seen in embryonic growth and development of the brain [Fundele et al., 1997]. Paternal genes are generally thought to act as growth enhancers, while maternal genes act as growth suppressors [Hurst and McVean, 1997]. This is seen in mice with induced UPD for specific chromosomes or subchromosomal regions, where maternal UPD typically leads to growth-retarded litters, and corresponding paternal UPD induces larger than normal litters [Cattanach and Kirk, 1985]. Comparison of growth between wild- type (wt) mice and chimeric mice with either an androgenetic (ag, paternal) or parthenogenetic (pg, maternal) cell lineage in addition to the wt cells showed increased cell proliferation in the ag-wt cells, whereas a decrease was observed in pg-wt cells [Fundele et al., 1997]. A similar increased proliferation of ag-wt cells and decreased proliferation of pg- wt cells is observed in skeletal muscle and most cartilages [Fundele et al., 1997].

The chimeric ag and pg mice also revealed that maternal genes appear to be in brain development [Barton et al., 1984; Fundele et al., 1989; Nagy et al., 1989]. Maternally derived genes are most abundant in the anterior parts of the brain and the cortex, while paternally derived genes are mostly observed in the hypothalamus [Keverne et al., 1996]. This different distribution of maternally and paternally derived genes in the developing brain may be confluent with the parent-of-origin-specific phenotypes observed in neurobehavioral and mental disorders [Nicholls, 2000]. Defective imprinting of genes in the cluster at 15q11-q13 leads to PWS or AS, which manifest as mental retardation and abnormal behavior (compulsive eating in PWS and inappropriate paroxysmal laughter in AS) [Nicholls et al., 1998]. Moreover, UBE3A, the gene mutated in AS, shows tissue-specific maternal expression in specific regions of the brain, hippocampus, and cerebellum [Kishino et al., 1997; Matsuura et al., 1997; Rougeulle et al., 1997; Vu and Hoffman, 1997]. The gene for PWS remains uncertain [Mann and Bartolomei, 1999]. Parent-of-origin-specific effects, such as a more severe disorder or earlier onset, are associated with parent-specific transmission for multiple mental disorders (e.g. maternal transmission for Tourette's syndrome or paternal transmission for schizophrenia), although specific imprinted genes have not been identified [Isles and Wilkinson, 2000; Nicholls, 2000]. Some disorders are clearly transmitted predominantly from one parent, e.g. maternal transmission of bipolar affective disorder [McMahon et al., 1995]. In addition, autism and epilepsy are associated with maternal transmission of the PWS/AS 25 region, suggesting effects of imprinted genes [Isles and Wilkinson, 2000]. Social functioning of Turner's syndrome girls, 45,X syndrome, appears to be poorer in girls who inherit their single X-chromosome from their mother as opposed to those who inherit it from their father [Skuse et al., 1997]. Inheritance of the paternal X-chromosome is also associated with higher verbal IQ and inhibitory skills [Skuse et al., 1997].

Cancer Loss of imprinting of H19 (maternally expressed) and especially IGF2 (paternally expressed) has been observed in the majority of heterozygous Wilms’ tumors [Ogawa et al., 1993; Rainier et al., 1993]. The Wilms’ tumor occurs more frequently in BWS than in the normal population [Wiedemann, 1983]. Furthermore, the maternally inherited chromosome 11 is specifically lost in Wilms’ tumors [Junien, 1992], indicating the tumorigenetic potential of paternal duplication of chromosome 11, especially at the 11p15 region which contains a cluster of imprinted genes associated with BWS. BWS patients have an increased predisposition to multiple childhood tumors, which might be caused by the disrupted imprinting pattern of IGF2. IGF2 has been suggested to be a candidate gene for tumor onset [Reeve et al., 1985; Scott et al., 1985].

De novo methylation of CpG islands leads to inactivation of tumor suppressor genes [Jones and Laird, 1999]. Abnormal methylation is attributed to one of the “hits” in Knudson’s hypothesis, in addition to loss of chromosomal material or mutations [Jones and Laird, 1999]. Methylation of CpG islands in the promotors of cancer-related genes is seen at least in retinoblastoma, Von Hippel Lindau disease, and colorectal tumors [Ohtani-Fujita et al., 1993; Prowse et al., 1997; Herman et al., 1998]. Chromosome aberrations are preferentially of maternal or paternal origin for some cancers, as in chromosome loss in tumors (preferential loss of the maternal chromosome in Wilms' tumors and osteosarcoma) and translocations (paternal chromosome 9 of the Philadelphia chromosome in chronic myeloid leukemia) [Toguchida et al., 1989; Haas et al., 1992].

Methods for identifying imprinting in humans Imprinted genes can be identified by a differently methylated region (DMR) or parent-of- origin-specific expression of a gene [Kaneko-Ishino et al., 1997]. Methods for studying imprinted genes are thoroughly reviewed by Kelsey and Reik [1998]. Parent-of-origin- specific methylation and expression patterns can be detected by comparing matUPD, patUPD 26 and a biallelic control, that shows both the maternal and paternal patterns. Obtaining both matUPD and patUPD samples is, however, quite difficult due to the scarceness of UPD cases, especially paternal UPDs, and UPD cases of both parental origins may not exist for all chromosomes. In situations where only matUPD or patUPD samples or no UPD samples are available, parental alleles can be distinguished by heterozygosity for a polymorphism. Production of somatic cell hybrids containing either a maternal or paternal human chromosome, as “synthetic UPDs”, is a potential method for studying imprinted genes [Gabriel et al., 1998].

Parent-of-origin-specific DNA methylation Methylation analyses based on methylation-sensitive restriction endonucleases (e.g. HpaII and HaeIII) and subsequent Southern hybridization or PCR are widely used [Kelsey and Reik, 1998; Rein et al., 1998]. The methylation-sensitive restriction enzyme is unable to cut its recognition sequence if the 5’cytosine is methylated, but will cut when it is unmethylated [Rein et al., 1998]. In Southern hybridization, different-sized fragments, based on the methylation status of the recognition sequences, can be visualized by hybridizing a gene- specific probe to the DNA samples. In the PCR based application, primers are designed on both sides of the restriction site [Rein et al., 1998]. A PCR product will be yielded if the site is methylated, i.e. uncut, but the DNA polymerase is unable to copy the template if it is digested, i.e. unmethylated [Rein et al., 1998]. Information using these two methods is restricted to the limited number of 5’methylcytosines located at the recognition sequence of the restriction endonuclease [Rein et al., 1998].

Sodium bisulfite treatment of DNA converts unmethylated cytosines to uracil, whereas methylated cytosines are resistant to bisulfite and remain intact [Frommer et al., 1992; Herman et al., 1996]. Subsequent PCR amplification is performed by primers specific for the methylated and unmethylated strands [Frommer et al., 1992]. In PCR, uracils are amplified as thymines and only the methylated cytosines are amplified as cytosines [Frommer et al., 1992]. Sequencing of the PCR products then discriminates between the corresponding thymines and cytosines, and thus, the methylation status of individual cytosines is ascertained [Frommer et al., 1992]. Alternatively, the allele-specific methylation can be detected by single-strand conformational polymorphism (SSCP) based on different mobility of PCR fragments according to methylation density, single nucleotide primer extension, or restriction 27 enzyme digestion [Sadri and Hornsby, 1996; Gonzalgo and Jones, 1997; Kinoshita et al., 2000].

Potential large-scale screening methods for parent-of-origin-specific methylation include restriction landmark genome scanning (RLGS) and arbitrarily primed PCR (AP-PCR) [Hatada et al., 1991; Gonzalgo et al., 1997; Kaneko-Ishino et al., 1997; Rein et al., 1998]. RLGS has been successfully used in identification of imprinted genes, and AP-PCR in detecting methylation differences in cancer [Plass et al., 1996; Kohno et al., 1998].

Parent-of-origin-specific expression RT-PCR (reverse transcriptase PCR) is widely used in studying parent-of-origin-specific gene expression (reviewed by Kelsey and Reik, 1998). PCR is performed on cDNA produced from RNA samples with specific primers. PCR fragments can be resolved on electrophoresis gels for comparison of expression levels between samples (e.g. reciprocal UPD samples). Sequence variants can be identified by direct sequencing of PCR products or digested with a restriction enzyme specific for a polymorphism. Northern hybridization performed on RNA from reciprocal UPD samples with gene-specific probes is also an applicable approach, although this method requires large quantities of RNA as compared with RT-PCR. cDNA microarrays may prove to be an efficient, novel method for screening parent-of-origin- specific expression simultaneously for thousands of genes in reciprocal UPD samples [Kelsey and Reik, 1998; Duggan et al., 1999]. However, low expression levels at single transcript levels will go undetected and inconsistency of hybridizations might be troubling [Kelsey and Reik, 1998]. No imprinting studies using array technologies have, to my knowledge, been reported.

Expression of imprinted genes may not only be tissue-specific but may also depend on the developmental stage [Bartolomei and Tilghman, 1997]. Therefore, multiple tissues from different developmental stages may need to be studied in order to detect imprinting. The choice of tissue is not as crucial in analyzing DMRs, since they are sustained in the majority of fetal and adult tissues [Kaneko-Ishino et al., 1997; Constancia et al., 1998]. 28

Location-based approach Because imprinted genes occur in clusters new imprinted genes are likely to be found in the vicinity of known ones [Reik and Maher, 1997]. Despite a few exceptions (e.g. U2AFBPL and IGF2R), imprinting is conserved between mouse and human genes [Kalscheuer et al., 1993; Pearsall et al., 1996]. Therefore, most human homologs to mouse imprinted genes are likely to be imprinted as well.

UNIPARENTAL DISOMY OF CHROMOSOME 7

MatUPD7 Over 30 cases of matUPD7 with growth retardation have been reported, suggesting that an imprinted growth-regulating gene(s) resides on chromosome 7 (Table 4). Furthermore, many matUPD7 cases have features typical of Silver-Russell syndrome (SRS), indicating that an imprinted gene(s) might explain, at least partly, the molecular etiology of SRS [Kotzot et al., 1995; Moore et al., 1999]. The first matUPD7 cases were observed in patients homozygous for maternal alleles of a recessive disease; cystic fibrosis (CF, MIM 219700) in two cases [Spence et al., 1988; Voss et al., 1989] and a COL1A2 (proα2(I) chain of type I procollagen) mutation in one [Spotila et al., 1992]. All three had severe growth retardation and isodisomy for the entire chromosome. The case presented by Spotila et al. [1992] had a region of heterozygous alleles on 7p compatible with maternal heterodisomy. Confinement of matUPD7 to only 7q and patUPD7 to 7p in a patient with postnatal growth retardation but no evidence of IUGR suggested that a paternally expressed gene(s) on 7p is required for normal intrauterine growth, and postnatal growth is regulated by paternally expressed genes on 7q [Eggerding et al., 1994]. The hypothesis of an imprinted gene underlying the observed growth retardation was strengthened by a case of maternal heterodisomy with prenatal and postnatal growth retardation [Langlois et al., 1995]. Reduction to homozygosity for a recessive allele could not be excluded in the initial cases of isodisomy. Moreover, no common isodisomic region was detected in five matUPD7 cases with mixed iso- heterodisomy, yielding further evidence for an imprinted gene rather than homozygosity for a recessive gene mutation underlying the matUPD7 phenotype [Preece et al., 1999]. 29

Finding three matUPD7 cases among 35 SRS patients suggests that a maternally imprinted gene(s) has a role in the etiology of SRS [Kotzot et al., 1995]. This was supported by observations of matUPD7 in five of 70 (7%) SRS cases screened [Eggermann et al., 1997; Preece et al., 1997]. An additional 12 cases of matUPD7 with SRS have been reported [Bernard et al., 1999; Price et al., 1999; Hehr et al., 2000; Russo et al., 2000] (Table 4). Screening for matUPD7 has been conducted for in a total of 145 SRS patients yielding matUPD7 in 10% (14/145) [Kotzot et al., 1995; Eggermann et al., 1997; Preece et al., 1997; Bernard et al., 1999; Russo et al., 2000]. Patients with pre- and postnatal growth retardation but not SRS have been included in two studies. Only one case of matUPD7 with pre- and postnatal growth retardation but not SRS was ascertained out of the 29 patients screened [Kotzot et al., 1995; Bernard et al., 1999]. Nevertheless, this patient did present with some features typical of SRS [Kotzot et al., 1995].

