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Exclusion of maternal uniparental disomy of chromosome 14 in patients referred for Prader-Willi syndrome using a multiplex methylation polymerase chain reaction assay
L G Dietz, A A Wylie, K A Rauen, S K Murphy, R L Jirtle and P D Cotter J. Med. Genet. 2003;40;46doi:10.1136/jmg.40.4.e46
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ELECTRONIC LETTER
L G Dietz, A A Wylie, K A Rauen, S K Murphy, R L Jirtle, P D Cotter
J Med Genet 2003;40:e46(http://www.jmedgenet.com/cgi/content/full/40/4/e46)
niparental disomy (UPD) is the inheritance of both homologues of a chromosome from one parent. For most of the autosomes, there is no denitive clinical consequence of this abnormal inheritance. However, UPDs of chromosomes 6, 7, 11, 14, and 15 are associated with abnormal phenotypes owing to overexpression or underexpression of imprinted genes on those chromosomes.Maternal UPD(14) (matUPD(14)) has been described in over 20 cases and is primarily characterised by intrauterine growth retardation and precocious puberty. Additional features can include hypotonia at birth, feeding difculties in early infancy, short stature, musculoskeletal ndings including small hands and feet and scoliosis, mild developmental delay, and early childhood obesity. Most patients with matUPD(14) are described with minor facial dysmorphism including frontal bossing, short philtrum, and high arched palate. Paternal UPD(14) (patUPD(14)) is less common, more severe, and is characterised by polyhydramnios, facial and skeletal anomalies, and severe developmental delay.Recently, Wylie et al5 described reciprocally imprinted genes DLK1 and MEG3, positioned 90 kb apart at 14q32, which are candidate genes for the UPD(14) phenotypes. DLK1, a cell surface transmembrane protein, is paternally expressed, and MEG3, which lacks an open reading frame, is maternally expressed.5 Dlk1 knockout mice show features of matUPD(14), providing evidence that many of the phenotypic consequences of matUPD(14) may be attributed to a lack of DLK1 expression in these patients.6 UPD(14) is usually ascertained through a combination of clinical features and a karyotype suggestive of UPD, such as conned placental mosaicism for trisomy 14, a nonhomologous Robertsonian or reciprocal translocation involving chromosome 14, or an isochromosome 14. These karyotypes are consistent with, or predispose to, monosomy or trisomy rescue events, which are the most common mechanisms leading to UPD.2 Recently, three patients with matUPD(14) were described who were originally referred for molecular analysis for Prader-Willi syndrome (PWS) based on clinical phenotypes suggestive of PWS. The authors suggested that there are enough phenotypic similarities between PWS and matUPD(14) such that some patients without PWS might have matUPD(14) syndrome.We recently described a rapid multiplex methylation polymerase chain reaction (mPCR) assay to identify UPD for chromosome 14 based on parent of origin differential methylation associated with the promoter region of the MEG3 gene, an imprinted gene on chromosome 14q32.9 In this communication, we report the analysis of 200 patients previously referred for PWS to determine if any were unrecognised as having matUPD(14) syndrome.
Key points Maternal UPD for chromosome 14 (matUPD(14)) shows some phenotypic overlap with PWS, notably hypotonia, obesity, and hypogonadism in some patients. Recently, three patients with matUPD(14) were reported who were originally referred for a possible diagnosis of PWS. The identification of matUPD(14) in these patients suggested that there may be some use in testing for matUPD(14) in patients referred for PWS, who were not confirmed by molecular analysis. In this study we selected 200 patients initially referred for molecular diagnosis of PWS based on their clinical phenotype and who were normal by Southern blot or mPCR analysis of the SNRPN region. Patients were screened with a rapid bisulphite modification/multiplex mPCR method based upon the differential methylation associated with the imprinted MEG3 gene on chromosome 14. All 200 samples from patients showed both the paternal and maternal specific PCR fragments, consistent with biparental inheritance of chromosome 14 and excluding matUPD(14). These data indicate that the incidence of matUPD(14) is likely to be low among patients referred for PWS.

