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Angelman syndrome associated with oculocutaneous albinism due to

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




Clinical Report
Angelman Syndrome Associated With
Oculocutaneous Albinism Due to
an 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
1
                                        ˜o         ˜o
  Department of Biology, University of Sa Paulo, Sa Paulo, Brazil
2
  Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan
3
  Maternal-Infantile Institute of Pernambuco, Neuropediatrics, PE, Brazil


    Angelman syndrome (AS) is a neurodevelop-                      associated with AS due to the loss of the
    mental disorder characterized by mental re-                    maternal chromosome 15 with the normal P
    tardation, speech impairment, ataxia, and                      allele, and the paternal deletion in the P
    happy disposition with frequent smiling.                       gene. As various degrees of hipopygmenta-
    AS results from the loss of expression of a                    tion are associated with PWS and AS
    maternal imprinted gene, UBE3A, mapped                         patients, the study of the P gene in a hemi-
    within 15q11-q13 region, due to different                      zygous state could contribute to the under-
    mechanisms: maternal deletion, paternal                        standing of its effect on human pigmentation
    UPD, imprinting center mutation, and                           during development and to disclose the
    UBE3A mutation. Deletion AS patients may                       presence of modifier pigmentation gene(s)
    exhibit hypopigmentation of skin, eye, and                     in the PWS/AS region. ß 2003 Wiley-Liss, Inc.
    hair correlating with deletion of P gene
    localized in the distal part of Prader-Willi                   KEY WORDS: hypopigmentation; albinism;
    (PWS)/AS region. Our patient presented                                    P gene; Angelman syndrome
    developmental delay, severe mental retarda-
    tion, absence of speech, outbursts of laugh-
    ter, microcephaly, ataxia, hyperactivity,
    seizures, white skin, no retinal pigmenta-                                     INTRODUCTION
    tion, and gold yellow hair. His parents were                   Angelman syndrome (AS) is characterized by neuro-
    of African ancestry. The SNURF-SNRPN                         developmental delay, severe mental retardation, speech
    methylation analysis confirmed AS diag-                       impairment, ataxia, happy disposition with frequent
    nosis and microsatellite studies disclosed                   laughter, macrostomia, wide-spaced teeth, and protrud-
    deletion with breakpoints in BP2 and BP3.                    ing tongue [Williams et al., 1995]. AS results from the
    All of the 25 exons and flanking introns of the               loss of expression of a maternal expressed imprinted
    P gene of the patient, his father, and mother                gene, UBE3A, mapped within the 15q11-q13 chromo-
    were investigated. The patient is hemizy-                    some region [Kishino et al., 1997; Matsuura et al., 1997;
    gous for the deleted exon 7 of the P gene                    Nicholls and Knepper, 2001]. Different mechanisms can
    derived from his father who is a carrier of the              lead to the AS phenotype and although the most common
    deleted allele. Our patient manifests OCA2                   genetic mechanism is a maternal deletion of the PWS/AS
                                                                 region which is responsible for about 70% of the cases,
                                                                 5% of the patients present paternal uniparental
                                                                 disomy (UPD), 1–2% show mutation in the imprinted
                                                                 center (IC), 10% of the patients present mutation in
  Grant sponsor: FAPESP; Grant sponsor: Ministry of Education,
                                                                 the UBE3A gene, and in the remaining cases the genetic
Science, and Culture of Japan; Grant number: 13670897; Grant     mechanism is still unknown [Nicholls and Knepper,
sponsor: Osaka City University Medical Research Foundation.      2001].
  *Correspondence to: C. Fridman, Departamento de Biologia,        Both PWS and AS deletion patients show a great
                   ˆ                             ˜
Instituto de Biociencias, USP, CEP: 05508-900, Sao Paulo, SP,    consistency in deletion size and identify three hotspots
Brazil. E-mail: cfridman@ib.usp.br                               for chromosome breakage in the 15q11-q13 region, re-
  Received 26 June 2001; Accepted 1 October 2002                 ferred to as BP1, BP2 (proximal breaks), and BP3 (distal
  DOI 10.1002/ajmg.a.20105                                       break) [Christian et al., 1999].

ß 2003 Wiley-Liss, Inc.
                                                                                         OCA2 in an AS Patient                       181

   The human P gene consists of 25 exons spanning
between 250 and 600 kb of the chromosome region
15q11-q13. The P protein appears to be an integral
membrane protein of melanosomes [Lee et al., 1995].
Many studies have demonstrated that mutations in
the P gene are associated with the tyrosinase-positive
oculocutaneous 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 maternal
deletion, respectively, exhibit hypopigmentation of the
skin, eyes, and hair when compared to their parents
that is correlated with the deletion of one copy of the
P gene localized in the distal part of the 15q11q-13
region [King et al., 1993; Spritz et al., 1997].
   AS patients may occasionally present the clinical
features of OCA2 due to a mutated P gene in the re-
maining paternal chromosome 15. Indeed, Saitoh
et al. [2000] described a patient with AS and OCA2 for
whom they found a paternal P gene missense muta-
tion (1441G!A, A481T) and deletion of the maternal
chromosome 15q11-q13. Durham-Pierre et al. [1994]
found that the 2.7 kb deletion spanning the whole exon
7 of the P gene is a frequent mutation among people of
African origin, and they briefly described an AS patient
who carries a single paternally derived P allele with the
2.7 kb deletion. Here we report a patient with Angelman
syndrome and OCA2 due to a 2.7 kb deletion present in
the remaining paternally inherited chromosome 15.