The phenotypes of matUPD7 patients do not differ significantly from non-matUPD7 SRS patients; however, the SRS features are regarded as mild, or even almost nondysmorphic, in some matUPD7 cases [Preece et al., 1997; Price et al., 1999]. Some SRS-like features are also described in the first cases of matUPD7 (Table 4) [Spence et al., 1988; Voss et al., 1989; Eggerding et al., 1994; Langlois et al., 1995], suggesting that mild SRS might be present in these patients as well [Kotzot et al., 1995]. Price et al. (1999) evaluated a cohort of 50 SRS patients, including four matUPD7 cases, and concluded that matUPD7 might be found in those patients for whom the diagnosis of SRS is contemplated but is discarded because of failure to meet stricter criteria. Delineation of the matUPD7 phenotype was conducted by Kotzot et al. (2000), who compared the phenotypes of nine matUPD7 subjects and reviewed the clinical findings of all reported matUPD7 patients. They concluded the matUPD7 phenotype to be characterized by pre- and postnatal growth retardation, retarded bone age, relative macrocephaly, a high and broad forehead, a pointed chin, and a slightly increased incidence (67%, 6/9) of psychomotor developmental delay.

A delimited candidate region for the imprinted SRS or growth-controlling gene could not be defined by the above-described matUPD7 cases. Maternal duplication at 7p12.1-p13 was reported in a child and mother, both with short stature and characteristics of SRS [Joyce et al., 1999]. The mother's de novo duplication was of paternal origin, which suggested that the phenotype was caused by an additional copy of the SRS gene(s) rather than imprinted genes in this region. Another SRS patient with a maternal de novo duplication of 7p11.2-p13 was Table 4. Characteristics of reported matUPD7 cases. Characteristics that have not been clearly described in reports have been designated as not reported and are depicted with no mark. += present, -= not present, +/-= slight. #Price et al. (1999) separated SRS facial features into classical and mild, without detailed specification. I= isodisomy, H= heterodisomy, I+H= mixed iso-/heterodisomy. *Preece et al. (1997) and Price et al. (1999) may include some of the same cases. Kotzot et al. (1995) includes reports by Kotzot et al. (1995) and Tariverdian et al. (1996). An additional 11 cases of matUPD7 have been reported by Preece et al. (1999), Cogliati et al. (1998), Dorr et al. (1999), and Mergenthaler et al. (1999). Spence et Voss et Spotila et Eggerding Langlois et Report al. (1988) al. (1989) al. (1992) et al. (1994) al. (1995) Eggermann et al. (1997) Preece et al. (1997) * Price et al. (1999) * Case number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sex FMM F F F // FF MFFF SRS +/- + + + + + + + + + IUGR //+- ++++// Birth weight SD <-2 <-2 P25-50 -3 SD

A SRS patient was reported with maternal isodisomy for the entire chromosome but with mosaicism for a paternal ring chromosome 7 r(7)(p13q11) in 27% of lymphocytes [Miyoshi et al., 1999]. Biparental inheritance of alleles was observed in the centromeric region, suggesting the exclusion of 7p13-q11 as the candidate region for SRS. However, only a paternally expressed imprinted gene can be excluded by this finding, and only if the ring chromosome is active [Wakeling et al., 2000a]. Activity on the ring chromosome was not evaluated. Furthermore, redefined mapping of the biparentally inherited markers verified the biparental region to be even smaller than 7p11.2-7q11.23 [Wakeling et al., 2000a]. Thus, the candidate SRS genes GRB10, IGFBP1,andIGFBP3 at 7p12-p13 were included in the matUPD7 region.

PatUPD7 Only two cases of patUPD7 have been reported and neither suggests the presence of paternally imprinted genes on chromosome 7 [Höglund et al., 1994; Pan et al., 1998]. The first patUPD7 patient was observed in a linkage study to localize a recessive disease gene, congenital chloride diarrhea (CLD, MIM 214700), with only paternal alleles being presented [Höglund et al., 1994]. Detailed genotyping revealed homozygosity in the patient for all markers studied and only paternal inheritance for ten alleles, thus indicative of paternal isodisomy for the entire chromosome 7. The patient had CLD and a mild, high-frequency sensorineural hearing loss extending to the speech range but was otherwise healthy. She was born appropriate for gestational age (AGA), after 34 weeks gestation (1900 g, 45 cm). Her subsequent development was normal and her adult height is -0.1 standard deviation scores (SDS), equal to a midparental height of (-0.1 SDS). The normal phenotype in this patient suggests that no paternally imprinted genes affecting the phenotype reside on chromosome 7 [Höglund, et al. 1994]. 33

The second patUPD7 case was reported in a male patient with CF, immotile cilia, and complete situs inversus, regarded as Kartagener syndrome (MIM 244400) [Pan et al., 1998]. He was also born AGA after 34 weeks gestation (2540 g), but at age 6 months was growth- retarded with signs of significant respiratory disease. Development was not described. It is probable that the reported growth retardation was due to the otherwise complex phenotype, not the effect of imprinted genes.

Candidate chromosome 7 genes for SRS and growth retardation Candidate genes for SRS on chromosome 7 include imprinted genes and genes that have known functions in growth regulation [Moore et al., 1999]. If the gene causing SRS is an imprinted gene, it is most likely maternally imprinted, and thus, a paternally expressed growth-promoting gene, in which matUPD7 patients are deficient, and thus, therefore being short statured [Preece and Moore, 2000]. It is also possible that it is a paternally imprinted and maternally expressed growth-suppressing gene, which is overexpressed in cases of matUPD7 [Preece and Moore, 2000].

Imprinted genes To date, three imprinted genes are known on chromosome 7: PEG1/MEST and COPG2 both at 7q32, and GRB10 at 7p11.2-p12 [Kobayashi et al., 1997; Riesewijk et al., 1997; Blagitko et al., 1999; Blagitko et al., 2000].

PEG1/MEST PEG1/MEST (paternally expressed gene 1/mesoderm-specific transcript) is maternally imprinted in both mice and humans [Kaneko-Ishino et al., 1995; Kobayashi et al., 1997; Riesewijk et al., 1997]. It maps to 7q32, which is homologous to a proximal part of mouse chromosome 6. Maternal duplication of this region causes growth retardation in the mouse [Beechey, 2000]. PEG1/MEST is monoallelically transcribed from the paternal allele in a wide number of fetal tissues [Kobayashi et al., 1997; Riesewijk et al., 1997]. Two different isoforms of PEG1/MEST distinguished by different first exons are found in humans [Kosaki et al., 2000a]. Only isoform 1 is imprinted (expressed from the paternal allele), but isoform 2 is biallelically expressed [Kosaki et al., 2000a]. Both isoforms are expressed concurrently in lymphocytes and lymphoblastoid cell lines, which explains the preliminary results of biparental expression in adult blood lymphocytes and lymphoblastoid cell lines and expression detected from the maternal allele in early embryos [Kobayashi et al., 1997; 34

Riesewijk et al., 1997]. In these studies, primers from the 3’ end of PEG1/MEST that identify both isoforms were used [Kosaki et al., 2000a]. A large 620-bp CpG island, including the promotor region and exon 1 extending into intron 1, showed parent-specific methylation in all tissues studied, with the maternal allele methylated and the paternal allele unmethylated [Riesewijk et al., 1997]. Tandem repeats are typical of imprinted genes and three imperfect direct repeats arranged in tandem are located upstream of the CpG island [Riesewijk et al., 1997]. PEG1/MEST consists of 12 exons and extends over 13 kb [Riesewijk et al., 1998]. Its function is unknown, but its sequence shares similarities with the α/β-hydrolase fold family.

PEG1/MEST is a good candidate for the SRS gene. It is paternally expressed in fetal tissues, thus missing from matUPD7 cases, and mutation of the paternal mouse Peg1/Mest allele, compatible with the situation in matUPD7, causes growth retardation and abnormal maternal behavior [Lefebvre et al., 1998]. However, its role in the etiology of SRS is unlikely [Riesewijk et al., 1998]. A search for mutations in 49 SRS patients and 9 patients with pre- and postnatal growth retardation revealed only one silent mutation and two novel polymorphisms, and no methylation alterations in the 5’ region in SRS patients were found; thus, defective imprinting was also ruled out [Riesewijk et al., 1998].

COPG2, γ2-COP COPG2 (γ2-COP, coatomer protein complex subunit γ 2) is located adjacent to PEG1/MEST in a tail-to-tail orientation with a common overlapping segment of 52 bp at their 3’-UTRs [Blagitko et al., 1999]. Thus, they seem to form the beginning of a novel cluster of imprinted genes at 7q32. COPG2 is expressed only from the paternal allele in the majority of fetal tissues tested, but biallelic expression is seen in fetal brain and liver and adult peripheral blood [Blagitko et al., 1999]. This initial report of imprinting was later debated, as biallelic expression was observed in multiple fetal and extraembryonic tissues and as well as in adult blood lymphocytes [Yamasaki et al., 2000]. The overlapping segment with PEG1/MEST was estimated to encompass over 2.7 kb including at least four exons and introns of COPG2 [Yamasaki et al., 2000]. Interestingly, monoallelic expression in fetal brain, liver, and lung of a paternally expressed antisense transcript from intron 20, CIT1, was found [Yamasaki et al., 2000]. An untranslated transcript, Mit1/Lb9, within intron 20 of mouse Copg2 was subsequently shown to be paternally imprinted [Lee et al., 2000]. In addition, a maternally imprinted antisense transcript, Copg-AS, spanning the 3’UTR’s of Peg1/Mest and Copg2 was identified [Lee et al., 2000]. A role for COPG2 is unlikely in SRS since only a mutation on 35 the silent maternal allele and seven polymorphisms have been found in SRS cases and patients with pre- and postnatal growth retardation [Blagitko et al., 1999; Mergenthaler et al., 2000a]. COPG2 extends over 50 kb, has 24 exons, and a 630 kb CpG island is located at the 5’ end [Blagitko et al., 1999]. A 27 bp repetitive element is located before the overlap with PEG1/MEST [Blagitko et al., 1999]. The function of COPG2 is unknown, but it possibly has a role in cellular vesicle trafficking between the Golgi and endoplastic reticulum [Blagitko et al., 1999].

MEG1/GRB10 MEG1/GRB10 (maternally expressed gene 1/growth factor receptor-bound protein 10) is located at 7p11.2-p12 [Jerome et al., 1997], which is homologous to mouse proximal chromosome 11. Maternal duplication of this region in the mouse results in prenatal growth retardation and paternal duplication in overgrowth [Cattanach and Kirk, 1985]. GRB10 has multiple splice variants and shows isoform-specific as well as tissue-specific imprinting in humans [Blagitko et al., 2000; Hitchins et al., 2001]. In contrast to mouse Grb10,whichis expressed exclusively from the maternal allele, human GRB10 is expressed from the maternal allele only in fetal skeletal muscle (one splice variant) [Blagitko et al., 2000]. While expression from the paternal allele is observed in the fetal brain and spinal cord, biallelic expression occurs in other fetal tissues [Miyoshi et al., 1998; Blagitko et al., 2000; Hitchins et al., 2001]. Maternal expression was also observed from lymphocytes in somatic cell hybrids [Yoshihashi et al., 2000]. Compatible with biallelic expression, the 5’ CpG islands are unmethylated on both parental alleles [Blagitko et al., 2000]. GRB10 consists of at least 22 exons and extends over 190 kb [Blagitko et al., 2000].

