Hypomethylation of the H19 Gene Causes Not Only Silver-Russell Syndrome (SRS) but Also Isolated Asymmetry or an SRS-Like Phenotype
2006; Elsevier BV; Volume: 78; Issue: 4 Linguagem: Inglês
10.1086/502981
ISSN1537-6605
AutoresJet Bliek, Paulien A. Terhal, Marie-José van den Bogaard, Saskia M. Maas, Ben C.J. Hamel, Georgette B. Salieb–Beugelaar, Marleen Simon, Tom G.W. Letteboer, Jasper van der Smagt, Hester Kroes, Marcel M.A.M. Mannens,
Tópico(s)Prenatal Screening and Diagnostics
ResumoThe H19 differentially methylated region (DMR) controls the allele-specific expression of both the imprinted H19 tumor-suppressor gene and the IGF2 growth factor. Hypermethylation of this DMR—and subsequently of the H19 promoter region—is a major cause of the clinical features of gigantism and/or asymmetry seen in Beckwith-Wiedemann syndrome or in isolated hemihypertrophy. Here, we report a series of patients with hypomethylation of the H19 locus. Their main clinical features of asymmetry and growth retardation are the opposite of those seen in patients with hypermethylation of this region. In addition, we show that complete hypomethylation of the H19 promoter is found in two of three patients with the full clinical spectrum of Silver-Russell syndrome. This syndrome is also characterized by growth retardation and asymmetry, among other clinical features. We conclude that patients with these clinical features should be analyzed for H19 hypomethylation. The H19 differentially methylated region (DMR) controls the allele-specific expression of both the imprinted H19 tumor-suppressor gene and the IGF2 growth factor. Hypermethylation of this DMR—and subsequently of the H19 promoter region—is a major cause of the clinical features of gigantism and/or asymmetry seen in Beckwith-Wiedemann syndrome or in isolated hemihypertrophy. Here, we report a series of patients with hypomethylation of the H19 locus. Their main clinical features of asymmetry and growth retardation are the opposite of those seen in patients with hypermethylation of this region. In addition, we show that complete hypomethylation of the H19 promoter is found in two of three patients with the full clinical spectrum of Silver-Russell syndrome. This syndrome is also characterized by growth retardation and asymmetry, among other clinical features. We conclude that patients with these clinical features should be analyzed for H19 hypomethylation. Silver-Russell syndrome (SRS [MIM 180860]) is a clinically heterogeneous syndrome, first described by Silver et al. (Silver et al., 1953Silver HK Kiyasu W George J Beamer WC Syndrome of congenital hemihypertrophy, shortness of stature, and elevated urinary gonadotropins.Pediatrics. 1953; 12: 368-376PubMed Google Scholar) and Russell (Russell, 1954Russell A A syndrome of intra-uterine dwarfism recognizable at birth with cranio-facial dysostosis, disproportionately short arms, and other anomalies (5 examples).Proc R Soc Med. 1954; 47: 1040-1044PubMed Google Scholar). Diagnosis can be difficult, and at least four of the following criteria should be present: intrauterine growth retardation (IUGR), poor postnatal growth, relatively normal head circumference, classic facial phenotype, and asymmetry (Price et al. Price et al., 1999Price SM Stanhope R Garrett C Preece MA Trembath RC The spectrum of Silver-Russell syndrome: a clinical and molecular genetic study and new diagnostic criteria.J Med Genet. 1999; 36: 837-842PubMed Google Scholar). At the genetic level, the syndrome is heterogeneous: although mostly sporadic, in familial cases, the syndrome can be transmitted in an autosomal dominant, autosomal recessive, and/or X-linked dominant way (Duncan et al. Duncan et al., 1990Duncan PA Hall JG Shapiro LR Vibert BK Three-generation dominant transmission of the Silver-Russell syndrome.Am J Med Genet. 