Prader-Willi Syndrome: Genetic Tests and Clinical ... - Genoma - USP

GENETIC TESTING
Volume 4, Number 4, 2000
Mary Ann Liebert, Inc.
Prader-Willi Syndrome: Genetic Tests and Clinical Findings
CINTIA FRIDMAN, 1 MONICA C. VARELA, 1 FERNANDO KOK, 2 NUVARTE SETIAN, 3
and CÉLIA P. KOIFFMANN 1
ABSTRACT
Here we describe the genetic studies performed in 53 patients with the suspected diagnosis of Prader-Willi
syndrome (PWS). PWS is characterized by neonatal hypotonia, hypogonadism, delayed psychomotor development,
hyperphagia, obesity, short stature, small hands and feet, learning disabilities, and obsessive-compulsive
behavior. Through the methylation analysis of the SNRPN gene, microsatellite studies of loci mapped
within and outside the PWS/AS region, and fluorescence in situ hybridization (FISH) study, we confirmed the
diagnosis in 35 patients: 27 with a paternal deletion, and 8 with maternal uniparental disomy (UPD). The clinical
comparisons between deleted and UPD patients indicated that there were no major phenotype differences,
except for a lower birth length observed in the UPD children. Our sample was composed of more girls than
boys; UPD patients were diagnosed earlier than the deleted cohort (2 10/12 vs. 7 9/12 years); and, in the deleted
group, the boys were diagnosed earlier than the girls (5 2/12 vs. 7 8/12 years, respectively).
INTRODUCTION
PRADER-WILLI SYNDROME (PWS) is a disease that occurs with
an incidence of , 1:15–20,000 births, and is associated with
developmental and behavioral problems. PWS children show
neonatal hypotonia, hypogonadism, failure to thrive, hyperphagia,
obesity, short stature, small hands and feet, mental retardation
with learning disability, and obsessive-compulsive behavior
(Prader et al., 1956; Cassidy, 1997).
Different mechanisms can lead to PWS, which is due to a
common genetic deficit (Glenn et al., 1997) that abolishes the
expression of imprinted paternal genes. The major genetic
mechanism giving rise to PWS is a paternal deletion of about
the same size in the 15q11–q13 region, that occurs in , 70%
of the cases. Maternal uniparental disomy (UPD) is the second
mechanism, occurring in , 25% of affected individuals. Because
the presence of two complete maternal chromosomes 15
cannot substitute for the absence of paternal alleles, it is suggested
that the 15q11–q13 region is imprinted (Nicholls et al.,
1989; Glenn et al., 1997). About 1–5% of patients with PWS
have biparental inheritance of chromosome 15, but show abnormal
methylation patterns and gene expression. These pa-
387
tients have a mutation in the imprinting process (Buiting et al.,
1995; Dittrich et al., 1996; Saitoh et al., 1996, 1997; Ohta et
al., 1999).
The test of the methylation pattern of SNRPN exon 1 detects
95% of the PWS and 80% of the Angelman Syndrome (AS) patients,
and the microsatellite analysis of loci within and outside
the PWS/AS region distinguishes between the genetic mechanisms
in each case (deletion, UPD, or an imprinting mutation).
G-band analysis is recommended to distinguish numerical and/or
structural chromosome aberrations, such as translocations, inversions,
or marker chromosomes; fluorescence in situ hybridization
(FISH) techniques are useful in detecting the deletion
cases, when parental DNA samples are not available, and
also to identify the specific chromosome aberrations detected.
Some genes (SNURF-SNRPN, IPW, ZNF127, NECDIN) and
transcripts (PAR1 and PAR5) are paternally expressed and map
to the PWS/AS critical region. Therefore, they are good candidates
to be involved in the etiology of PWS (Glenn et al., 1997;
MacDonald and Wevrick, 1997; Saitoh et al., 1996). However,
only two of them (SNRPN and NECDIN) have a known protein
that is completely absent in PWS patients. The SNRPN protein
(SmN) is involved in RNA splicing, whereas NECDIN is
1 Department of Biology, Institute of Bioscience, University of São Paulo, São Paulo, Brazil.
2 Child Neurology Service, Department of Neurology, Hospital das Clínicas, School of Medicine, University of São Paulo, São Paulo, Brazil.