GRB10 initially appeared an excellent candidate for the SRS gene. It functions as a growth suppressor via inhibitory interactions with IGF-1, the GH receptor, and insulin [O'Neill et al., 1996; Moutoussamy et al., 1998]. Furthermore, GRB10 is included in a maternally duplicated region (7p11.2-p13) reported in two separate SRS patients, which was suggested to harbor a maternally expressed imprinted gene [Joyce et al., 1999; Monk et al., 2000]. A maternally inherited transition leading to a proline-serine substitution at codon 95 (P95S) in exon 3 of GRB10 was identified initially in two unrelated Japanese SRS patients but in none of 100 controls [Yoshihashi et al., 2000]. However, further evaluation revealed no other P95S mutations in 105 SRS patients and over 400 controls, suggesting that it is only a rare Japanese polymorphism [Mergenthaler et al., 2001; Yoshihashi et al., 2001]. Thus, a role for 36

GRB10 in SRS is unlikely since it is biparentally expressed in the majority of fetal tissues and only multiple polymorphisms, but no mutations, have been detected [Blagitko et al., 2000; Hitchins et al., 2001; Mergenthaler et al., 2001; Yoshihashi et al., 2001].

Imprinted genes occur in clusters [Reik and Maher, 1997]. Therefore, genes in the vicinity of GRB10, PEG1/MEST, and COPG2, such as CPA1, CPA2, CATR1, UBE2H,andNRF1 [Hayashida et al., 2000], are of special interest with regard to imprinting. However, mouse homologs of UBE2H, IRF5, CALU, KIAA0265, and Cda15a02 located close to PEG1/MEST and TTC4, near GRB10, exhibit biparental expression in many mouse tissues [Lee et al., 2000].

Genes not imprinted Several potentially good candidate genes for SRS have been proven not to be imprinted in humans, making them unlikely SRS genes [Wakeling et al., 1998b; Mergenthaler et al., 2000b; Wakeling et al., 2000b]. EGFR (epidermal growth factor receptor) at 7p12 and IGFBP-1 and IGFBP-3 (insulin-like growth factor binding proteins 1 and 3) at 7p12-13 are located in a region homologous to proximal mouse chromosome 11, known to harbor imprinted genes and to have parent-of-origin-specific effects on growth [Cattanach and Kirk, 1985; Cattanach and Beechey, 1990]. Mutant Egfr mice manifest with IUGR and minor abnormalities, however, no evidence exists for Egfr imprinting in the mouse [Wakeling et al., 1998b]. EGFR is widely expressed in fetal tissues and has a significant role during fetal development [Wakeling et al., 1998b]. Expression of EGFR is biallelic in multiple fetal tissues and the placenta as well as in fibroblasts and lymphoblasts of SRS patients with and without matUPD7 [Wakeling et al., 1998b]. Thus, no evidence is available for EGFR imprinting, and it is not likely to be the cause of the matUPD7 phenotype. EGFR is a tyrosine protein kinase and functions as a mediator of epidermal growth factor and transforming growth factor alpha. IGFBP-1 and IGFBP-3 both regulate fetal growth through the insulin- like growth factor axis. Imprinting in humans is unlikely for either gene, since biallelic expression of IGFBP-1 and IGFBP-3 was found in fetal tissues and in matUPD7 patients [Wakeling et al., 2000b].

PAX4 (paired box gene 4) is located at 7q32, close to PEG1/MEST and COPG2. PAX genes function in differentiation and organogenesis, and PAX4-deficient mice are growth-retarded and die shortly after birth [Mergenthaler et al., 2000b]. Biallelic expression of PAX4 in 37 peripheral blood lymphocytes and expression in a matUPD7 sample indicate that it is not maternally imprinted [Mergenthaler et al., 2000b]. No other tissues were analyzed. Mutation screening failed to show relevance to SRS in 50 SRS patients studied, making its role in SRS improbable [Mergenthaler et al., 2000b].

Human chromosome 7 and mouse homology Human chromosome 7 shares homologous regions with mouse chromosomes 2, 3, 5, 6, 7, 11, 12, 13, and 17 [Mouse Genome Database, 2001]. Of these, imprinting phenotypes and imprinted genes have been found on chromosomes 2, 6, 7, 11, 12, and 17 [Beechey et al., 2000]. Maternal duplication of mouse chromosomes 6 and 11 affects growth, which makes the human homologous regions at 7q and 7p, respectively, very plausible for harboring imprinted genes influencing the SRS and matUPD7 phenotype [Cattanach and Beechey, 1990; Moore et al., 1999].

SILVER-RUSSELL SYNDROME

Clinical manifestation Silver-Russell syndrome (SRS) is a syndrome of severe pre- and postnatal growth retardation (as the main manifestation) and slight dysmorphic features (MIM 180860). In 1953, Silver et al. reported two patients with hemihypertrophy, short stature, elevated urinary gonadotropins, café au lait skin pigmentations, a small face in relation to the skull, incurved fifth fingers, and normal development (Table 5). A year later, Russell (1954) reported five patients with intrauterine and postnatal growth retardation, a triangular face with a high bossed forehead, a hypoplastic receding chin, a small face relative to the skull, a shark-mouth with downturned mouth corners, incurved fifth fingers, a high-pitched squeaky voice, and a lean appearance with hypoplastic muscles (Table 5). Later, it was agreed that Silver and Russell had in fact described the same syndrome, although each emphasized different features [Black, 1961; Tanner et al., 1975]. However, due to the great variation of clinical characteristics in SRS patients, much debate has occurred about whether the syndrome should be divided into two syndromes: Silver’s syndrome; patients with congenital short stature and asymmetry, and Russell’s syndrome; patients without asymmetry [Tanner and Ham, 1969]. Such a division 38

Table 5. Features observed in the initial reports [Silver et al., 1953; Russell, 1954] and later in comprehensive studies of SRS patients. += present, blank= not present/reported. Numbers are given as percentages. *, #, §= features are included in the same percentage. General diagnostic criteria are highlighted in bold [Tanner et al., 1975; Escobar, et al. 1978; Saal, 2001].

Silver et Russell Silver Marks et Escobar et Saal et Wollmann al. (1953) (1954) (1964) al. (1977) al. (1978) al. (1985) et al. (1995) n= 2 n=5 n=29 n=140 n=90 n=15 n=386 IUGR + + 93 80 94 Postnatal growth retardation + + 93 97 100 100 99 Triangular face + + 52* 83 73 79 Small face in relation to skull + + 66 Downturned mouth corners +62 74 46 Bossed forehead + * 26 65 Micrognathia + * 43 33 Asymmetry ++2/57957743351 Short 5th fingers + 79 45 80§ 68 Curved 5th fingers + + 76 51 § 60 48 Café au lait spots + + 45 40 28 19 Altered pattern of sexual +341922013 development Development normal + + 71 Poor feeding in infancy + 27 Slender build + + Disproportionately short upper +112220 limbs Delayed anterior fontanelle +18 closure Muscle hypoplasia + Squeaky voice + 7 22 Cryptorchidism + 15 32 # Syndactyly of toes (II-III) 24 14 20 19 Delayed bone age 41 51 Developmental delay frequent 37 18 40 45 Relative macrocephaly 30 67 64 High arched palate 47 Scoliosis-lordosis 6 17 7 Low set/ abnormal ears 20 53 Irregular teeth 28 Hypospadias 8 # Cleft/high arched palate 16 Hypoglycemia 1 11 Simian crease 4 25 Excessive sweating 7 Renal/urethral anomalies 6 8 Pes cavus 7 Anteverted nares 5 Blue sclerae 5 Cardiac anomalies 5 Hypertrichosis 5 Calcaneovalgus deformities 4 Hepatomegaly 4 Splenomegaly 4 Clitoromegaly 3 Ambiguous genitalia 2 Eyebrows meeting at midline 2 Pectus carinatum 2 Epicantal folds 1 Mongoloid slant 1 39 has not been made, although it has been suggested that SRS consists of various subgroups separated by clinical manifestations [Price et al., 1999].

Diagnosis of SRS is based on the typical clinical features since the etiology is still unknown and no specific tests are available [Saal, 2001]. SRS features grow increasingly milder throughout adolescence, and the diagnosis is easily missed in adults [Escobar et al., 1978]. The diagnostic characteristics of SRS are regarded as growth retardation of prenatal onset, a typical triangular face with a bossed forehead, small lower jaw, and downturned mouth corners (shark’s mouth or inverted V), clino- and/or brachydactyly of the fifth fingers, and asymmetry or hemihypertrophy of the face, trunk, and/or limbs [Tanner et al., 1975; Escobar et al., 1978; Saal, 2001]. Numerous other anomalous features are less frequently observed and are considered as mainly confirmatory, not obligatory for the diagnosis (Table 5). The severity of features and the number of anomalies observed in individual patients show extreme variation [Saal et al., 1985]. Therefore, the syndrome is extremely heterogenous, with no single feature being consistently reported in all patients [Saal et al., 1985]. Severe IUGR and postnatal growth retardation are the only features that are observed in nearly all patients (Table 5).

Estimates for the incidence of SRS vary from 1/3 000 or 1/50 000 to 1/100 000 live births [Stanhope et al., 1998; Moore et al., 1999]. SRS is observed in all racial groups equally and in both sexes, however, boys (58%) have been slightly more frequently reported than girls (42%) [Wollmann et al., 1995].

Growth SRS patients are predominantly born at term after normal labor and pregnancy, complicated only by progressive IUGR [Tanner et al., 1975]. Birth weight and length are both severely retarded, but head circumference is in the low normal range [Wollmann et al., 1995]. In a study of 386 SRS patients, the mean birth length for boys born at full term was –4.3 SDS (43.2 cm, range 32-48 cm) and birth weight –3.8 SDS (1940 g, range 1150-2850 g) [Wollmann et al., 1995]. Mean birth length for SRS girls was –3.9 SDS (43 cm, range 34-48 cm) and birth weight –3.6 SDS (1897 g, range 1100-2630 g). The mean birth head circumference was –0.6 SDS (33.3 cm, range 31.5-35.1 cm) [Wollmann et al., 1995]. Postnatal growth velocity is slightly below normal, and in the first three years of life, a reduction in height SDS is seen in both boys and girls, with means of –4.6 SDS and –4.9 40

SDS, respectively [Tanner et al., 1975; Wollmann et al., 1995]. From 3 to 11 years, slight catch-up growth can be seen in both sexes [Wollmann et al., 1995]. A relative improvement in weight is seen in both boys and girls, from –4.1 SDS in boys and –4.3 SDS in girls for the first three years to approximately –3 SDS in both sexes at age 4-12 years [Wollmann et al., 1995]. The pubertal growth spurt has been reported to be normal in its timing and peak velocity [Tanner et al., 1975], early and subnormal [Davies et al., 1988], and completely absent [Wollmann et al., 1995]. The average adult height of SRS patients is approximately 150 cm (-4.4 SDS) for males and 139 cm (-4.8 SDS) for females [Patton, 1988; Wollmann et al., 1995].

Head circumference is not as severely affected as growth of the body and it falls within normal limits or is slightly below normal [Wollmann et al., 1995]. This might give a false impression of hydrocephaly, especially in the neonatal period, but is regarded as relative macrocephaly (a large head relative to the body). A head circumference well below normal values has been reported for a few SRS patients, however, it was concordant with their heights [Saal et al., 1985].