1990; 35: 245-250Crossref PubMed Scopus (68) Google Scholar; Teebi Teebi, 1992Teebi AS Autosomal recessive Silver-Russell syndrome.Clin Dysmorphol. 1992; 1: 151-156Crossref PubMed Scopus (26) Google Scholar; Al-Fifi et al. Al-Fifi et al., 1996Al-Fifi S Teebi AS Shevell M Autosomal dominant Russell-Silver syndrome.Am J Med Genet. 1996; 61: 96-97Crossref PubMed Scopus (24) Google Scholar; Ounap et al. Ounap et al., 2004Ounap K Reimand T Magi ML Bartsch O Two sisters with Silver-Russell phenotype.Am J Med Genet A. 2004; 131: 301-306Crossref PubMed Scopus (18) Google Scholar). In most patients, the molecular pathology of SRS is unknown; however, abnormalities have been described for chromosome 7 (especially maternal uniparental disomy [mUPD], in ∼10% of cases) (Monk et al. Monk et al., 2002Monk D Bentley L Hitchins M Myler RA Clayton-Smith J Ismail S Price SM Preece MA Stanier P Moore GE Chromosome 7p disruptions in Silver Russell syndrome: delineating an imprinted candidate gene region.Hum Genet. 2002; 111: 376-387Crossref PubMed Scopus (71) Google Scholar). Although, in general, complete mUPD 7 is found in these cases, a single case was reported with a partial mUPD 7q31-qter (Hannula et al. Hannula et al., 2001Hannula K Lipsanen-Nyman M Kontiokari T Kere J A narrow segment of maternal uniparental disomy of chromosome 7q31-qter in Silver-Russell syndrome delimits a candidate gene region.Am J Hum Genet. 2001; 68: 247-253Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Analysis of genes in this region did not lead to the identification of a candidate gene involved in the syndrome. Various other chromosomal abnormalities have been described, including trisomy 1q32.1-q42.1 (van Haelst et al. van Haelst et al., 2002van Haelst MM Eussen HJ Visscher F de Ruijter JL Drop SL Lindhout D Wouters CH Govaerts LC Silver-Russell phenotype in a patient with pure trisomy 1q32.1-q42.1: further delineation of the pure 1q trisomy syndrome.J Med Genet. 2002; 39: 582-585Crossref PubMed Scopus (20) Google Scholar), deletion of chromosome 15q or ring chromosome 15 (Rogan et al. Rogan et al., 1996Rogan PK Seip JR Driscoll DJ Papenhausen PR Johnson VP Raskin S Woodward AL Butler MG Distinct 15q genotypes in Russell-Silver and ring 15 syndromes.Am J Med Genet. 1996; 62: 10-15Crossref PubMed Scopus (40) Google Scholar), 18p− (Christensen and Nielsen Christensen and Nielsen, 1978Christensen MF Nielsen J Deletion short arm 18 and Silver-Russell syndrome.Acta Paediatr Scand. 1978; 67: 101-103Crossref PubMed Scopus (20) Google Scholar), translocations involving breakpoints 17q25 (Ramirez-Duenas et al. Ramirez-Duenas et al., 1992Ramirez-Duenas ML Medina C Ocampo-Campos R Rivera H Severe Silver-Russell syndrome and translocation (17;20) (q25;q13).Clin Genet. 1992; 41: 51-53Crossref PubMed Scopus (48) Google Scholar) and 17q23–24 (Dorr et al. Dorr et al., 2001Dorr S Midro AT Farber C Giannakudis J Hansmann I Construction of a detailed physical and transcript map of the candidate region for Russell-Silver syndrome on chromosome 17q23-q24.Genomics. 2001; 71: 174-181Crossref PubMed Scopus (30) Google Scholar), and, finally, a paternally inherited deletion of the CSH1 gene at chromosome 17q22-24 (Eggermann et al. Eggermann et al., 1998Eggermann T Eggermann K Mergenthaler S Kuner R Kaiser P Ranke MB Wollmann HA Paternally inherited deletion of CSH1 in a patient with Silver-Russell syndrome.J Med Genet. 1998; 35: 784-786Crossref PubMed Scopus (25) Google Scholar). In 2002, Fisher et al. (Fisher et al., 2002Fisher AM Thomas NS Cockwell A Stecko O Kerr B Temple IK Clayton P Duplications of chromosome 11p15 of maternal origin result in a phenotype that includes growth retardation.Hum Genet. 