American Journal of Medical Genetics 119A:180–183 (2003)

Clinical Report

Angelman Syndrome Associated WithOculocutaneous Albinism Due toan Intragenic Deletion of the P Gene

C. Fridman,1* N. Hosomi,2 M.C. Varela,1 A.H. Souza,3 K. Fukai,2 and C.P. Koiffmann1

1Department of Biology, University of Sao Paulo, Sao Paulo, Brazil2Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan3Maternal-Infantile Institute of Pernambuco, Neuropediatrics, PE, Brazil

Angelman syndrome (AS) is a neurodevelop-mental disorder characterized by mental re-tardation, speech impairment, ataxia, andhappy disposition with frequent smiling.AS results from the loss of expression of amaternal imprinted gene, UBE3A, mappedwithin 15q11-q13 region, due to differentmechanisms: maternal deletion, paternalUPD, imprinting center mutation, andUBE3A mutation. Deletion AS patients mayexhibit hypopigmentation of skin, eye, andhair correlating with deletion of P genelocalized in the distal part of Prader-Willi(PWS)/AS region. Our patient presenteddevelopmental delay, severe mental retarda-tion, absence of speech, outbursts of laugh-ter, microcephaly, ataxia, hyperactivity,seizures, white skin, no retinal pigmenta-tion, and gold yellow hair. His parents wereof African ancestry. The SNURF-SNRPNmethylation analysis confirmed AS diag-nosis and microsatellite studies discloseddeletion with breakpoints in BP2 and BP3.All of the 25 exons and flanking introns of theP gene of the patient, his father, and motherwere investigated. The patient is hemizy-gous for the deleted exon 7 of the P genederived from his father who is a carrier of thedeleted allele. Our patient manifests OCA2

associated with AS due to the loss of thematernal chromosome 15 with the normal Pallele, and the paternal deletion in the Pgene. As various degrees of hipopygmenta-tion are associated with PWS and ASpatients, the study of the P gene in a hemi-zygous state could contribute to the under-standing of its effect on human pigmentationduring development and to disclose thepresence of modifier pigmentation gene(s)in the PWS/AS region. � 2003 Wiley-Liss, Inc.

KEY WORDS: hypopigmentation; albinism;P gene; Angelman syndrome

INTRODUCTION

Angelman syndrome (AS) is characterized by neuro-developmental delay, severe mental retardation, speechimpairment, ataxia, happy disposition with frequentlaughter, macrostomia, wide-spaced teeth, and protrud-ing tongue [Williams et al., 1995]. AS results from theloss of expression of a maternal expressed imprintedgene, UBE3A, mapped within the 15q11-q13 chromo-some region [Kishino et al., 1997; Matsuura et al., 1997;Nicholls and Knepper, 2001]. Different mechanisms canlead to the AS phenotype and although the most commongenetic mechanism is a maternal deletion of the PWS/ASregion which is responsible for about 70% of the cases,�5% of the patients present paternal uniparentaldisomy (UPD), 1–2% show mutation in the imprintedcenter (IC), �10% of the patients present mutation intheUBE3A gene, and in the remaining cases the geneticmechanism is still unknown [Nicholls and Knepper,2001].

Both PWS and AS deletion patients show a greatconsistency in deletion size and identify three hotspotsfor chromosome breakage in the 15q11-q13 region, re-ferred to as BP1, BP2 (proximal breaks), and BP3 (distalbreak) [Christian et al., 1999].

Grant sponsor: FAPESP; Grant sponsor: Ministry of Education,Science, and Culture of Japan; Grant number: 13670897; Grantsponsor: Osaka City University Medical Research Foundation.

*Correspondence to: C. Fridman, Departamento de Biologia,Instituto de Biociencias, USP, CEP: 05508-900, Sao Paulo, SP,Brazil. E-mail: cfridman@ib.usp.br

Received 26 June 2001; Accepted 1 October 2002

DOI 10.1002/ajmg.a.20105

� 2003 Wiley-Liss, Inc.

The human P gene consists of 25 exons spanningbetween 250 and 600 kb of the chromosome region15q11-q13. The P protein appears to be an integralmembrane protein of melanosomes [Lee et al., 1995].Many studies have demonstrated that mutations inthe P gene are associated with the tyrosinase-positiveoculocutaneous albinism (OCA, type II), the most pre-valent type of albinism worldwide [Ramsay et al., 1992;Rinchik et al., 1993; Durham-Pierre et al., 1994, 1996;Lee et al., 1994; Stevens et al., 1997].