Bone age is generally retarded relative to chronological age, particularly in early childhood [Tanner et al., 1975; Wollmann et al., 1995]. However, as puberty approaches, bone age tends to catch-up, and at the verge of puberty, it is usually only slightly or not at all delayed [Tanner et al., 1975; Wollmann et al., 1995].

Growth hormone (GH) therapy is widely used to increase the final adult height in SRS patients, even though GH deficiency is seldom diagnosed [Tanner et al., 1975; Cassidy et al., 1986; Patton, 1988]. The response to GH therapy is usually good, with an increased height velocity similar to responses seen in SGA children without SRS [Ranke and Lindberg, 1996; Stanhope et al., 1998]. The overall benefit of GH therapy in SRS remains obscure [Saal, 2001].

Dysmorphic features The face is typically triangular with a wide protruding (bossing) forehead and a small lower jaw (Table 5). The corners of the mouth are turned downwards and the palate is highly arched, giving the impression of a shark’s mouth or inverted V. Due to the small mandible, teeth do not fit properly into the jaws and are placed irregularly. Ears often appear low set 41 and slightly posteriorly rotated, and slight ear anomalies have been reported [Escobar et al., 1978; Wollmann et al., 1995]. Clinodactyly and brachydactyly of the fifth digits are evident on inspection, and radiologically, a short fifth middle metacarpal is often observed. Syndactyly of the 2nd and 3rd toes is also a frequent finding [Silver, 1964; Escobar et al., 1978]. Asymmetry of the face, trunk, or limbs is noted independently or conjointly with hemihypertrophy. Only one region may be affected, but in more severe dysmorphic cases, many body parts are asymmetric or hemihypertrophic simultaneously. In addition, a multitude of small anomalies have been reported in singular cases (Table 5).

Development and behavior Alterations in sexual development, indicated by elevated urinary gonadotropins, precocious puberty, premature estrogenization of the vaginal or urethral mucosa, or a markedly retarded skeletal age in relation to sexual development, were a central finding in the initial reports [Silver et al., 1953; Silver, 1964]. However, normal timing of puberty in a cohort of 17 SRS patients argued against precocious puberty [Tanner et al., 1975]. While genital anomalies are rare in girls, cryptorchidism is frequently observed in SRS boys, along with hypospadias. Single cases of ambiguous genitalia have also been reported [Marks and Begeson, 1977; Sujansky and Riccardi, 1978]. Fertility of at least SRS females appears to be normal [Abramowicz and Nitowsky, 1977], but comprehensive studies on fertility of SRS adults have not been reported.

SRS patients are generally considered to have normal psychomotor development and not to exhibit behavioral problems, but a higher than normal incidence of developmental problems has been noted in some SRS patient series [Silver, 1964; Saal et al., 1985]. Studies on the intellectual performance and development of 20 SRS patients between the ages of six and twelve, however, revealed that approximately half of SRS patients actually have impaired cognitive abilities [Lai et al., 1994]. Special educational needs were assessed in 36% of the patients with difficulties especially in reading and arithmetic. Speech therapy for articulation problems or developmental speech problems was received by 48% of the SRS children. Their mean IQ was nearly 1 SD below normal, with 20% scoring at a borderline range (IQ 70-84) of mental retardation and 32% in a range (IQ <70) indicating mild to moderate learning disabilities. The attention span of SRS was normal. 42

Differential diagnosis Due to the nonspecificity of the SRS features and the heterogeneity of SRS, differential diagnosis includes all disorders with IUGR, prenatal short stature, and SRS-like features including chromosomal abnormalities [Saal, 2001]. These disorders include Turner’s syndrome mosaicism, the 3-M syndrome, and fetal alcohol syndrome [Saal, 2001]. One of the most important disorders for differential diagnosis of SRS is Mulibrey nanism (Muscle-liver- brain-eye nanism, MIM 253250), which is a rare autosomal recessive disorder belonging to the Finnish disease heritage [Perheentupa et al., 1970; Lipsanen-Nyman, 1986; Avela et al., 2000]. It manifests with growth retardation of prenatal onset and typical facial features including a triangular face with a prominent forehead, telecanthus, and a low nasal bridge [Perheentupa et al., 1970; Lipsanen-Nyman, 1986]. Other typical Mulibrey nanism features include a J-shaped cella turcica, slender long bones with cortical thickening, fibrous dysplasia of long bones, hepatomegaly, pericardial constriction, prominent jugular veins, pigmented yellow dots in ocular fundi, muscle hypotonia, a high-pitched voice, and naevus flammeus skin pigmentations [Lipsanen-Nyman, 1986]. Both Mulibrey nanism and SRS patients are slender and have a triangular face with a bossed forehead [Lipsanen-Nyman, 1986]. Nevertheless, these two syndromes are distinguishable by typical features, which are rarely or never observed in the other [Lipsanen-Nyman, 1986]. Micrognathia, downturned mouth corners, and clinodactyly are seen in SRS but not in Mulibrey nanism, while hepatomegaly is confined to Mulibrey nanism [Lipsanen-Nyman, 1986]. Asymmetry is more frequent in SRS [Lipsanen-Nyman, 1986].

The Mulibrey nanism gene, MUL, is located at 17q22-q23 [Paavola et al., 1999], and it encodes a RING-B-box-Coiled-coil zinc finger protein [Avela et al., 2000]. Its function is not yet known, but several members of the zinc finger protein family are involved in development and oncogenesis [Avela et al., 2000]. The MUL region maps close to the reported translocation at 17q25 in two SRS patients [Ramirez-Duenas et al., 1992; Midro et al., 1993] and a deletion in the 17q22-q24 region in one SRS case [Eggermann et al., 1998]. UPD for chromosome 17 has not been observed in SRS patients. Only one case of matUPD17 with normal growth and development has been reported, suggesting that no maternally imprinted genes with phenotypic effects are present on chromosome 17 [Genuardi et al., 1999]. 43

Etiology The etiology of SRS is unknown and intrauterine, chromosomal, and genetic causes have all been proposed [Wakeling et al., 1998a]. In addition, aberrations of chromosomes 8, 15, 17, and 18 have been reported in patients with features resembling SRS [Christensen and Nielsen, 1978; Roback et al., 1991; Niklaus et al., 1992; Ramirez-Duenas et al., 1992; Midro et al., 1993; Tamura et al., 1993; Schinzel et al., 1994; Rogan et al., 1996; Eggermann et al., 1998]. Recently, the observation of matUPD7 in approximately 10% of SRS patients has suggested imprinted genes in its etiology. MatUPD7 was discussed in the previous section (p. 28-32). The majority of SRS cases are sporadic, but some familial cases have been reported [Escobar et al., 1978; Duncan et al., 1990; Wakeling et al., 1998a].

Familial SRS Autosomal dominant inheritance of SRS has been reported in seven patients in two 3- generation families and later in seven members of two families, one 3-generation and one 2- generation [Duncan et al., 1990; Al-Fifi et al., 1996]. Duncan et al. (1990) reviewed 190 previously reported SRS cases and found that 38 patients in 23 families had SRS with a likely autosomal dominant or X-linked dominant transmission. In 17 of the families, multiple maternal relatives were of short stature or had signs of SRS. A short-statured mother was reported in 14 and a mother with SRS in 3 families [Duncan et al., 1990]. X-linked inheritance was also suggested in a family with males possessing a more severe phenotype of short stature, SRS-like features, and multiple small pigmentations [Partington, 1986]. However, the skin pigmentations were unlike the café au lait spots seen in SRS [Partington, 1986].

Autosomal recessive inheritance is likely in four families with normal parents and at least two siblings with SRS [Gray and Evans, 1959; Callaghan, 1970; Fuleihan et al., 1971; Robichaux et al., 1981; Partington, 1986]. Autosomal recessive transmission of SRS was later reported in two brothers born to nonconsanguineous parents and six siblings with SRS of consanguineous parents [Chitayat et al., 1988; Teebi, 1992]. The few reports of monozygotic twins discordant for SRS suggest that SRS might be caused by a postzygotic mutation or by nongenetic, mainly uteroplacental, factors [Samn et al., 1990; Bailey et al., 1995]. 44

Chromosome aberrations Deletions of the distal long arm of chromosome 15 (15q26-qter, and more specifically 15q26.3) and ring chromosome 15 have been associated with SRS features [Roback et al., 1991; Tamura et al., 1993; Rogan et al., 1996]. Although many similarities exist between the 15q deletion and ring 15 patients and SRS patients, phenotypic delineation of chromosome 15 aberration patients and SRS patients is suggested [Wilson et al., 1985]. Ring 15 cases have distinct features not typical of SRS (microcephaly, cardiac defects, limb anomalies, and mental retardation), and they lack some classical SRS features such as relative macrocephaly and asymmetry [Wilson et al., 1985]. However, due to the heterogeneity among SRS cases, it is possible that ring 15 and 15q26-qter patients represent another yet to be defined subgroup of SRS. Interestingly, the insulin-like growth factor receptor gene (IGFR1), which is important in normal growth, is located at 15q26.3 [Roback et al., 1991]. Loss of one IGFR1 copy was observed in patients with severe growth retardation, suggesting that IGFR1 mutations could explain the short stature of SRS patients [Roback et al., 1991; Tamura et al., 1993]. However, biallelic expression of IGFR1 was seen in SRS patients, making its role in SRS unlikely [Abu-Amero et al., 1997].

Translocations at 17q25 have been reported in two patients, one with a paternally inherited t(17;20)(q25;q13) and the other with a de novo t(1;17)(q31;q25) [Ramirez-Duenas et al., 1992; Midro et al., 1993]. Both patients presented with pre- and postnatal growth retardation, relative macrocephaly, a small triangular face, thin lips, microglossia, micrognathia, anomalous ears, bilateral 5th finger clinodactyly, and asymmetry. In addition, the patient with t(17;20)(q25;q13) presented with hypoplastic genitalia and SRS was designated as severe. She walked at 27 months but otherwise had normal cognitive and speech development. The other patient had learning difficulties and delayed speech development. The occurrence of both translocations at 17q25 renders this a candidate region for a SRS gene. Inheritance of the translocation from a normal father in the patient reported by Ramírez-Dueñas et al. (1992) suggested that a maternally imprinted gene caused the phenotype observed in the child. A paternally inherited deletion of the chorionic somatomammotrophin hormone (CSH1)gene located in the GH gene cluster at 17q22-q24 in a SRS patient focused more interest on the 17q chromosome region for a SRS gene in at least some SRS patients [Eggermann et al., 1998]. A screening for mat/patUPD17 in seven SRS patients yielded no cases of UPD17 [Ayala-Madrigal et al., 1996]. Furthermore, a case of matUPD17 with normal growth and 45 development suggests that no maternally imprinted genes are present on chromosome 17 [Genuardi et al., 1999].

SRS-like features, including pre- and postnatal growth retardation, a triangular face with a prominent forehead, small and protruding ears, clinodactyly and brachymesophalangy of the fifth fingers, mild developmental delay, and muscular hypotrophy, were observed in a girl with an interstitial deletion of proximal 8q11-q13 [Schinzel et al., 1994]. However, no asymmetry or micrognathia were observed. Trisomy 18 mosaicism and deletions of 18p have been reported in isolated patients with SRS features and psychomotor developmental delay [Chauvel et al., 1975; Christensen and Nielsen, 1978; Niklaus et al., 1992]. Although similarity to SRS might be coincidental in these cases, it is possible that genes on 8q11-q13 or 18p might be responsible for a portion of SRS cases. UPD8 screening in a total of 44 SRS patients revealed no cases of mat/patUPD8 [Schinzel et al., 1994; Ayala-Madrigal et al., 1996].