3 Pediatric Endocrinology Unit, Department of Pediatrics, Hospital das Clínicas, School of Medicine, University of São Paulo, São Paulo,
Brazil.

388
a nuclear protein that acts, mainly, in cerebral development
(Glenn et al., 1996; Jay et al., 1997). These genes show paternal
expression in humans and mice, and map to the PWS/AS
critical region in humans and to the syntenic conserved region
in the central part of chromosome 7 in mice (Glenn et al., 1996;
Jay et al., 1997; MacDonald and Wevrick, 1997; Watrin et al.,
1997; Gray et al., 1999).
PWS is presumed to be a contiguous gene disease because
no patients with mutations in a single gene have so far been detected.
Phenotype characterization is, therefore, important to understand
the possible roles of genes in the critical region, with
manifestations of the syndrome. Until now, two main clinical
differences have been observed between the different classes of
PWS patients: hypopigmentation, most common in deleted patients,
is due to the loss of the P gene (Spritz et al., 1997),
whereas increased maternal age has been associated with UPD
cases (Butler, 1989; Gillessen-Kaesbach et al., 1995).
In this report we studied PWS patients diagnosed through
methylation analysis of the SNRPN exon 1, microsatellites, and
FISH. Clinical features of 27 deleted PWS patients are described
and compared with those of 8 patients with maternal
UPD.
Patients
MATERIALS AND METHODS
Fifty-three patients suspected clinically of having PWS were
referred to our laboratory for genetic tests. The majority of these
patients were seen by at least one of us, but in some cases only
a blood sample was sent to us, with a short list of clinical characteristics.
The main features that prompted physicians to ask
for these studies were hypotonia in infants, and obesity and
mental retardation in children and adolescents.
Methods
Cytogenetic studies: Chromosome studies of patients and
their parents were performed on peripheral blood lymphocytes
to investigate structural and numerical alterations. The chromosomes
were examined by the GTG banding technique.
FISH was performed using the SNRPN probe, specific for
the 15q11–q13 region, and control chromosome 15 marker cosmids,
which detect specific sequences in 15q22. The hybridization
and immunochemical detection were carried out according
to the manufacturer’s instructions (Oncor, Inc.). Twenty
cells were examined for each FISH slide.
DNA analysis: Methylation analysis. DNA was extracted
from peripheral blood leukocytes by standard procedures. The
methylation status of the PWS/AS region was assessed by
Southern blotting (Southern, 1975). Genomic DNA was digested
with XbaI 1 NotI, separated by size on a 1.0% agarose
gel, transferred to a nylon membrane, and hybridized using the
probe that corresponds to a 0.6-kb EcoRI–NotI fragment that
contains exon 1 of SNRPN (Glenn et al., 1996).
Dinucleotide repeat (CA) n polymorphisms within the
PWS/AS critical region. Microsatellite analysis was performed
with three markers within the critical region, 15q11–q13, 4-
3RCA (D15S11); LS6-1CA (D15S113); and GABRB3CA
(GABRB3). Two loci outside the PWS/AS region, D15S131 and
D15S984, were also studied to distinguish between deletion and
UPD. Deletion is suggested if there is biparental inheritance of
these loci; isodisomy, if one maternal allele is present; and heterodisomy,
if two different maternal alleles are evident. For
UPD patients, we also analyzed the loci D15S41 and D15S42,
localized close to the centromere, making it possible to identify
the meiotic origin of the non-disjunction (Robinson et al.,
1998). Multiplex PCR and polyacrylamide gel electrophoresis
of 32 P-end-labeled amplification products followed the protocols
described by Mutirangura et al. (1993).
Statistical analysis
Statistical analysis was performed using the Fisher test, Student’s
t-test, and Mann-Whitney’s test. Confidence levels of
a 5 0.05 were used to indicate a statistically significant difference
between the different classes of PWS patients (with
deletion or UPD).
RESULTS
Of 53 patients suspected of having PWS who were referred
for genetic tests, the syndrome was confirmed in 35 (20 girls
and 15 boys, with ages ranging from 3 months to 18 years) (Fig.