PWS and AS patients with paternal or a maternaldeletion, respectively, exhibit hypopigmentation of theskin, eyes, and hair when compared to their parentsthat is correlated with the deletion of one copy of theP gene localized in the distal part of the 15q11q-13region [King et al., 1993; Spritz et al., 1997].

AS patients may occasionally present the clinicalfeatures of OCA2 due to a mutated P gene in the re-maining paternal chromosome 15. Indeed, Saitohet al. [2000] described a patient with AS and OCA2 forwhom they found a paternal P gene missense muta-tion (1441G!A, A481T) and deletion of the maternalchromosome 15q11-q13. Durham-Pierre et al. [1994]found that the 2.7 kb deletion spanning the whole exon7 of the P gene is a frequent mutation among people ofAfrican origin, and they briefly described an AS patientwho carries a single paternally derived P allele with the2.7 kb deletion. Here we report a patient with Angelmansyndrome and OCA2 due to a 2.7 kb deletion present inthe remaining paternally inherited chromosome 15.

CLINICAL REPORT

The propositus is a Brazilian boy who was born tohealthy non-consanguineous parents. At birth, themother was 24 and the father 32 years of age. He wasborn in the 31st week of gestation, with birth weight of1750 g, and apgar score of 3 (1 min) and 6 (5 min).Developmental milestones were delayed: he sat withsupport at 4 years of age and unsupported after theage of 5 years, walked alone between 6 and 7 years ofage. He was referred for genetic studies at age 7 5=12 todisclose the diagnosis of AS as he presented neuro-psychomotor developmental delay, severe mental re-tardation, absence of speech, happy disposition withoutbursts of laughter, microcephaly, brachycephaly,ataxia, hyperactivity, seizures with onset at the ageof 1 6=12 years, and albinism with gold yellow hair(Figure 1A,B). His father and mother are of Africanancestry and the father has darker skin than themother. The mother indicated that a maternal grand-mother’s sister had albinism.

MATERIALS AND METHODS

Southern Blotting

The methylation status of the PWS/AS region wasassessed by Southern blotting. The genomic DNA of thepatient was extracted from peripheral blood leukocytesby standard procedures, digested with XbaIþNotI, runon a 0.8% agarose gel, transferred to a nylon membrane,and hybridized as described [Nicholls et al., 1989]. The

probe used was a 0.6 kb EcoRI-NotI fragment, whichcontains exon 1 of SNURF-SNRPN [Glenn et al., 1996;Gray et al., 1999].

Analysis of Microsatellite Markers

Microsatellite analyses were also performed on DNAfrom the patient and his parents in order to identifythe genetic mechanism envolved. Three microsatellite

Fig. 1. The patient at the age of (A) 2 6=12 years, (B) 7 years. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com]

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markers within the critical region 15q11-q13, 4-3RCA(D15S11), LS6-1CA (D15S113), and GABRB3CA(GABRB3) were typed in a multiplex PCR reactionas previously described [Mutirangura et al., 1993], andone marker outside the critical region was also tested(D15S131). To detect the extent of the deletion, weused markers D15S542, D15S543, D15S1002, andD15S1048 as described by Amos-Landgraf et al. [1999].

Mutation Study of the P Gene

All of the 25 exons and flanking introns of the P genewere PCR-amplified by the primer pairs describedpreviously [Lee et al., 1995]. The products were agarosegel-purified by GFX PCR and Gel Band Purification Kit(Amersham Pharmacia Biotech, Uppsala, Sweden), anddirect-sequenced with the ABI PRISM Big Dye Termi-nator Cycle Sequencing Ready Reaction Kit. Sequencereactions were run on Perkin-Elmer 310 Automaticsequencer and analyzed by use of LASERGENE soft-ware (DNASTAR, Madison, WI). The three primer assaysystem developed by Durham-Pierre et al. [1994]was employed to detect the 2.7 kb intragenic deletionof the P gene. The sequences of the primers were50-TTTGCTCTAGGTTTCAGGCGAG-30 (MHB51), 50-GCGGTGGCTGTCATGGC-30 (MHB72), and 50-GGAG-GGTGCATTCATTCTTCAG-30 (MHB71). The primerpair MHB51 and MHB71 gives an 820 bp PCR productfor the 2.7 kb intragenic deleted allele, whereas theprimer pair MHB72 and MHB71 gives a 240 bp PCRproduct for the normal allele.