GROWTH RETARDATION

Many syndromes with or without known genetic defects manifest with pre- or postnatal onset growth retardation [Cowell, 1995]. Short stature is regarded as an individual’s height being at least 2.5 SDS below the mean height for age and gender or midparental height [Rosenthal and Wilson, 1994]. Genetic, endocrine, and environmental factors as well as chronic diseases may cause growth retardation [Rosenthal and Wilson, 1994; Cowell, 1995]. The most common cause of short stature is normal variation due to familial short stature and/or constitutional delay [Rosenthal and Wilson, 1994]. Endocrine abnormalities are diagnosed in a minority of short-statured children [Rosenthal and Wilson, 1994]. Chronic diseases, malnutrition, drugs, and psychosocial deprivation are other common causes, and skeletal dysplasias, such as achondroplasia and diastrophic dysplasia, contribute a small but distinct portion of short stature diagnoses [Rosenthal and Wilson, 1994]. Despite the abundance of different etiologic factors, up to one-fifth of short-statured children remain without an explanation for their growth retardation [Cowell, 1995]. 46

Intrauterine growth retardation Prenatal growth is principally controlled by fetal genetic factors, but it is dependent on maternal factors and placental functions and is highly vulnerable to exogenous insults [Wollmann, 1998]. The placenta not only provides nutrients and oxygen to the fetus but also produces many hormones, such as placental GH and lactogen, which are significant controllers of fetal growth [Wollmann, 1998]. The most crucial endocrine factors for fetal growth are insulin-like growth factors (IGF-1 and IGF-2) and insulin [Fisher, 1998]. Fetal GH has only a small influence on fetal growth [Fisher, 1998]. Fetal growth is caused mainly by multiple cell divisions [Blizzard and Johanson, 1994].

Intrauterine growth retardation (IUGR) is generally defined as a birth length/weight below 2 SDS [Chatelain et al., 1998]. IUGR is attributed to environmental factors (i.e. maternal, placental, or exogenous, such as infections and teratogens) in the majority of cases (60%) and to genetic factors in about 30% of cases [Wollmann, 1998]. Maternal causes include pregnancy-induced hypertension, preeclampsia, severe chronic diseases and infections, smoking, alcohol, substance abuse, and some specific drugs [Cowell, 1995; Wollmann, 1998]. Other maternal factors associated with IUGR are small maternal size, young maternal age, previous IUGR, and short interpregnancy intervals [Cowell, 1995; Wollmann, 1998]. Restriction of space for the developing fetus due to uterus abnormalities or multiparous pregnancy also limits fetal growth [Wollmann, 1998]. Placental insufficiency caused by reduced placental blood flow, placental metabolic disturbance, and chromosomal aberrations, such as mosaicism, leads to fetal distress because of deprivation of nutrients and oxygen [Wollmann, 1998]. Fetal causes of IUGR are relatively rare but include infections, genetic causes, and malformations [Wollmann, 1998]. IUGR is frequently observed conjointly with genetic diseases, leading to inborn metabolic errors, chromosomal abnormalities, and congenital syndromes and malformations [Wollmann, 1998]. Despite the vast number of different growth-restricting factors even up to 40% of IUGR cases remain without a clear cause [Wollmann, 1998].

Most children with IUGR have catch-up growth beginning at two to three months after birth, leading to normal height [Cowell, 1995; Albertsson-Wikland et al., 1998; Chatelain et al., 1998]. This occurs especially in cases where IUGR is caused by other than fetal factors and the child is freed of the restricting factor after birth (e.g. placental insufficiency or maternal disease). Sometimes catch-up growth is seen later, after the age of two years [Cowell, 1995; 47

Albertsson-Wikland et al., 1998]. However, some IUGR children retain their growth retardation and some show even further regression [Cowell, 1995].

Postnatal growth retardation Postnatal growth is greatly dependent on normal endocrine functions. Hypothyroidism, GH deficiency, glucocorticoid excess, diabetes mellitus, hypogonadism, and premature puberty are all causes of postnatal growth retardation [Cowell, 1995]. Chronic diseases, such as colic disease, renal tubular acidosis, cardiopulmonary diseases, hematological disorders, infections, and cancer, as well as many drugs e.g. corticosteroids, may decrease growth [Rosenthal and Wilson, 1994]. Deprivation of vitamin D (rickets), zinc, and general malnutrition are still common causes of short stature, especially in third world countries [Rosenthal and Wilson, 1994; Cowell, 1995]. Psychosocial deprivation caused by abuse, neglect, or emotional deprivation is an unfortunate cause [Cowell, 1995]. Prenatal onset growth retardation may sustain also be sustained postnatally, and thus, a common cause may be found for both. 48

AIMS OF THE STUDY

The principal aim of this study was to investigate matUPD7 and its role in Silver-Russell syndrome (SRS) and growth retardation in general. This was achieved by the specific aims to:

1. identify cases of matUPD7;

2. evaluate the phenotype of matUPD7 patients and to define its discrepancies from SRS patients without matUPD7;

3. determine the role of matUPD7 in growth retardation of unknown etiology;

4. delineate the candidate region for imprinted genes in the etiology of SRS; and

5. study parent-of-origin-specific methylation of genes and ESTs on chromosome 7 between matUPD7 and patUPD7 DNA. 49

MATERIALS AND METHODS

This study was approved by the Ethics Review Board of the Hospital for Children and Adolescents, Helsinki University Central Hospital.

PATIENTS

Patients were recruited from the outpatient clinic for growth disorders at the Hospital for Children and Adolescents, University of Helsinki, Finland, during 1996-2000. Ten SRS patients were from the University Central Hospitals of Oulu, Tampere, and Kuopio, Finland. All patients and/or their parents gave written informed consent, and blood samples were obtained from the patients and both parents (when available).

Silver-Russell syndrome patients Patients with a SRS diagnosis were initially collected from the outpatient clinic for growth disorders at the Hospital for Children and Adolescents, University of Helsinki, Finland. The diagnoses of SRS were confirmed for all patients by either a pediatric endocrinologist or a medical geneticist. An additional ten SRS patients were referred by pediatric endocrinologists and clinical geneticists from the Clinical Genetics Unit, University of Helsinki and the University Central Hospitals of Oulu, Tampere, and Kuopio, Finland.

Further evaluation of all SRS patients was performed (by M. L-N. and K.H.) by reviewing medical records and growth charts. Nine patients were additionally evaluated by physical examination and structured interviews. Patients were included in the SRS group when they fulfilled the following criteria: 1) intrauterine growth retardation and/or born small for gestational age, 2) postnatal growth retardation with height SDS below -2.5, and 3) at least three of the following facial characteristics: triangular face, micrognathia, frontal bossing, craniofacial disproportion in early infancy, and relative macrocephaly, and 4) at least one of the following relative criteria: asymmetry, hemihypertrophy, clinodactyly and/or brachydactyly of fifth digits, low-set ears, and hypospadias/cryptorchidism. Patients were also required to have normal karyotype, growth hormone secretion, and thyroid function. Two SRS patients with GH deficiency were included. 50

Detailed evaluation of matUPD7 patients 1-4 was conducted by physical examination of patients and structured interviews with patients and parents (M.L-N. and K.H.). MatUPD7 patients 5 and 6 were closely evaluated from medical records (M.L-N. and K.H.) and by personal communication with the attending pediatricians and patients' parents (M.L-N.). Dental age and craniofacial structures of matUPD7 patients 1-3 were examined clinically and from photographs, lateral cephalograms, and orthopantomograms by an experienced orthodontist (S.P.) by comparison with a cohort of 19 SRS patients [Kotilainen et al., 1995].

Growth-retarded patients Patients with growth retardation were recruited systematically from the outpatient clinic for growth disorders at the Hospital for Children and Adolescents, University of Helsinki, Finland, during 1996-2000. The evaluation of patients was conducted by reviewing medical dataandgrowthcharts.

Patients were chosen by the following criteria: 1) intrauterine growth retardation (assessed by calculating the birth weight and length SDS for the appropriate weeks of gestation in each individual; patients who had a birth weight and/or length more than 2.0 SDS below the mean weight/length for age [equivalent to 2.5 percentiles] were regarded as IUGR), and/or postnatal growth retardation (height at least 2.5 SDS below the mean for age or midparental height), 2) normal thyroid function and cortisol secretion, and 3) no chronic illnesses known to stunt growth (i.e. juvenile rheumatoid arthritis, kidney or liver failure, colic disease, or cancer). Dysmorphic features and psychomotor developmental problems were accepted. Small groups of patients with familial short stature and growth hormone deficiency were also selected. Patients with an abnormal karyotype, metabolic disorders, or short stature syndromes other than SRS were excluded. Statistical assessment of patient groups (IV) was performed by the Student T-Test.

PatUPD7 patient Fresh blood samples were obtained from a previously identified patUPD7 patient [Höglund et al., 1994]. 51

GENOTYPING

DNA was isolated from peripheral blood samples by standard procedures [Lahiri and Nurnberger, 1991]. Initial screening for matUPD7 cases was performed by genotyping patients and their parents with a set of 13 (III) or 14 (I, II) chromosome 7-specific fluorescent tetra- and dinucleotide repeat microsatellite markers (D7S2201, D7S1808, D7S817, GATA31A10, D7S1818, GATA73D10 (not in study III), D7S2212, D7S821, D7S1799, D7S1804, D7S1824, D7S2195, D7S1826, and D7S559) by PCR and an automated sequencer (ABI). The markers spanned the entire chromosome 7 at 4-25 cM (0.4-33.7 Mb) intervals. Analysis of genotyping data was performed with Genotyper software (Perkin-Elmer). PCRs were performed in 15 ul reactions containing 50 ng DNA, 1xDyNAzyme II buffer, 1.5 mM

MgCl2, 300 uM of each dNTP, 0.3 uM of each primer, 1% DMSO, and 0.3 U DyNAzyme II DNA Polymerase (Dynazyme). Amplification was performed with an initial denaturation of 5 min at 94°C, followed by 30-35 cycles each of 30 s at 94°C, 75 s at 55°C,and60sat72°C, with a final extension at 72°C for 10 min.

When one or more markers suggested matUPD7, additional microsatellite markers were genotyped to verify matUPD7. Criteria for possible matUPD7 were: 1) no paternal allele found; 2) consecutive homozygous markers or heterozygous markers where both alleles were present in the mother. The 2nd criterium was especially designed to increase our sensitivity to detect segmental matUPD7 cases and/or possible deletions. PCRs were performed in 10 µl reactions containing 20 ng DNA, 1x Amplitaq Buffer II, 2.0 uM MgCl2, 100 µM of each dNTP, 4.0 µM of each primer, and 5 U AmpliTaq Gold polymerase (Perkin Elmer). Amplification was done with an initial denaturation of 10 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 59°C, and 30 s at 72°C, with a final extension at 72°C for 10 min. The PCR products were resolved on 6% polyacrylamide gels by electrophoresis and silver- stained, and the results were read visually.