testing for suspected PWS based on their clinical phenotype. All samples had normal chromosomes (46,XX or 46,XY) and had tested normal by Southern blot or mPCR analysis, excluding changes in methylation at the SNRPN locus associated with PWS. Southern blot analysis Genomic DNA was extracted from peripheral blood samples with a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA) according to the manufacturers instructions. Methylation analysis for PWS by Southern blotting with the PW71B (D15S63) probe was performed as described,10 except that the nal posthybridisation wash was in 0.5 SSC/1% SDS at 55C for 20 minutes. Methylation PCR analysis Bisulphite modication of genomic DNA was performed as described.11 For PWS analysis, methylation specic PCR

MATERIALS AND METHODS

Patient samples Two hundred samples were selected from patients who were referred to our laboratory between 1995 and 2002 for DNA
Abbreviations: CVS, chorionic villus sampling; matUPD(14), maternal UPD(14); mPCR, methylation polymerase chain reaction; patUPD(14), paternal UPD(14); PWS, Prader-Willi syndrome; UPD, uniparental disomy

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Figure 1 Methylation PCR of the differentially methylated region upstream of the MEG3 promoter showed the presence of both maternal and paternal alleles in all 200 patients. The paternal and maternal UPD(14) controls showed only paternal or maternal alleles, respectively. Lane 1 was the molecular weight marker X174 (Pharmacia, Piscataway, NJ); lane 2, control DNA without bisulphite modification; lane 3, representative patient sample; lane 4, matUPD(14) control; lane 5, patUPD(14) control; lane 6, normal control.
reactions with maternal and paternal oligonucleotide primers for the CpG island of the SNRPN gene were performed as described.12 Methylation specic PCR reactions with maternal and paternal oligonucleotide primers for the differentially methylated region 5 of the MEG3 promoter on chromosome 14q32 were performed as described,9 except that the methylation specic forward oligonucleotide used was ve nucleotides shorter at the 5 end: 5-GGTAGTAATCGGGTTTGTCGGC-3. Annealing temperatures were ve cycles at 65C for 30 seconds, ve cycles at 60C for 30 seconds, and 31 cycles at 55C for 30 seconds. Products of the PCR were separated on a 3% Nusieve agarose or a 6% non-denaturing acrylamide gel, stained with ethidium bromide, and visualised under UV illumination. Controls included normal samples, maternal UPD(14) (unpublished data), and paternal UPD(14).3

RESULTS

Samples from patients were selected from those referred to the laboratory for analysis for PWS, based on clinical phenotype. Two hundred patients were selected that had normal karyotypes (46,XX or 46,XY) and showed inheritance of both maternal and paternal alleles for chromosome 15 after methylation analysis by Southern blot or methylation PCR analysis. Methylation PCR analysis diagnostic for UPD(14) was used to determine if any of these patients were unrecognised matUPD(14). The matUPD(14) control sample showed only the 115 bp maternal PCR product, the patUPD(14) control showed only the 160 bp paternal PCR product, and the normal control showed both maternal and paternal specic PCR fragments as predicted (g 1). All 200 patient samples showed both the 160 bp paternal and 115 bp maternal allele specic PCR fragments, consistent with biparental inheritance (g 1).