RESULTS

The methylation analysis of exon 1 of the SNURF-SNRPN bicistronic gene showed a typical methylationpattern for AS, thus confirming the diagnosis. Micro-satellite analysis of loci mapped within and outside thePWS/AS region revealed that the genetic mechanisminvolved is a maternally derived deletion and the break-points are in BP2 (between the markers D15S542 andD15S543) and BP3 (between the markers D15S1002 andD15S1048) [Amos-Landgraf et al., 1999; Christian et al.,1999] (data not shown).

To analyse the molecular basis for the albinism in thisAS patient, all of the exons and flanking introns of theP gene were direct sequenced. All of the exons andflanking introns were normal except exon 7 that was notamplified. Therefore the patient could have an intra-genic deletion of the P gene. PCR analysis for detect-ing the deletion using the primer pairs described byDurham-Pierre et al. [1994] were used. The patient ishemizygous for the deleted exon 7 of the P gene that isderived from the father who is a carrier of this allele; themother has a normal allele (Fig. 2).

DISCUSSION

Our patient manifests recessively inherited OCA2associated with AS because he has lost the maternalchromosome 15 carrying one P allele and has a mutatedP allele in the paternal 15. The distal breakpoint ofthe chromosome deletion is in BP3, which is distal of

the P gene, and is thus included in the deletion. Theprevalence of the OCA2 resulting from the 2.7 kbdeletion of exon 7 of the P gene is 1 out of 1,800 in indi-viduals of African origin [Durham-Pierre et al., 1994].Theoretically, about 1 out of 20 persons of African origincarries this mutation.

The intragenic deletion of the P gene results in aframeshift mutation in the first luminal loop, predictinga truncation of the polypeptide and hence a non-functional gene product [Durham-Pierre et al., 1994].Because the maternal P gene is lost and the paternalP gene has intragenic deletion, the patient has nofunctional P gene product. This is reflected in his whiteskin, gold hair, and no retinal pigmentation. The color ofthe skin and hair of the patient has not changed so far atleast by the age of 7 6=12 years old. This is different fromthe patient described by Saitoh et al. [2000] whose haircolor changed from gold to black after some years due tothe hemizygous A481T missense mutation that appearsto result in a partially functional protein. These casescan help us to understand the full spectrum of the AScondition associated with P mutations.

AS patients can present hypopigmentation resultingfrom reduced expression of the P gene, due to mutationsin promoter region or some polimorphic changes thatinfluence theP gene expression. Other unknown gene(s)in PWS/AS region can also be deleted and also be re-sponsible for pigmentation. Besides, AS patients pre-sent the gene in hemizygous state while normal carriers

Fig. 2. PCR analysis for detection of the intragenic deletion of P generevealed that the affected child (lane 1) and the father (lane 3) have thedeleted allele. The affected child does not have normal allele (lane 2),whereas the father has the normal allele (lane 4). Lanes 1, 2: Affected child;lanes 3, 4: father; lanes 5, 6: mother; lanes 1, 3, 5: PCR with a primer pairMHB51 and MHB71 to work for deleted allele with the expected size, 820 bp.Lanes 2, 4, 6: PCR with a primer pair MHB72 and MHB71 to work fornormal allele with the expected size, 240 bp. Lane 7 is the size marker:X174HaeIIIdigest.

182 Fridman et al.

of OCAII mutation are not hypopigmentated becausethey still have a normal allele.

As various degrees of hypopigmentation are asso-ciated with PWS and AS deletion patients [Spritz et al.,1997], the study of the P gene present in a hemizygousstate could contribute to the understanding of its effecton human pigmentation during development, and todisclose the presence of additional or modifier pigmen-tation gene(s) in the PWS/AS region.

ACKNOWLEDGMENTS

We thank Dr. Robert Nicholls for valuable commentsof the manuscript. This work was supported by FAPESP(CF, MCV, CPK) and grant-in-aid #13670897 from theMinistry of Education, Science, and Culture of Japan(KF) and the fund for medical research of Osaka CityUniversity Medical Research Foundation (KF).

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