Correct paternity was verified for all matUPD7 patients by genotyping microsatellite markers specific for chromosomes other than chromosome 7 in the above-described ways. 52

SOUTHERN HYBRIDIZATION

Southern hybridization was used in study IV to investigate methylation differences between matUPD7 (patients 1-3) samples and the patUPD7 sample. DNA isolation was performed from fresh blood samples and early-passage (under five doublings) EBV-transformed lymphoblast (LB) cell lines by standard procedures [Lahiri and Nurnberger, 1991]. DNA samples of 5 ug each isolated from blood and LB were digested overnight at 37°C with HindIII alone, HindIII+MspI, or HindIII+HpaII (New England Biolabs). The digestion products were resolved on 0.8% agarose gels (Seakem) by electrophoresis at 30 V for 16-17 h, then transferred by alkaline blotting to Hybond N+ membranes (Amersham). DNA was fixed to membranes by either UV crosslinking for 30 s or baking at 80°C for 2 h. Membranes were washed in 3XSSC prior to prehybridization. Prehybridization was carried out at 65°C for 4 h and hybridization of probes at 65°C overnight in 10% dextran sulfate, 1% SDS (sodium dodecyl sulphate), 1M NaCl, and 160 ug/ml denatured herring sperm DNA. Random primed alpha-32P-dCTP labeled (Rediprime DNA labeling system, Amersham) gene or EST probes were used. Posthybridization washes were performed in 3xSSC, and 0.1% SDS (1x3 min, 2x3 min) at 65°C. Autoradiograms were exposed at 4°Cfor1to14days.

The cDNA probes were chosen based on criteria that they perform as good probes in blot- hybridization analysis and that as a group they are distributed evenly across chromosome 7. Nonchromosome 7 probes were selected from three different chromosomes based on good performance in previous hybridizations. A 4.7 kb PEG1/MEST probe was produced to match the probe used by Riesewijk et al. (1997). 53

RESULTS AND DISCUSSION

IDENTIFICATION OF CASES OF MATUPD7 IN SRS (I, II, III)

MatUPD7 was ascertained in six SRS patients (15%, 6/39) by the presence of maternal alleles only (I, II, III) (Figure 3). Five SRS patients (patients 1-4 and 6) had matUPD7 for the entire chromosome (I, III), and one patient (patient 5) segmental matUPD7 confined to 7q31- qter (II). MatUPD7 patients 1 and 6 had complete isodisomy, and patients 2-4 mixed isodisomy and heterodisomy (Figure 3). MatUPD7 cases 3 and 4 have most likely originated from meiosis I nondisjunction, leading to heterozygosity of pericentromeric markers, while case 2 is derived from meiosis II nondisjunction, resulting in homozygosity of pericentromeric markers [Engel, 1980]. The intervening isodisomic and heterodisomic regions have occurred by additional recombinations [Engel, 1980]. Whether gametic complementation or trisomy rescue resulted in the UPD in these patients is unknown. Isodisomy consisted of the entire chromosome in patient 1 and was most likely caused by either a meiosis II or a mitotic nondisjunction. A normal karyotype was found in lymphocytes of all matUPD7 cases, as well as in chorionic villus sampling in patient 2 and amniocentesis in patient 5.

The segmental maternal isodisomy in patient 5 must have arisen from a rare postzygotic mitotic recombination. Crossing-over in (possibly) the first zygotic division, with subsequent loss of one daughter cell is the most likely explanation. Had recombination occurred in a later cell division or both daughter cells been viable, mosaic UPD would have been the outcome. Mosaicism was not evident in blood DNA analysis; however, it cannot be excluded from other tissues. Other tissues were not available for analysis. A normal karyotype was seen in amniocentesis as well as in a blood sample from the patient. Somatic recombination resulting in segmental UPD is quite rare and has been reported for only chromosomes 2p16 [Stratakis et al., 2001], 4q21-q35 [Yang et al., 1999], patUPD6q24-qter [Das et al., 2000], patUPD11p15 in BWS [Henry et al., 1993], and matUPD14q23-q24.2 [Martin et al., 1999]. Small UPD segments are easily missed in low-density screenings if monoparental inheritance is not evident for a marker. Identical alleles in continuous markers might suggest segmental UPD, which should be verified by a more detailed analysis. case case case case case case 1 P M F 2 P M F 3 P M F 4 P M F 5 P M F 6 P M F

22 D7S2201 I b/b b/c a/b I/H b/b b/b a/b H b/c b/c a/b I/H a/a a/a a/b ho a/a a/a a/a I b/b b/b a/a

21 D7S1808 I a/a a/a b/c H b/c b/c a/a H a/c a/c a/b H a/c a/c b/c he b/c a/b ND I a/a ND ND 15.3 D7S817 I b/b a/b ND H b/c b/c a/d H a/c a/c b/b H a/b a/b b/c ho b/b a/b b/b I a/a a/a a/b 15.1 14 GATA31A10 I b/b b/b a/a H a/c a/c a/b H b/c b/c a/a ND ND ND ND ND a/c c/d a/b I b/b b/b a/b 13 D7S1818 he a/b a/b a/b a/b 12 I b/b b/b a/b I/H b/b b/b a/b H b/c b/c a/c I/H a/a a/a a/b I a/a a/b 11.2

11.21 11.22 D7S2212 I b/b a/b a/b I a/a a/b b/b H a/c a/c b/c H a/b a/b a/b ho b/b a/b a/b I b/b b/b a/b 11.23 D7S821 I a/a a/b b/c I/H c/c c/c a/b H c/c a/c a/b H a/c a/c a/b he a/b b/b a/c I a/a a/b a/a 21.1

21.2 D7S1799 I c/c ND a/b H a/b a/b a/b ND ND a/b c/d I a/a a/b b/c ho a/a a/a a/a I b/b a/b a/c 21.3

22 D7S1804 I c/c b/c a/b H b/c b/c a/d I/H a/a ND ND H a/d a/d b/c I a/a a/a a/b I b/b a/b b/c

31.1 D7S1824 I c/c a/c a/b I b/b b/d a/c I a/a a/c b/b H a/b a/b a/b I c/c c/d a/b I c/c b/c a/c 31.2

31.3 D7S2195 I c/c b/c a/d I/H a/a a/a b/c I b/b a/b a/c H a/d a/d b/c I c/c b/c a/c I a/a a/c b/c

32.1 D7S1826 I/H a/a a/a ND H b/c b/c a/d I/H a/a a/a a/a H a/c a/c b/c I b/b a/b ND I a/a a/b b/b 33 34 D7S559 I c/c a/c a/b I/H c/c c/c a/b H b/c b/c a/b H b/c b/c a/a I c/c b/c a/c I d/d a/d b/c 35

36

Figure 3. Genotyping results of matUPD7 cases. Isodisomy for the entire chromosome was observed in patients 1 and 6, mixed iso-/heterodisomy in patients 2-4, and isodisomy confined to 7q31-qter in patient 5. P=patient, M= mother, F= father, I=isodisomy, H=heterodisomy, I/H= iso-or heterodisomy, ho= homozygous in biparental region, he= heterozygous in biparental region, ND= no data. Informative alleles for matUPD7 are in bold. 55

No common isodisomic region exists in these six patients nor among five previously reported mixed iso-/heterodisomic matUPD7 patients, rendering the possibility of a recessive chromosome 7 gene in the matUPD7 phenotype very unlikely [Preece et al., 1999].

CHARACTERIZATION OF THE MATUPD7 PHENOTYPE (I)

In study I, the phenotypes of four matUPD7 patients (1-4) were compared with each other and as a group with 28 SRS patients without matUPD7. We concluded that the uniformity of characteristics noted in four SRS patients with matUPD7 raises the possibility that matUPD7 patients might comprise a distinct phenotypic entity among SRS patients with mild dysmorphic features. The characteristics distinctive of this subgroup include 1) pre- and postnatal growth retardation, 2) mild or absent SRS craniofacial dysmorphic features: slight or absent facial triangularity, no micrognathia, no downturned mouth corners, 3) speech delay, 4) strikingly poor feeding throughout childhood, 5) excessive sweating, without evidence of hypoglycaemic episodes, and 6) increased parental age at birth.

Further screening produced two additional matUPD7 cases, patients 5 and 6 (II, III), concordant phenotypically with the four earlier cases (Table 6). The similarity of characteristics in patient 5, with matUPD7q31-qter only, to the other matUPD7 patients indicates that matUPD of this small region is sufficient to cause the observed matUPD7 phenotype.

All matUPD7 patients had only mild SRS characteristics and the dysmorphic facial features were in fact so minor that their appearance might well have been regarded as normal. Previously reported matUPD7 patients have also been noted to have a mild or incomplete SRS phenotype [Kotzot et al., 1995; Preece et al., 1997; Price et al., 1999] and features have been described as slight (triangular face, asymmetry of limbs, clinodactyly of fifth digits). Thorough characterization of other matUPD7 cases is needed to verify these criteria. Clinical data of previous matUPD7 patients (Table 4) is insufficient to conduct a detailed comparison with our patients.

Dental examination verified the lack of micrognathia in matUPD7 patients 1-3. Dental examination of patients 4-6 was not sufficient for a valid comparison. A broader lower part of 56 the mandible, and thus, less lower dental arch crowding distinguished the matUPD7 patients clearly from other SRS patients [Kotilainen et al., 1995]. Delayed dental development, especially in early childhood, was markedly more pronounced in matUPD7 patients 1-3 than in SRS patients in general [Kotilainen et al., 1995].

Significant speech delay was noted in all five matUPD7 patients (1-4, 6) evaluated for verbal skills (patient 5 was too young for verbal evaluation). Reports of speech delay in matUPD7 cases are sparse, but a similar finding of a delay, particularly in expressive speech, has been noted in a matUPD7 patient in addition to selective learning disabilities [Bernard et al., 1999]. Compared with non-matUPD7 SRS patients, matUPD7 cases had a higher incidence of speech delay, diagnosed in only 18% of non-matUPD7 SRS patients. It is generally recognized that SRS patients do not have a higher than average incidence of cognitive problems, although recently, an increased need for speech therapy and special education as compared with the population in general has been reported [Saal et al., 1985; Lai et al., 1994]. Our observation of developmental delay in 41% of all SRS patients (III) is consistent with these findings.

In addition, common features for all matUPD7 patients include failure to thrive with strikingly gross feeding difficulties and excessive sweating, both beginning in infancy and continuing into childhood. Feeding difficulties are quite common in SGA children, but usually only shortly after birth. Almost half of non-matUPD7 SRS patients also suffered from poor feeding in the first weeks of life. This is in concordance with a reported 56% (28/50) of SRS patients having feeding difficulties [Price et al., 1999]. In contrast, matUPD7 SRS patients have extremely poor feeding still at 3 to 4 years. Feeding difficulties have also been noted in four other matUPD7 cases [Eggerding et al., 1994; Bernard et al., 1999; Price et al., 1999]. Excessive sweating was observed in matUPD7 patients, particularly shortly after falling asleep or eating. Hypoglycemic symptoms, such as fatigue and irritability, were not reported to occur concurrently with sweating in any of the matUPD7 patients. A history of feeding difficulties and excessive sweating is easily missed if they are not specifically enquired about from the parents. Therefore, it is likely that the actual number of SRS patients with these symptoms is higher than reported. Recently, in a cohort of 32 SRS patients, a total of 90% were reported to suffer from feeding difficulties such as slow feeding, food fussiness, and poor appetite [Blissett et al., 2001]. Table 6. Characteristics of matUPD7 patients. +, characteristic present; - characteristic absent; NE; not evaluated; SD, standard deviation. Initially cases 1-4 were reported in study I, case 5 in study II, and case 6 in study III. * Weight % = percentage in relation to the mean weight for height. matUPD7 case 123456Mean Growth Birth weight/length SD -4.0/-3.6 -3.4/-2.2 -3.5/-2.6 -4.6/-3.4 -4.3/-5.2 -2.2/-3.2 -4.6/-3.1 SD Gestation wks 36 35 39 39 38 39 38 wks Height 2 yrs SD -4.3 -3.0 -3.8 -6.0 -3.0 -4.8 -4.4 SD Weight 2 yrs % * -20 -28 -20 -28 -24 -13 -22 % Height at last evaluation (age) -3.1 (8.5) -3.1 (3.2) -3.6 (2.8) -4.8 (4.4) -2.9 (1.4) -4.8 (6.4) -3.6 SD Weight at last evaluation % * +46 -22 -20 -28 -22 -12 -10% Midparental height +0.6 +0.9 -0.4 -0.4 +0.4 -0.6 0 SD Years bone age delayed (age) 2.3 (4.8) 1.3 (3.2) 0 (2.0) 2.6 (3.8) NE 1.7 (6.3) 1.6 yrs Face Triangular face - - - - +/- +/- 0% (33%) Frontal bossing + + + + + + 100% 6/6 Micrognathia ------0% Downturned mouth corners - - - - +/- +/- 0% Low-set/posteriorly rotated ears + + + + + + 100% Craniofacial disproportion + + + + + + 100% Relative macrocephaly + + + + + + 100% Limbs and Clino-/brachydactyly + + + - + + 83% body Asymmetry/hemihypertrophy + + + + - facial 83% SyndactylyoftoesII/III-----+17% Dimples ----++33% Development Psychomotor development delay + - - + - - 33% and behavior Speech difficulties + + + + NE + 100% High-pitched voice - - - - + + 33% Excessive sweating + + + + NE + 100% Feeding difficulties + + + + + + 100% 58