                CLINICAL REPORT
  The propositus is a Brazilian boy who was born to
healthy non-consanguineous parents. At birth, the
mother was 24 and the father 32 years of age. He was
born in the 31st week of gestation, with birth weight of
1750 g, and apgar score of 3 (1 min) and 6 (5 min).
Developmental milestones were delayed: he sat with
support at 4 years of age and unsupported after the
age of 5 years, walked alone between 6 and 7 years of
age. He was referred for genetic studies at age 7 5=12 to
disclose the diagnosis of AS as he presented neuro-
psychomotor developmental delay, severe mental re-
tardation, absence of speech, happy disposition with
outbursts of laughter, microcephaly, brachycephaly,
ataxia, hyperactivity, seizures with onset at the age
of 1 6=12 years, and albinism with gold yellow hair
(Figure 1A,B). His father and mother are of African
ancestry and the father has darker skin than the
mother. The mother indicated that a maternal grand-           Fig. 1. The patient at the age of (A) 2 6=12 years, (B) 7 years. [Color figure
                                                            can be viewed in the online issue, which is available at www.interscience.
mother’s sister had albinism.                               wiley.com]


           MATERIALS AND METHODS                            probe used was a 0.6 kb EcoRI-NotI fragment, which
                                                            contains exon 1 of SNURF-SNRPN [Glenn et al., 1996;
                 Southern Blotting
                                                            Gray et al., 1999].
  The methylation status of the PWS/AS region was
assessed by Southern blotting. The genomic DNA of the
                                                                       Analysis of Microsatellite Markers
patient was extracted from peripheral blood leukocytes
by standard procedures, digested with XbaI þ NotI, run        Microsatellite analyses were also performed on DNA
on a 0.8% agarose gel, transferred to a nylon membrane,     from the patient and his parents in order to identify
and hybridized as described [Nicholls et al., 1989]. The    the genetic mechanism envolved. Three microsatellite
182      Fridman et al.