DISCUSSION

Prader-Willi syndrome is a well recognised genetic disorder with a variable and evolving phenotype.13 Major criteria include hypotonia in infancy with associated feeding difculties and failure to thrive. This is followed by rapid weight gain usually after 1 year of age resulting in notable obesity if uncontrolled. Other major criteria include hypogonadism and delay in motor and speech development. Characteristic facial features include bitemporal narrowing, almond shaped palpebral ssures, strabismus, narrow nasal bridge, and downturned corners of the mouth with a thin upper lip.
Musculoskeletal ndings may include small hands and feet, which become more pronounced in mid-childhood, and scoliosis or kyphosis or both. Patients with PWS may have considerable behavioural issues which are quite characteristic to this syndrome, including tantrums, manipulative behaviour, and obsessive-compulsive tendencies. Interestingly, 17% of patients who tested positive by mPCR for PWS did not meet the diagnostic criteria, highlighting the phenotypic variability in this syndrome.13 Several aneusomies and Mendelian disorders can present with phenotypes that overlap with PWS. Patients with functional disomy for regions of the X chromosome, either from a duplication or supernumerary ring X chromosome, have phenotypic similarities to PWS, including polyphagia, neonatal growth retardation, and obesity in older children.1416 Deletions of 6q also present with a PWS-like phenotype, including hypotonia, polyphagia, facial features, and obesity.Chudley et al19 described a family with an X linked disorder in whom the male patients presented with mental retardation, short stature, obesity, and hypogonadism, suggestive of PWS. Also, a group of patients with fragile X syndrome was reported with phenotypic overlap with PWS.20 Recently, three patients were reported with matUPD(14) who were described as having a phenocopy of PWS, and who were originally referred for PWS testing on the basis of their clinical phenotype. The authors proposed that there was an overlap between the phenotypes of these two syndromes, and that some patients referred for PWS may be unrecognised as matUPD(14).We used a rapid multiplex mPCR assay for UPD(14)9 to screen 200 patients originally referred for PWS testing based on their clinical phenotype and found to be normal for PWS by molecular analysis. All 200 patients showed an mPCR prole consistent with biparental inheritance of chromosome 14 (g 1), excluding UPD(14). Thus, the incidence of unrecognised matUPD(14) among PWS referrals is likely to be low. None the less, the clinical ndings for the two conditions have several similarities that merit further consideration. Many of the patients reported with matUPD(14) had phenotypic features overlapping with PWS to the extent that some were originally referred with a clinical diagnosis suggestive of PWS.A review of clinical data of 17 patients with matUPD(14) showed several features seen in PWS: hypotonia in 11/14, feeding difculties in 9/10, childhood obesity in 6/15, motor delay in 12/15, and mental delay in 5/15.21 As noted by Sanlaville et al,21 the obesity in matUPD(14) was not as severe as in PWS and behavioural disorders were not as consistent in matUPD(14). Conventional cytogenetic analysis is important in the diagnosis of UPD(14). Most patients with matUPD(14) reported to date have had rearrangements suggestive of UPD, that is, Robertsonian translocations or isochromosomes.21 Indeed, the two PWS-like patients with matUPD(14) described by Berends et al7 had a Robertsonian translocation and a chromosome 14 isochromosome, respectively, both karyotypes that would suggest a UPD(14) study in the context of phenotypic abnormalities. UPD(14) testing should be performed where cytogenetic analysis identies a Robertsonian translocation involving chromosome 14 or isochromosome for chromosome 14,a supernumerary marker chromosome 14 (unpublished data), or in amniocytes secondary to identication of conned placental mosaicism for trisomy 14 in CVS.24 Additional studies to test the hypothesis that there are unrecognised patients with matUPD(14) among referrals for PWS will ultimately determine the use of matUPD(14) testing in patients with PWS. The availability of a rapid multiplex mPCR test that does not require parental samples will facilitate these studies.

Electronic letter. 3 of 3
10 Dittrich B, Robinson WP, Knoblauch H, Buiting K, Schmidt K, Gillessen-Kaesbach G, Horsthemke B. Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q1113. Hum Genet 1992;90:31315. 11 Herman JG, Baylin SB. Determination of DNA methylation patterns by methylation-specific PCR. In: Dracopli NC, Haines JK, Korf BR, eds. Current protocols in human genetics online edition. John Wiley, 2002. 12 Kubota T, Das S, Christian SL, Baylin SB, Herman JG, Ledbetter DH. Methylation-specific PCR simplifies imprinting analysis. Nat Genet 1997;16:167. 13 Gunay-Aygun M, Schwartz S, Heeger S, ORiordan MA, Cassidy SB. The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria. Pediatrics 2001;108:E92. 14 Lammer EJ, Punglia DR, Fuchs AE, Rowe AG, Cotter PD. Inherited duplication of Xq27.2>qter: phenocopy of infantile Prader-Willi syndrome. Clin Dysmorphol 2001;10:1414. 15 Manea SR, Gershin IF, Babu A, Willner JP, Desnick RJ, Cotter PD. Mosaicism for a small supernumerary ring X chromosome in a dysmorphic, growth-retarded male: mos47,XXY/48,XXY, +r(X). Clin Genet 1997;52:4325. 16 Stratakis CA. Prader-Willi syndrome phenotype in X chromosome anomalies: evidence for a distinct syndrome. Am J Med Genet 1998;80:2945, 3001. 17 Gilhuis HJ, van Ravenswaaij CM, Hamel BJ, Gabreels FJ. Interstitial 6q deletion with a Prader-Willi-like phenotype: a new case and review of the literature. Europ J Paediatr Neurol 2000;4:3943. 18 Stein CK, Stred SE, Thomson LL, Smith FC, Hoo JJ. Interstitial 6q deletion and Prader-Willi-like phenotype. Clin Genet 1996;49:30610. 19 Chudley AE, Lowry RB, Hoar DI. Mental retardation, distinct facial changes, short stature, obesity, and hypogonadism: a new X-linked mental retardation syndrome. Am J Med Genet 1988;31:74151. 20 de Vries BB, Fryns JP, Butler MG, Canziani F, Wesby-van Swaay E, van Hemel JO, Oostra BA, Halley DJ, Niermeijer MF. Clinical and molecular studies in fragile X patients with a Prader-Willi-like phenotype. J Med Genet 1993;30:7616. 21 Sanlaville D, Aubry MC, Dumez Y, Nolen MC, Amiel J, Pinson MP, Lyonnet S, Munnich A, Vekemans M, Morichon-Delvallez N. Maternal uniparental heterodisomy of chromosome 14: chromosomal mechanism and clinical follow up. J Med Genet 2000;37:5258. 22 Shaffer LG, Agan N, Goldberg JD, Ledbetter DH, Longshore JW, Cassidy SB. American College of Medical Genetics statement of diagnostic testing for uniparental disomy. Genet Med 2001;3:20611. 23 Silverstein S, Lerer I, Sagi M, Frumkin A, Ben-Neriah Z, Abeliovich D. Uniparental disomy in fetuses diagnosed with balanced Robertsonian translocations: risk estimate. Prenat Diagn 2002;22:64951. 24 Towner DR, Shaffer LG, Yang SP, Walgenbach DD. Confined placental mosaicism for trisomy 14 and maternal uniparental disomy in association with elevated second trimester maternal serum human chorionic gonadotrophin and third trimester fetal growth restriction. Prenat Diagn 2001;21:3958.