2002; 111: 290-296Crossref PubMed Scopus (81) Google Scholar) reported three patients with a phenotype that includes growth retardation. These patients presented with maternal duplications of chromosome 11p15. In 2005, Eggermann et al. (Eggermann et al., 2005Eggermann T Meyer E Obermann C Heil I Schuler H Ranke MB Eggermann K Wollmann HA Is maternal duplication of 11p15 associated with Silver-Russell syndrome?.J Med Genet. 2005; 42: e26Crossref PubMed Scopus (81) Google Scholar) described two patients with maternal duplications of 11p15 and SRS. Recently, Gicquel and coworkers (Gicquel et al., 2005Gicquel C Rossignol S Cabrol S Houang M Steunou V Barbu V Danton F Thibaud N Le MM Burglen L Bertrand AM Netchine I Le BY Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome.Nat Genet. 2005; 37: 1003-1007Crossref PubMed Scopus (393) Google Scholar) published a series of patients with SRS who had hypomethylation of the H19 region at 11p15 (MIM 103280). The 11p15 region is also associated with the Beckwith-Wiedemann syndrome (BWS [MIM 130650]), characterized by macroglossia, omphalocele, fetal gigantism, and other abnormalities, including various childhood tumors (DeBaun et al. DeBaun et al., 2002DeBaun MR Niemitz EL McNeil DE Brandenburg SA Lee MP Feinberg AP Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects.Am J Hum Genet. 2002; 70: 604-611Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In many cases, the overgrowth is asymmetric, producing hemihypertrophy, which is probably more accurately termed "hemihyperplasia." The syndrome can be caused by various molecular defects, which lead to altered expression of imprinted genes on chromosome 11p15, including H19 and IGF2 (MIM 147470) (Bliek et al. Bliek et al., 2001Bliek J Maas SM Ruijter JM Hennekam RC Alders M Westerveld A Mannens MM Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.Hum Mol Genet. 2001; 10: 467-476Crossref PubMed Scopus (139) Google Scholar) (fig. 1a). Given the opposite phenotypes of SRS and BWS, Chitayat et al. hypothesized, as long ago as 1988, that the growth abnormality and asymmetry in both disorders might be caused by disregulation of the same gene (Chitayat et al. Chitayat et al., 1988Chitayat D Friedman JM Anderson L Dimmick JE Hepatocellular carcinoma in a child with familial Russell-Silver syndrome.Am J Med Genet. 1988; 31: 909-914Crossref PubMed Scopus (15) Google Scholar). Here, we report support for that hypothesis from a series of patients with H19 hypomethylation and clinical features ranging from isolated asymmetry to the full clinical spectrum of SRS. Methylation indices of both H19 and KCNQ1OT1 (MIM 604115) were measured in blood lymphocytes as described elsewhere (Bliek et al. Bliek et al., 2001Bliek J Maas SM Ruijter JM Hennekam RC Alders M Westerveld A Mannens MM Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.Hum Mol Genet. 2001; 10: 467-476Crossref PubMed Scopus (139) Google Scholar). In brief, DNA was digested overnight with a methylation-sensitive restriction enzyme (NotI for KCNQ1OT1 and SmaI for the H19 promoter), was precipitated, and was digested overnight with a second restriction enzyme (BamHI for KCNQ1OT1 and PstI for H19). Completion of digestion of the methylation-sensitive enzyme was monitored by the use of control probes that recognize the nonmethylated restriction site. After Southern blotting, the filters were hybridized with the probes listed below. Hybridization was measured in a phosphoimager (Amersham). The Imagequant program (Amersham) was used to measure the intensity of the radioactive