markers within the critical region 15q11-q13, 4-3RCA
(D15S11), LS6-1CA (D15S113), and GABRB3CA
(GABRB3) were typed in a multiplex PCR reaction
as previously described [Mutirangura et al., 1993], and
one marker outside the critical region was also tested
(D15S131). To detect the extent of the deletion, we
used markers D15S542, D15S543, D15S1002, and
D15S1048 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 gene
were PCR-amplified by the primer pairs described
previously [Lee et al., 1995]. The products were agarose
gel-purified by GFX PCR and Gel Band Purification Kit
(Amersham Pharmacia Biotech, Uppsala, Sweden), and
direct-sequenced with the ABI PRISM Big Dye Termi-
nator Cycle Sequencing Ready Reaction Kit. Sequence
reactions were run on Perkin-Elmer 310 Automatic
sequencer and analyzed by use of LASERGENE soft-
ware (DNASTAR, Madison, WI). The three primer assay
system developed by Durham-Pierre et al. [1994]
was employed to detect the 2.7 kb intragenic deletion
of the P gene. The sequences of the primers were
50 -TTTGCTCTAGGTTTCAGGCGAG-30 (MHB51), 50 -
GCGGTGGCTGTCATGGC-30 (MHB72), and 50 -GGAG-                      Fig. 2. PCR analysis for detection of the intragenic deletion of P gene
GGTGCATTCATTCTTCAG-30 (MHB71). The primer                      revealed that the affected child (lane 1) and the father (lane 3) have the
                                                               deleted allele. The affected child does not have normal allele (lane 2),
pair MHB51 and MHB71 gives an 820 bp PCR product               whereas the father has the normal allele (lane 4). Lanes 1, 2: Affected child;
for the 2.7 kb intragenic deleted allele, whereas the          lanes 3, 4: father; lanes 5, 6: mother; lanes 1, 3, 5: PCR with a primer pair
primer pair MHB72 and MHB71 gives a 240 bp PCR                 MHB51 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 for
product for the normal allele.                                 normal allele with the expected size, 240 bp. Lane 7 is the size marker:
                                                               X174HaeIIIdigest.
                       RESULTS
  The methylation analysis of exon 1 of the SNURF-             the P gene, and is thus included in the deletion. The
SNRPN bicistronic gene showed a typical methylation            prevalence of the OCA2 resulting from the 2.7 kb
pattern for AS, thus confirming the diagnosis. Micro-           deletion of exon 7 of the P gene is 1 out of 1,800 in indi-
satellite analysis of loci mapped within and outside the       viduals of African origin [Durham-Pierre et al., 1994].
PWS/AS region revealed that the genetic mechanism              Theoretically, about 1 out of 20 persons of African origin
involved is a maternally derived deletion and the break-       carries this mutation.
points are in BP2 (between the markers D15S542 and               The intragenic deletion of the P gene results in a
D15S543) and BP3 (between the markers D15S1002 and             frameshift mutation in the first luminal loop, predicting
D15S1048) [Amos-Landgraf et al., 1999; Christian et al.,       a truncation of the polypeptide and hence a non-
1999] (data not shown).                                        functional gene product [Durham-Pierre et al., 1994].
  To analyse the molecular basis for the albinism in this      Because the maternal P gene is lost and the paternal
AS patient, all of the exons and flanking introns of the        P gene has intragenic deletion, the patient has no
P gene were direct sequenced. All of the exons and             functional P gene product. This is reflected in his white
flanking introns were normal except exon 7 that was not         skin, gold hair, and no retinal pigmentation. The color of
amplified. Therefore the patient could have an intra-           the skin and hair of the patient has not changed so far at
genic deletion of the P gene. PCR analysis for detect-         least by the age of 7 6=12 years old. This is different from
ing the deletion using the primer pairs described by           the patient described by Saitoh et al. [2000] whose hair
Durham-Pierre et al. [1994] were used. The patient is          color changed from gold to black after some years due to
hemizygous for the deleted exon 7 of the P gene that is        the hemizygous A481T missense mutation that appears
derived from the father who is a carrier of this allele; the   to result in a partially functional protein. These cases
mother has a normal allele (Fig. 2).                           can help us to understand the full spectrum of the AS
                                                               condition associated with P mutations.
                                                                 AS patients can present hypopigmentation resulting
                     DISCUSSION
                                                               from reduced expression of the P gene, due to mutations
  Our patient manifests recessively inherited OCA2             in promoter region or some polimorphic changes that
associated with AS because he has lost the maternal            influence the P gene expression. Other unknown gene(s)
chromosome 15 carrying one P allele and has a mutated          in PWS/AS region can also be deleted and also be re-
P allele in the paternal 15. The distal breakpoint of          sponsible for pigmentation. Besides, AS patients pre-
the chromosome deletion is in BP3, which is distal of          sent the gene in hemizygous state while normal carriers
                                                                                                         OCA2 in an AS Patient                     183

of OCAII mutation are not hypopigmentated because                            King RA, Wiesner GL, Townsend DW, White JG. 1993. Hypopigmentation
they still have a normal allele.                                                in Angelman syndrome. Am J Med Genet 46:40–44.
  As various degrees of hypopigmentation are asso-                           Kishino T, Lalande M, Wagstaff J. 1997. UBE3A/E6AP mutations cause
                                                                                Angelman syndrome. Nat Genet 15:70–73.
ciated with PWS and AS deletion patients [Spritz et al.,
                                                                             Lee S-T, Nicholls RD, Schnur RE, Guida LC, Lu-Kuo J, Spinner NB,
1997], the study of the P gene present in a hemizygous                          Zackai EH, Spritz RA. 1994. Diverse mutations of the P gene among
state could contribute to the understanding of its effect                       African-Americans with type II (tyrosinase-positive) oculocutaneous
on human pigmentation during development, and to                                albinism (OCA2). Hum Mol Genet 3:2047–2051.
disclose the presence of additional or modifier pigmen-                       Lee S-T, Nicholls RD, Jong MTC, Fukai K, Spritz RA. 1995. Organization
tation gene(s) in the PWS/AS region.                                            and sequence of the human P gene and identification of a new family of
                                                                                transport proteins. Genomics 26:354–363.
                                                                             Matsuura T, Sutcliffe JS, Fang P, Galjaard R-J, Jiang YH, Benton CS,
                                                                                Rommens JM, Beaudet AL. 1997. De novo truncating mutations in
                   ACKNOWLEDGMENTS                                              E6-AP ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome.
                                                                                Nat Genet 15:74–77.
   We thank Dr. Robert Nicholls for valuable comments
                                                                             Mutirangura A, Greenberg F, Butler MG, Malcolm S, Nicholls RD,
of the manuscript. This work was supported by FAPESP                           Chakravarti A, Ledbetter DH. 1993. Multiplex PCR of three dinucleotide
(CF, MCV, CPK) and grant-in-aid #13670897 from the                             repeats in the Prader-Willi/Angelman critical region (15q11-13): Mole-
Ministry of Education, Science, and Culture of Japan                           cular diagnosis and mechanism of uniparental disomy. Hum Mol Genet
(KF) and the fund for medical research of Osaka City                           2:143–151.

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