The delineation of a unique subgroup of matUPD7, among SRS patients would be of use in diagnosing this mild phenotype of SRS. In a recent study of 50 SRS patients attempting to set further clinical criteria for SRS, a subgroup was observed presenting with the following homogenous characteristics: 1) classical dysmorphic facial features, 2) higher frequency of asymmetry, 3) hand anomalies, 4) birth length below or equal to -2 SDS from the mean, 5) poor postnatal growth (height SDS below or equal to -2 SDS), and 6) preservation of head circumference [Price et al., 1999]. Patients included in this group generally had four of these criteria. No matUPD7 cases filled these criteria, but the matUPD7 cases were noted to have a generally milder SRS phenotype.

The mild dysmorphic features observed in matUPD7 SRS patients are concordant with mild phenotypes observed in chromosome 15 UPD patients as compared with deletion patients. Angelman syndrome (AS) patients with patUPD15 have consistently been observed with a milder phenotype than AS cases with a deletion [Bottani et al., 1994; Moncla et al., 1999]. The patUPD15 cases generally lack the typical facial features of AS and have only mild epilepsy and ataxia, in contrast to the typical AS phenotype. PatUPD15 patients are also reported to manifest with atypical early-onset prepubertal overweight, walk at an earlier age, and have short stature less frequently than AS cases with a deletion. Prader-Willi syndrome (PWS) patients with matUPD15 were found to have an atypical face for PWS more frequently than patients with a paternal deletion of chromosome 15q11-q13 [Allanson et al., 1999].

DELINEATION OF THE CANDIDATE SRS REGION TO 7q31-QTER BY SEGMENTAL matUPD7 (II)

The finding of segmental matUPD7 confined to only 7q31-qter in a SRS patient significantly narrowed down the candidate imprinted gene region. The patient was homozygous for 23 consecutive markers with maternal-only inheritance of alleles for nine markers. Biparental inheritance was evident for markers on the rest of chromosome 7. The matUPD7 segment spanned a maximum of 43 Mb and a minimum of 35 Mb.

This region encompasses the previously identified imprinted genes PEG1/MEST and COPG2 at 7q32. Further interest is thus focused on them as well as on the genes adjacent to them, Table 7. Putative candidate genes for SRS located in matUPD7 segment 7q31-qter. Localization of genes is according to GeneMap’99, LDB, and OMIM. Only genes with a possible or known function in regulating growth and development, cell cycle, signalling pathways, or a phenotype similar to characteristics noted in SRS are listed. The segment also includes other genes not listed.

Gene OMIM Mouse chr. HYAL4 Hyaluronoglucosaminidase 4 604510 GPR37 G protein-coupled receptor-37 602583 SPCH1 Speech-language disorder-1 602081 WNT2 Wingless-type MMTV integration site family, member 2 147870 6 CAV1 Caveolin-1 601047 CAV2 Caveolin-2 601048 6 LEP Leptin 164160 6 PTPRZ1 Protein-tyrosine phosphatase receptor-type, zeta-1, polypeptide 176891 IMPDH1 Inosine-5'-monophosphate dehydrogenase, type I 146690 CATR1 CATR tumorigenic conversion 1 600676 NRF1 Nuclear respiratory factor 1 600879 CPA1 Carboxypeptidase A1 114850 6 CPA2 Carboxypeptidase A2 600688 UBE2H Ubiquitin-conjugating enzyme E2H 601082 PEG1/MEST Paternally expressed gene 1/Mesoderm-specific transcript 601029 6 COPG2 Coatomer protein complex, subunit gamma-2 604355 PAX4 Paired box homeotic gene-4 167413 6 EPHA1 Eph tyrosine kinase 1 ephrin receptor EphA1 179610 PTN Pleiotrophin (heparin-binding growth factor 8, neurite growth-promoting factor 1) 162095 6 EPHB6 Ephrin receptor EPHB6 602757 ZYX Zyxin 602002 CASP2 Caspase2 600639 SMARCD3 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 3 601737 CDK5 Cyclin-dependent kinase 5 123831 5 INSIG1 Insulin-induced gene 1 602055 GPX1 Gastrulatin brain homeo box 1 603354 5 HLXB9 Homeo box-HB9 142994 NOS3 Nitric oxide synthase 3, endothelial cell 163729 5 RHEB2 Ras homolog enriched in brain 2 601293 SCRA1 Sacral agenesis, autosomal dominant (Currarino triad) 176450 CUL1 Cullin1 603134 60 which might expand the imprinted gene cluster even more. Genes mapped close to PEG1/MEST and COPG2 include CPA1, CPA2, CATR1, UBE2H,andNRF1 [Hayashida et al., 2000]. Mouse homologs of UBE2H and NRF1 exhibit biparental expression, which makes it unlikely that the human homologs are imprinted [Lee et al., 2000]. Other intriguing genes in this region for the matUPD7 phenotype include those with a known function in regulating growth, the cell cycle, or gene expression (Table 7). Genes where a mutation leads to a phenotype with features of SRS are also likely candidates; especially good candidates are CULLIN1 and NOS3. CULLIN1 regulates mammalian G1/S transitions [Yu et al., 1998] and mutation in C. elegans causes hyperplasia of all tissues and abnormally small cells [Kipreos et al., 1996]. NOS3 synthesizes nitric monoxide, which regulates vasomotor tone and blood flow through the intrauterine artery to the fetus. Nitric monoxide deficiency leads to fetal growth retardation in pregnant rats [Yallampalli and Garfield, 1993].

7q31-qter is homologous to mouse chromosomes 5, 6, and 12. The proximal region of chromosome 6 contains Peg1/Mest and Copg2, and maternal duplication of this region is associated with growth retardation and early embryonic lethality in mice [Beechey et al., 2000]. Chromosome 12 also harbors imprinted genes Dlk and Meg3/Gtl2, but these localize to human chromosome 14q [Beechey et al., 2000]. Chromosome 5 is not known to harbor imprinted genes [Beechey et al., 2000].

THE ROLE OF MATUPD7 IN GROWTH RETARDATION (III)

MatUPD7isconfinedtoSRS In addition to 39 SRS patients, matUPD7 was screened in 176 patients with prenatal and/or postnatal growth retardation of unknown etiology but no SRS. MatUPD7 was confined to patients fulfilling the diagnostic criteria of SRS (3%, 6/205). MatUPD7 was not observed in patients from the other groups studied, and therefore it does not appear to be a general cause for growth retardation.

The patient cohort consisted of patients with 1) growth retardation of prenatal (IUGR) or postnatal onset (PNGR), 2) the presence or absence of dysmorphic features, and 3) delayed psychomotor development or normal development. These characteristics are not only 61 compatible with features reported in matUPD7 patients and in association with abnormal imprinting [Tilghman, 1999], but they also represent typical clinical findings in children evaluated for abnormal growth. Therefore, cases of matUPD7 would likely also have been found in patients other than exclusively SRS patients if matUPD7 had a significant role in growth retardation.

Cases of matUPD7 had significantly higher maternal (38 years) and paternal (40 years) ages at birth than non-matUPD7 SRS (29 and 31 years) and IUGR and PNGR patients (30 and 32 years in both groups, respectively). Paternal age of the patUPD7 patient studied was also increased, at 44 years, and maternal age, at 39 years, at birth. Increased parental ages, especially paternal, are also found in cases of matUPD15 and patUPD15 [Robinson et al., 1993]. Increased paternal age might raise the risk for nondisjunctive events in spermatogenesis, similar to maternal age in oogenesis, and thus, increase the risk for UPD [Robinson et al., 1993]. MatUPD7 and SRS patients' birth lengths were significantly shorter than IUGR patients', and matUPD7 patients had a higher prevalence of speech delay than SRS and other patients. Otherwise, all patient groups were indistinguishable by multiple clinical parameters.

Imprinted gene effects on growth failure and other characteristics seen in patients without matUPD7 cannot be excluded. Our coarse screening method would have missed, for example mutations, small deletions, small UPD segments, and imprinting defects, all of which could cause silencing of the expressed allele of an imprinted gene.

MatUPD7 screening as a diagnostic tool It is often difficult to find an appropriate diagnosis for a IUGR child with growth failure and dysmorphic features. Thus, screening for matUPD7 in IUGR children with postnatal growth retardation and mild SRS features, may be a valuable diagnostic tool. From a clinical perspective, it is not, however, worthwhile to screen all growth-retarded children, or even all IUGR patients, for matUPD7. MatUPD7 would be found in only 3% (6/205) or 5% (6/130) of children, respectively. We propose that screening for matUPD7 be directed at especially on IUGR patients with persistent short stature, dysmorphic features indicative of SRS, (however, not compulsorily the classical facial dysmorphic features), speech difficulties, poor feeding, and excessive sweating. 62

PARENT-OF-ORIGIN-SPECIFIC METHYLATION OF CHROMOSOME 7 GENES IN LYMPHOBLAST DNA AND BLOOD DNA (IV)

Parent-of-origin-specific methylation of 41 chromosome 7 genes and ESTs was studied by comparing DNA samples from three matUPD7 and one patUPD7 case. DNA was extracted from both fresh blood and from low-passage (under five cell divisions) EBV-transformed lymphoblast (LB) cell lines. Up to 41% of the genes and ESTs studied showed parent-of- origin-specific methylation in LB DNA, while methylation differences were not detected in blood DNA. The paternal chromosome showed more frequent and stronger alterations in methylation patterns when comparing blood DNA with LB DNA; methylation changes were much less frequent and weaker on the maternal chromosome and the control with biparental contribution of the chromosomes. Reduced methylation occurred for 64% and de novo methylation for 18% of probes in the patUPD7 subject, whereas in the matUPD7 subject, reduced methylation was observed for only 27% and de novo methylation for only 5% of probes. The controls consistently shared the same methylation pattern as the matUPD7 subject. This indicates that chromosome-wide methylation is possibly maintained differently on the maternal and paternal chromosomes 7. Furthermore, it is possible that the maternal chromosome 7 maintains methylation patterns on both the maternal and paternal chromosomes by trans-acting factors which might regulate either a maintenance methyltransferase or act by suppressing or enhancing a de novo methylase/demethylase [Reik and Walter, 1998].