bands. The mean methylation index (intensity of the measured band divided by the intensity of both bands) (±SD) for control individuals for H19 is 0.5 (±0.03) and for KCNQ1OT1 is 0.51 (±0.025). Normal methylation was defined as the mean ±2 SD. Deletion studies were performed on patient DNA digested with ApaI. After Southern blotting, the filter was hybridized with the following probes and primer sets: deletion detection probe (probe 1 in fig. 1a), forward 5′-ATTTCCTGAGTCTCCCCTTGG-3′ and reverse 5′-TCGGCAAACCCTCTGTTCC-3′; methylation detection probe (in first exon of H19) (probe 2 in fig. 1a), forward 5′-GTGGGAGCCAAGGAGCACCTTGGACATCTG-3′ and reverse 5′-TCCTGGTGACGTCCTGCTGCAACTCCCCGA-3′; and methylation detection probe (KCNQ1OT1), forward 5′-CCAGGTGAGAGGTAGTGGTAGAAGTC-3′ and reverse 5′-TCTTTGCATTCCTAGAGCAATCC-3′. DNA containing CA repeats was amplified using standard PCR methods. Markers were taken from the ABI PRISM Linkage Mapping Sets version 2.5 kit or were retrieved from the National Center for Biotechnology Information Web site. The PCR was done with Cy5-dCTP, and the fragments were analyzed by polyacrylamide gel electrophoresis with ALFexpress (Amersham Pharmacia). The PCR products generated with the ABI PRISM Linkage Mapping Sets version 2.5 kit were analyzed by capillary electrophoresis by use of Genetic Analyzer ABI310 (Applied Biosystems). Three primer sets were used to amplify all seven CTCF-binding sites, described elsewhere (Bell and Felsenfeld Bell and Felsenfeld, 2000Bell AC Felsenfeld G Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene.Nature. 2000; 405: 482-485Crossref PubMed Scopus (1295) Google Scholar), in the differentially methylated region (DMR) between the IGF2 and the H19 genes. The first three sites were amplified with the primers CTCFS1-3F, 5′-GCCCATCTTGCTGACCTCAC-3′, and CTCFS1-3R, 5′-AGAAGACCTCCGAGAACCCTG-3′. For CTCF site 4–6, the following primers were used: CTCFS4-6F, 5′-GGTAGGACCCTTGTACGAGCC-3′, and CTCFS4-6R, 5′-GACCTGAAGATCTGGTGCGG-3′. For CTCF site 4, we used the following primer set: CTCFS7F, 5′-ATTTCCTGAGTCTCCCCTTGG-3′, and CTCFS7R, 5′-TCGGCAAACCCTCTGTTCC-3′. Two additional sequence primers were used: CTCFS site 2R, 5′-AATGTGGCTCCCATGAGTG-3′, for CTCF site 2, and CTCF site 5R, 5′-AGAAGGGTTTCACACTAGGGCCG-3′, for CTCF site 5. PCR reactions were done in a total volume of 25 μl with MgCl2 (1.5 μM), dNTP (0.24 mM), Betaine (50 mM), primers (1.2 μM), and 0.3 U Amplitaq Gold (Applied Biosysytems), with an annealing temperature of 60°C. After purifying the PCR product with QIAquick PCR Purification Kit 250 (QIAGEN), a sequence reaction was performed using the BigDye Terminator v1.1. Cycle Sequencing Kit (Applied Biosystems), with an annealing temperature of 60°C. Samples were analyzed on an ABI 310 genetic analyzer after ethanol precipitation. The patient was the third child of healthy parents. She was born after a pregnancy of 40 wk and 2 d. Birth weight was 1,870 g (−3.6 SD), birth length was 41 cm (−4.6 SD), and skull circumference was 33.5 cm (−1 SD). In the first weeks, nasogastric tube feeding was necessary, and, in the first months, there was a failure to thrive. At age 7 mo, a ventrical septal defect was detected. At age 8 mo, she developed circulatory and respiratory insufficiency after gastroenteritis, followed by multiorgan failure and postanoxic encephalopathy. Because of severe feeding problems, she was fed by percutaneous gastrostomia from age 2.5 years onward. Mental development is retarded because of encephalopathy. Physical examination at age 2 years and 9 mo showed a height of 87 cm (−2 SD). Unfortunately, skull circumference was not measured. She had dysmorphic features suggestive of SRS. An