Authors affiliations

L G Dietz, P D Cotter, Division of Medical Genetics and Department of Pathology, Childrens Hospital and Research Center at Oakland, 747 Fifty Second Street, Oakland, CA 94609, USA A A Wylie, S K Murphy, R L Jirtle, Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA K A Rauen, P D Cotter, Division of Medical Genetics, Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143, USA K A Rauen, Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA 94115, USA P D Cotter, Division of Genetics, US Labs, 2601 Campus Drive, Irvine, CA 92612, USA Correspondence to: Dr P D Cotter, Division of Genetics, US Labs, 2601 Campus Drive, Irvine, CA 92612, USA; pcotter@itsa.ucsf.edu

REFERENCES

1 Kotzot D. Abnormal phenotypes in uniparental disomy (UPD): fundamental aspects and a critical review with bibliography of UPD other than 15. Am J Med Genet 1999;82:26574. 2 Engel E, Antonarakis SE. Genomic imprinting and uniparental disomy in medicine. Clinical and molecular aspects. New York: Wiley-Liss, 2002. 3 Cotter PD, Kaffe S, McCurdy LD, Jhaveri M, Willner JP, Hirschhorn K. Paternal uniparental disomy for chromosome 14: a case report and review. Am J Med Genet 1997;70:749. 4 Kurosawa K, Sasaki H, Sato Y, Yamanaka M, Shimizu M, Ito Y, Okuyama T, Matsuo M, Imaizumi K, Kuroki Y, Nishimura G. Paternal UPD14 is responsible for a distinctive malformation complex. Am J Med Genet 2002;110:26872. 5 Wylie AA, Murphy SK, Orton TC, Jirtle RL. Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation. Genome Res 2000;10:171118. 6 Moon YS, Smas CM, Lee K, Villena JA, Kim KH, Yun EJ, Sul HS. Mice lacking paternally expressed Pref-1/Dlk1 display growth retardation and accelerated adiposity. Mol Cell Biol 2002;22:558592. 7 Berends MJ, Hordijk R, Scheffer H, Oosterwijk JC, Halley DJ, Sorgedrager N. Two cases of maternal uniparental disomy 14 with a phenotype overlapping with the Prader-Willi phenotype. Am J Med Genet 1999;84:769. 8 Hordijk R, Wierenga H, Scheffer H, Leegte B, Hofstra RM, Stolte-Dijkstra I. Maternal uniparental disomy for chromosome 14 in a boy with a normal karyotype. J Med Genet 1999;36:7825. 9 Murphy SK, Wylie AA, Coveler KJ, Cotter PD, Papenhausen PR, Sutton VR, Shaffer LG, Jirtle RL. Epigenetic detection of human chromosome 14 uniparental disomy. Hum Mutat (in press).