CpG islands are known to undergo de novo methylation and demethylation after long periods and multiple passages in cell culture [Wilson and Jones, 1983; Antequera et al., 1990]. Methylation status may change due to the transformation performed in cell cultures or because of reagents used in culture [Doherty et al., 2000]. The methylation changes observed in our studies occurred after fewer than five cell divisions and remained unchanged in subsequent cell divisions. Global alteration of methylation patterns was not seen for three other chromosomes tested, therefore, cell culture in itself does not provide a sufficient explanation for the observed methylation patterns. Similar alterations in methylation between blood DNA and LB cell line have been noted for PW71 [Kubota et al., 1996]. This observation cautions against overinterpreting results of imprinting studies obtained from cultured tissues since methylation alterations appear to be significant. Expression patterns 63 may also vary to a great extent. Whether or not the methylation differences observed in our study portray actual imprinted genes warrants further studies. 64

CONCLUSIONS AND FUTURE PROSPECTS

The observation of what appears to be a distinct phenotype in matUPD7 patients is an interesting finding, but is still only a small step in elucidating the etiology of SRS. For the first time, a diagnostic test is available for at least a small portion of SRS patients. Detailed descriptions of several additional matUPD7 patients are needed to clearly delineate the matUPD7 phenotype. This initial observation of a distinct phenotype will hopefully serve as a guideline in this task.

Since matUPD7 is observed in only 10-15% of SRS cases, the majority of these remain without an etiologic explanation. It is possible that some non-matUPD7 cases might have loss of function of the paternal allele due to a deletion, mutation, or another mechanism disrupting normal imprinting. This would lead to the same situation as in matUPD7, but the loss of activity would be more specific since only one or a few genes would be affected instead of the whole chromosome. Milder and atypical phenotypes are observed in AS patients with patUPD15 and PWS patients with matUPD15 as compared with patients with deletions. In light of these findings, a deletion or mutations in a specific imprinted chromosome 7 locus might explain some of the more severe SRS cases. This might be one explanation for the discrepancy of characteristics observed in SRS cases.

Our observation of a segmental matUPD7 region at 7q31-qter notably narrows down the candidate gene region. Even though the imprinted gene GRB10 is located outside the matUPD7q31-qter segment at 7p11-p13, GRB10 and other genes in its vicinity cannot be completely excluded from the search for the SRS gene. Two SRS patients with maternal duplication encompassing this region make it very intriguing.

Multiple modes of inheritance have been postulated to underlie SRS and SRS has been suggested to consist of multiple subgroups instead of one sound entity. These subgroups may fall under different molecular etiologies portraying distinct but similar characteristics. Therefore, it is possible that genes in other chromosomes might be responsible for the remaining 90% of SRS cases, such as the translocations and deletion observed in 17q25 in three SRS patients and the SRS-related aberrations of chromosomes 8, 15, and 18. The delineation of additional subgroups among SRS cases would greatly facilitate the studies on the molecular etiology of SRS and also help in clinical evaluation of SRS patients. 65

Additional imprinted genes to the ones currently known on chromosome 7 are needed to identify the gene(s) responsible for the matUPD7 phenotype. Moreover, disrupted imprinting or mutations in a gene leading to loss of expression of both alleles in patients without matUPD7 is required to verify the SRS gene. In-depth understanding of the mechanisms governing imprinting and the magnitude of imprinted genes on chromosome 7 are essential for uncovering at least part of the etiology of SRS. Obviously, the clarification of the role of a single imprinted gene in the pathology of SRS, or any disorder, requires thorough consideration of various parameters such as the origin of the tissues studied (fresh versus cultured), possible isoforms, tissue-specific imprinting, studying embryonic tissues during a range of developmental stages instead of only adult tissues, and revealing the mechanisms controlling imprinting of a specific gene and the genes around it.

The precise molecular etiology of not only SRS but of a large number of short-statured children remains unclear. MatUPD7 does not appear to be a general cause of growth retardation, but the role of a general growth-controlling imprinted gene on chromosome 7 cannot be excluded. Future studies should shed more light on the effects of UPD and imprinted genes on human growth and development in general. 66

SUMMARY

The inheritance of both chromosomes 7 from only the mother, i.e. maternal uniparental disomy of chromosome 7 (matUPD7), is observed in approximately 10% of cases of Silver- Russell syndrome (SRS). SRS is a syndrome of unknown etiology manifesting as severe pre- and postnatal growth retardation and slightly dysmorphic features. Paternal UPD7 (patUPD7) has no effect on growth and development, which suggests that an imprinted gene(s) on chromosome 7 is connected with growth regulation and SRS. Imprinted genes are active only on the maternally or paternally inherited chromosome. The genetic imprints are established differently during oogenesis and spermatogenesis, most likely by DNA methylation. Imprinted genes generally have crucial roles during embryonic development, and thus, inheritance of both maternal and paternal genomes are required for normal development. UPD disrupts the normal balance of imprinted genes and may in many cases lead to abnormalities in growth, development, and behavior. The aim of this study was to investigate matUPD7 and its role in SRS and growth retardation in general by clarifying the molecular genetic and phenotypic effects of matUPD7 and by delineating the candidate region for imprinted genes in the etiology of SRS.

In an initial screening for cases of matUPD7, DNA samples from 32 SRS patients and their parents were screened for matUPD7 (I). Four cases of matUPD7 were identified, all of which resembled each other and had distinct characteristics that distinguished them from non- matUPD7 SRS patients. The matUPD7 patients had very mild SRS features, lacked many typical SRS characteristics, and presented with additional characteristics not typical of SRS. Excessive sweating, severe feeding difficulties, and speech delay with problems especially in articulation and production of speech were pronounced in all matUPD7 patients. It was concluded that matUPD7 patients appear to form a distinct entity among SRS patients.

Further screening for matUPD7 in SRS patients revealed a patient with matUPD7 for only 7q31-qter (II). This was the first report of segmental matUPD7 in a patient with SRS, which significantly narrowed the candidate region for imprinted genes in the etiology of SRS. This segment includes two imprinted genes, PEG1/MEST and COPG2, both at 7q32, thus rendering further interest in their roles in SRS. Other candidate genes for SRS in this segment include those with known functions in growth regulation or those which when mutated cause 67 a SRS-like phenotype. Because imprinted genes tend to occur in clusters, especially genes in close proximity to PEG1/MEST and COPG2 are strong candidates.

The significance of matUPD7 in growth retardation in general was evaluated by screening 205 children with either SRS or intrauterine and/or postnatal growth retardation of unknown etiology for matUPD7 (III). Six cases of matUPD7 were observed (3%, 6/205), all among the SRS patients (15%, 6/39). The confinement of matUPD7 to patients with features of SRS suggests that matUPD7 is not a general cause of growth retardation. Thus, screening for matUPD7 should be focused on children with severe pre- and postnatal growth retardation, features indicative of SRS, speech difficulties, and poor feeding.

The identification of matUPD7 cases (I) enabled methylation differences between matUPD7 patients and a previously identified case of patUPD7 [Höglund et al., 1994] to be studied (IV). Southern hybridizations were performed with EST/gene cDNA probes spanning the entire chromosome 7. Parent-of-origin-specific methylation was observed for up to 41% of ESTs/genes studied in DNA from lymphoblast (LB) cell culture. No methylation differences were observed in DNA isolated from blood. The paternal chromosomes 7 were more prone to alterations in methylation in LB DNA than the maternal chromosomes. These findings indicate that methylation patterns may change in a parent-of-origin-specific manner during cell culture, which cautions against overinterpreting results of imprinting studies on cultured cell lines. The results suggest that methylation is differently maintained on maternal and paternal chromosomes 7, which may give insight on how methylation and possibly imprinting is governed on chromosome 7.

In conclusion, the matUPD7 phenotype has been further characterized by the identification of six new cases of matUPD7 that seem to form a distinct phenotypic entity among SRS patients. Segmental matUPD7 confined to 7q31-qter in one SRS patient delineates the candidate region for imprinted genes in the etiology of SRS. Methylation analyses of ESTs/genes between matUPD7 and patUPD7 cases revealed many parent-of-origin-specific differences. These results will aid in identifying new imprinted genes on chromosome 7 and in unraveling the molecular etiology of SRS. 68

ACKNOWLEDGMENTS

This study was carried out at the Department of Medical Genetics and the Hospital for Children and Adolescents, University of Helsinki, during 1996-2001. I wish to thank the former and present heads of the Department of Medical Genetics, Professors Albert de la Chapelle, Juha Kere, Leena Palotie, and Pertti Aula, for providing me with excellent research facilities. My sincere gratitude to everyone who made this study possible.

I am grateful to Professor Juha Kere, the supervisor of my thesis, for his skillful guidance throughout this study. I have been fortunate to have had him as my supervisor and also to have had such an expert guide in to the exciting world of molecular genetics. His never- ending optimism is remarkable. I truly admire his endless enthusiasm towards science in addition to his cornucopia of new ideas.

Dr. Marita Lipsanen-Nyman, my "clinical supervisor", is profoundly thanked for her expertise in all the clinical aspects. Her contribution to this study is immeasurable. I have been privileged to share in her vast knowledge of pediatric endocrinology, especially in growth disorders and the Silver-Russell syndrome.

Professor Christer Holmberg is warmly thanked for his collaboration and interest in this study. His insight into the outlines of each part of this thesis is gratefully acknowledged.

Professor Jaakko Perheentupa is acknowledged for providing excellent resources at the outpatient clinic for growth disorders at the Hospital for Children and Adolescents for this study. All physicians and the entire staff at the clinic are thanked for their help in collecting patients for this study.

My sincere thanks to all collaborators in this study: Dr. Stephen W. Scherer for, providing the EST/gene probes and for fruitful collaboration; Doctors Tero Kontiokari, Päivi Tapanainen, Ilkka Kaitila, Hanna Liisa Lenko, and Kalle Simola, for referring SRS patients; Dr. Paula Kristo, for expert assistance in genotyping; Professor Sinikka Pirinen, for dental evaluation of patients and for her interest in my work; Dr. Pia Höglund for all her advice and support.

The official referees Docent Helena Kääriäinen and Professor Raimo Voutilainen are thanked for reviewing this thesis and for their constructive criticism and valuable comments.

Docents Anna-Elina Lehesjoki and Leo Dunkel, members of my thesis committee for the Helsinki Biomedical Graduate School, are thanked for their interest in my work.

Carol Ann Pelli is thanked for revising the English language of this thesis.

My gratitude to everyone at the Department of Medical Genetics for creating such a pleasant and fruitful working environment. Sinikka Lindh is acknowledged for assistance in collecting blood samples. Minna Maunula and Ilpo Vilhunen are thanked for all their help in practical matters. Maija Wolf is especially thanked for her friendship in and outside of the laboratory.

I warmly thank everyone formerly and presently in Juha’s group for their friendship and helpfulness. Ms. Riitta Lehtinen is especially thanked for her skillful assistance in the laboratory. My heartfelt thanks to Siru, not only for sharing the lab bench but for the many 69 laughs we shared during the long evenings and weekends in the lab during medical school. I’m also grateful for her help in scientific matters.

I am fortunate to have so many wonderful friends, all of whom are warmly thanked for their friendship and all the extracurricular activities throughout the years. Siru and Laura are entitled to a special thanks for all the numerous relaxing lunchs and coffee breaks we've shared.

My mother and father are dearly thanked for their love and support. You’ve brought me up to pursue the things that are important to me. My sister Johanna and brother-in-law Jussi are thanked for their continuous encouragement. You showed me how this is done.

My dear Juha, thank you for bringing music to my life. Your support and faith are invaluable.

My sincere thanks to all the patients and their families taking part in this study.

This work was financially supported by the Päivikki and Sakari Sohlberg Foundation, the Finnish Medical Foundation, the Sigrid Juselius Foundation, the Foundation for Pediatric Research, the Research and Science Foundation of Farmos, the Paulo Foundation, the Finnish Cultural Foundation, the Helsinki Biomedical Graduate School, and the Academy of Finland.

Helsinki May 2001, 70

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