iris coloboma was noted in the left eye. There was asymmetry of the arms and legs in (left side larger) and bilateral mild ulnar deviation of the third, fourth, and fifth fingers (fig. Figure 2, Figure 2). The patient was born after a spontaneous gemelli pregnancy. The twin sister is healthy. Because of IUGR of the patient, a cesarean section was performed at 35 wk and 6 d of pregnancy. The proband had a birth weight of 1,310 g (−2.4 SD) and a skull circumference of 32 cm (−0.8 SD). Length at age 2 wk was 38 cm (−6.5 SD). Pathologic examination showed dichorionic, diamniotic placentas. The placenta of the patient was smaller than the placenta of her healthy twin sister (1:2). DNA analysis confirmed dizygosity. Postpartum, features compatible with arthrogryposis multiplex congenita were present. The proband had ulnar deviation of the hands; flexion deformity of the fingers, especially on her right hand; and camptodactyly of the index finger. A luxation of the left hip, bilateral knee luxation, and bilateral pes equinovarus deformity were noted. In the first years, she had a severe failure to thrive. Partial nasogastric tube feeding was necessary until age 1 year and 9 mo. Mental development was normal. Physical examination at age 4 years and 1 mo showed a height of 84.5 cm (−5 SD), a head circumference of 49.5 cm (−0.3 SD), and a weight of 9.6 kg (−2.4 SD for her length). She had facial features that suggested SRS. Two café-au-lait spots were present. Total hand length, leg length, and leg circumference were smaller on the right side than on the left side (fig. Figure 2, Figure 2). Cardiac evaluation showed two small ventricular septal defects. Metabolic studies were normal except for a temporary, isolated elevation of pipecolinic acid. An electromyography showed no abnormalities compatible with neuropathy. The patient was born after a pregnancy of 39 wk and 3 d, with a birth weight of 1,780 g (−3.6 SD), a birth length of 40 cm (−5.5 SD), and a skull circumference of 33 cm (−2 SD). After birth, she underwent surgery because of ambiguous genitalia with a large clitoris. In the first weeks, she was fed partially by nasogastric tube. At age 14 years, her development is completely normal. Because of absence of breast development and hypergonadotropic hypogonadism, magnetic resonance imaging of the abdomen was performed and showed absence of ovaries and a hypoplastic uterus. She has been treated with growth hormone; her height is currently 1.60 m. Physical examination at age 2 years showed a slender girl with a height of 77 cm (−3.3 SD), head circumference of 47.5 cm (−0.3 SD), and a weight of 8,050 g (−2.6 SD for her length). She had facial characteristics of SRS. There was asymmetry of the body (left side larger). Physical examination at age 14 years was not allowed. Patient 4 was born after a pregnancy of 41 wk, with a birth weight of 1,800 g (−4.2 SD), a birth length of 42 cm (−5 SD), and a skull circumference of 34 cm (−1 SD). He had ambiguous genitalia consisting of severe periscrotal hypospadias and a small introitus. An atrial septal defect was detected. Hepatosplenomegaly and a conjugated hyperbilirubinemia with progressive liver function disturbance was present. Liver biopsy at age 3.5 mo showed nonspecific fibrosis and ductular proliferation. The patient had a severe failure to thrive. Nasogastric tube feeding was necessary. He died at age 7 mo, probably as a result of an influenza pneumoniae. Physical examination shortly after birth showed several dysmorphic features suggestive of SRS (fig. 2g). Metabolic and endocrine investigations showed no abnormalities. Obduction showed mild asymmetry of the arms and legs (left side larger), severe liver cirrhosis without biliary atresia, and fibrous material in the pericardiac sac. Patient 5 was born after a pregnancy of 39 wk and 2 d, with a birth weight of 2,260 g (−2.5 SD) and a birth length of 47.5 cm (−1.75 SD). Skull circumference was 32.2 cm (−2.2 SD). No feeding problems were present in the neonatal period. Psychomotor development was normal. At age 10 years, she was referred to the orthopedic surgeon because of leg length discrepancy and asymmetry of the arms (right side larger). No clinical features of SRS were present. The parents of patient 6 are of Turkish descent. The patient was born after a pregnancy of 41 wk and 3 d, with a birth weight of 2,800 g (−1.8 SD) and a birth length of 50 cm (−1 SD). Skull circumference was 35.5 cm (+0.4 SD). Feeding problems started at age ∼4 mo, when she often refused fluids. At age 8 years, she was referred to the Department of Medical Genetics because of growth retardation in combination with discrepancy of leg length and circumference (left side larger). Mental development was normal. Physical examination at age 8 years showed a height of 119.2 cm (−2 SD according to the growth curve for Turkish children). Head circumference was 51 cm (−0.5 SD). No facial abnormalities were present except a slightly narrow face with thin lips. She had three small café-au-lait spots. This patient was born after a pregnancy of 41 wk and 3 d, complicated with a beginning HELLP syndrome (hemolysis, elevated liver enzyme levels, and a low platelet count), which prompted inducing labor. She had a birth weight of 2,560 g (−2 SD). Birth length was 46 cm (−3 SD). The neonatal period was uneventful. At age 7 wk, her occipital-frontal circumference was 38.3 cm (0 SD). Asymmetry was noticed from birth and involved limbs and face, which clinically looked more like a rightsided hemihypotrophy than hemihypertrophy. At age 1 year, her height was 72 cm (−1.5 SD), and, at age 4 years and 9 mo, it was 109 cm (0 to −1 SD). Apart from bilateral fifth finger clinodactyly, no SRS-like features were present. The right hand showed a variant simian crease. Otherwise, she is a healthy young girl of mixed Dutch-Angolese descent. Her mental and motor development has been completely normal. Methylation studies were performed for this patient because of body asymmetry. This patient was born after a pregnancy of 38 wk, with a birth weight of 1,500 g (−3.4 SD). Information about his birth length and skull circumference was not available. An asymmetry (left side larger) of the body was present. Since age 23 years, he has suffered from progressive muscle weakness in his right arm. At age 38 years, he developed insulin-dependent diabetes. Recently, it was discovered that his creatine kinase (CK) is elevated (1,264 U); the explanation of the elevated levels is still unknown. Intelligence is normal. Height currently is 1.70 m. He has no dysmorphic features suggestive of SRS. This pregnancy was achieved after intracytoplasmic sperm injection. The patient was born after a pregnancy of 40 wk and 3 d, with a birth weight of 2,500 g (−2 SD) and a birth length of 49 cm (−1 SD). No feeding problems were present in the neonatal period. Shortly after birth, a leg length discrepancy of 3 cm was noted (right leg larger). Height was 90.5 cm at age 3 years and 3 mo (−2.2 SD). Physical examination showed mild dysmorphic features (fig. Figure 2, Figure 2). Her mental development has been normal. For patients 1 and 3, a diagnosis of SRS was made. For patients 2 and 4, the clinical features were suggestive of SRS; however, the additional features of severe arthrogryposis multiplex congenital and liver cirrhosis have not been described before in patients with SRS. In the remaining patients, the diagnosis of SRS could not be made. Chromosome analysis was performed for patients 1, 2, 3, 4, 6, 8, and 9 and showed a normal karyotype. Comparative genome hybridization was performed for patient 2 and patient 4, and no abnormalities were detected. Methylation studies of the H19 gene on chromosome 11p15 are routinely performed in our laboratory as part of a diagnostic test for BWS (Bliek et al. Bliek et al., 2001Bliek J Maas SM Ruijter JM Hennekam RC Alders M Westerveld A Mannens MM Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.Hum Mol Genet. 2001; 10: 467-476Crossref PubMed Scopus (139) Google Scholar). We noticed that, in seven patients with asymmetry, the H19 promoter was hypomethylated rather than hypermethylated (fig. 1b, patients 2, 4, 5, 6, 7, 8, and 9). All seven patients also exhibited IUGR or postnatal growth retardation (table 1 and fig. 2). This suggested to us that the asymmetry in the patients could be due to hemihypotrophy rather than hemihypertrophy. In addition, patients 2 and 4 had an SRS-like phenotype (table 1). Therefore, we examined patients 1 and 3 and one additional patient, whose diagnoses were clearly SRS, according to the criteria of Price et al. (Price et al., 1999Price SM Stanhope R Garrett C Preece MA Trembath RC The spectrum of Silver-Russell syndrome: a clinical and molecular genetic study and new diagnostic criteria.J Med Genet. 1999; 36: 837-842PubMed Google Scholar). Among these patients, patients 1 and 3 had a complete hypomethylation of the H19 gene (fig. 1). The third patient with SRS had no methylation defect.Table 1Clinical Features of Patients with Hypomethylation of H19Clinical FeaturesP1P2P3P4P5P6P7P8P9Neonatal period: Pregnancy40 wk 2 d35 wk 6 d (twins)39 wk 3 d41 wk39 wk 2 d41 wk 3 d41 wk d38 wk40 wk 3 d ≤−2 SD, birth weight (g),+, 1,870+, 1,310+, 1,780+, 1,800+, 2,260−, 2,800+, 2,560+, 1,500+, 2500 ≤−2 SD, birth length (cm)+, 41+, 38 (at age 2 wk)+, 40+, 42−, 47.5−, 50+, 46Unknown−, 49 Skull circumference (cm)33.532333432.235.538.3 (at age 7 wk)UnknownUnknown Hypoglycemia−−−−−Unknown−Unknown−Facial features: Triangular face++++−−−−+ Frontal bossing++++−−−−+ Micro- or rethrognathia++++−−+−− Downturned corners of the mouth++++−−−−+ Thin lips++++−+−−+ Crowded teeth/enamel defects−−−Unknown−−−− Delayed closure or large fontanel anterior+−++−−+Unknown−Delayed development: Motor+++ (mild)+−−−−− Mental+−−Unknown−−−−−Skeletal: Postnatal growth retardation++++−+−++ Asymmetry+++++++++ Clinodactyly digit 5+++++−+−+ (mild) Joint luxation++−−−−−−− Camptodactyly−+−−−−−−−Gastrointestinal: Feeding problems++++−+−−− Liver anomalies+, Post-anoxic+,aTemporary increased pipecolinic acid.−Cirrhosis−−−−−Urogenital: Abnormal genitalia−bNeonatal vaginal prolapse.−+cAmbiguous genitalia with clitoral hypertrophy, absent ovaries, and hypoplastic uterus.+dAmbiguous genitalia and periscrotal hypospadias with small introitus.−−−−− Renal abnormalities+eSmall kidneys and postanoxic acute tubular necrosis.−−+fSubcortical renal cysts.−−−−−Congenital cardiovascular anomaliesgVSD = ventrical septal defect; ASD = atrial septal defect.+ (VSD)+ (VSD)−+ (ASDhAnd fibrinous layer pericard.)−−−−−Skin findings: Café-au-lait spots−++−−+−−− Hyperhidrosis+−++−Unknown−−−OphthalmologicalColoboma−−−−−−−−Cancer−−−−−−−−−Other−−−−−−−+iElevated CK, diabetes mellitus, and muscle weakness.−Note.—A plus sign (+) = present; a minus sign (−) = absent.a Temporary increased pipecolinic acid.b Neonatal vaginal prolapse.c Ambiguous genitalia with clitoral hypertrophy, absent ovaries, and hypoplastic uterus.d Ambiguous genitalia and periscrotal hypospadias with small introitus.e Small kidneys and postanoxic acute tubular necrosis.f Subcortical renal cysts.g VSD = ventrical septal defect; ASD = atrial septal defect.h And fibrinous layer pericard.i Elevated CK, diabetes mellitus, and muscle weakness. Open table in a new tab Note.— A plus sign (+) = present; a minus sign (−) = absent. Hypermethylation of H19 is often seen in patients with BWS in conjunction with hypomethylation of the imprinted KCNQ1OT1 gene, as a result of a UPD, mostly in a mosaic form (Bliek et al. Bliek et al., 2001Bliek J Maas SM Ruijter JM Hennekam RC Alders M Westerveld A Mannens MM Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS.Hum Mol Genet. 2001; 10: 467-476Crossref PubMed Scopus (139) Google Scholar). All H19 hypomethylated patients had a normal methylation pattern at the KCNQ1OT1 locus (fig. 1), so we exclude mUPD of a large part of chromosome 11p15 as an explanation for the methylation defects seen in these patients. To further exclude a small mUPD around the H19 gene, we analyzed a series of polymorphic markers in this region (fig. 3 and table 2). In all patients, biallelic signals with normal intensity were found, excluding uniparental isodisomy in all cases. For patients 1, 2, 3, 6, and 7, parents were available and uniparental heterodisomy could be excluded. Additionally, we found no evidence of UPD-7 in any of our patients (table 2). We conclude that UPD around H19 is not the mechanism that leads to the H19 hypomethylation in our patient group.Table 2UPD Screening ResultsAlleles by Patient (Methylation Index ofH19)Chromosome and Marker (Location)P1 (.00)P2 (.18)P3 (.00)P4 (.14)P5 (.32)P6 (.36)P7 (.29)P8 (.23)P9 (.39)7p14-q21: D7S484 (35.1 Mb)NDNI3/73/63/53/73/43/4NI D7S510 (38.9 Mb)ND3/53/43/53/63/64/51/2NI D7S519 (45.9 Mb)ND3/44/53/54/64/94/52/85/7 D7S502 (66.0 Mb)ND3/82/5NI4/45/10ND2/35/7 D7S669 (77.5 Mb)ND2/71/35/93/75/6ND7/107/8 D7S630 (88.0 Mb)ND1/57/81/7NI3/7674/76/77q32-36: D7S640 (132 Mb)ND2/72/44/6NI4/5125/82/6 D7S684 (138 Mb)ND6/7NINI2/31/647NI1/7 D7S661 (143 Mb)ND6/85/62/84/66/7283/5NI D7S636 (150 Mb)ND7/124/114/68/95/8386/113/9 D7S798 (152 Mb)5/6NI5/64/63/64/6NI5/63/611p15: D11S2071 (0.24 Mb)1/3NI3/82/4ND1/537NIND D11S922 (1.5 Mb)NI5/67/121/4ND4/7596/9ND D11S4046 (1.9 Mb)NDNI7/93/86/83/8251/107/8 TH (2.1 Mb)NDNINI1/5ND2/312NDND D11S1318 (2.3 Mb)6/82/81/7NDND3/7NINDND D11S988 (4.4 Mb)6/94/107/96/9ND1/9266/7ND D11S1338 (5.9 Mb)1/4NI3/41/4NDNINI2/4ND D11S902 (17 Mb)ND7/8ND5/63/71/3NI7/8NI D11S904 (27 Mb)ND2/62/56/71/34/6232/56/7Note.—When parents are available, the paternal allele is underlined and the maternal allele is in boldface italics. ND = not done; NI = not informative. Open table in a new tab Note.— When parents are available, the paternal allele is underlined and the maternal allele is in boldface italics. ND = not done; NI = not informative. Sparago et al. (Sparago et al., 2004Sparago A Cerrato F Vernucci M Ferrero GB Silengo MC Riccio A Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome.Nat Genet. 2004; 36: 958-960Crossref PubMed Scopus (229) Google Scholar) and Prawitt et al. (Prawitt et al., 2005Prawitt D Enklaar T Gartner-Rupprecht B Spangenberg C Oswald M Lausch E Schmidtke P Reutzel D Fees S Lucito R Korzon M Brozek I Limon J Housman DE Pelletier J Zabel B Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor.Proc Natl Acad Sci USA. 2005; 102: 4085-4090Crossref PubMed Scopus (114) Google Scholar) demonstrated that maternal inheritance of small deletions (2–3 kb) at the H19 DMR could cause hypermethylation of H19 in patients with BWS. In our patient group, we could exclude the existence of such deletions (fig. 1); therefore, it is unlikely that paternal inheritance of an H19 DMR deletio
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