1), through methylation pattern analysis.
Microsatellite analysis of loci within and outside the
PWS/AS region was performed in 30 of the 35 PWS patients,
because parental samples were not available in 5 patients. We
identified 18 patients with a paternal deletion, 8 with maternal
UPD, and 4 patients with the absence of paternal alleles, but
who were noninformative with regard to the specific genetic
mechanism. In those 9 patients in which microsatellite studies
were not available or informative, FISH showed a deletion at
the PWS critical region. Seven cases with heterodisomies and
one with complete isodisomy were identified among the 8 UPD
patients. In summary, we obtained 27 deletion cases (77.2%)
and 8 maternal UPD patients (22.8%).
The main clinical features of the UPD and deleted patients
are summarized in Table 1.
DISCUSSION
FRIDMAN ET AL.
In this study, we diagnosed 35 PWS cases in 53 patients referred
for genetic evaluation. Clinical characteristics of 27 individuals
with a paternal deletion (14 girls and 13 boys), and 8
patients with maternal UPD (6 girls and 2 boys), were compared
(Table 1).
Previous clinical comparisons between deleted and UPD
PWS patients pointed only to differences in pigmentation, with
hypopigmentation more frequent in deleted patients, and in maternal
age, which has been reported to be increased in the UPD
patients (Butler, 1989; Robinson et al., 1991; Gillessen-Kaesbach
et al., 1995). Nevertheless, in our data (Table 1) a significant
difference in the age at diagnosis between the two classes
of PWS patients was observed: our children with UPD were diagnosed
earlier than the deleted ones (2 10/12 vs. 7 5/12 years, respectively),
different from the studies of Mitchell et al. (1996)

PWS SYNDROME
c
a
d e
b
f g h i j
FIG. 1. Prader-Willi patients. (a) patient at 24/12 years; (b) patient at 15 years; (c) patient at 9 months; (d) patient at 118/12 years; (e) patient at 18 years; (f) patient at 29/12 years;
(g) patient at 118/12 years; (h) patient at 5 years; (i) patient at 5 years; (j) patient at 10 months. Patients a, c, h, and i are UPD cases; patients b, d, e, f, g, and j are deletion cases.
389

390
TABLE 1. CLINICAL FINDINGS IN PWS PATIENTS WITH DELETION AND UPD
and Gunay-Aygun et al. (1997a). Within the deleted group, the
boys were diagnosed earlier than the girls (5 2/12 vs. 7 8/12 years,
respectively).
As expected, mean maternal age in the UPD group was
higher than in the deleted group, because increased maternal
age is associated with the meiotic non-disjunctive events that
originate UPD zygotes (Robinson et al., 1993, 1998). Mean paternal
age was also elevated in the UPD group; not unexpected
because paternal age is usually related to maternal age.
Only half of our UPD sample had hyperphagia and showed
weights above the 97%. Children that were not in a hyperphagic
phase had weights below the 25% and did not have the characteristic
PWS behavioral features.
PWS UPD patients showed lower birth lengths than the
deleted patients, as observed in other studies (Mitchell et al.,
1996; Gunay-Aygun et al., 1997a). In the 7 patients with heterodisomy,
placental insufficiency due to the trisomy preceding
maternal chromosome 15 UPD could explain such differences
(Gunay-Aygun et al., 1997a). No differences in facial
features, such as narrow bifrontal diameter, almond-shaped
eyes, and strabismus, as suggested by Cassidy et al. (1997),
were observed.
Among patients with normal genetic results, obesity and
mental retardation in children and adolescents, and hypotonia
FRIDMAN ET AL.
UPD Deletion
(8 patients) (27 patients) P a
Mean age of diagnosis (y) (range) 2 10/12 7 9/12 0.0152 (1)
(3/12 to 6) (9/12 to 18)
Sex ratio (Female; Male) 6; 2 14; 13
Mean maternal age (y) (range) 36 10/12 27 2/12 0.0022 (1)
(19 to 47) (20 to 38)
Mean paternal age (y) (range) 38 6/12 29 8/12 0.0088 (1)
(21 to 50) (23 to 43)
Reduced fetal activity 71,43% (5/7) 77,77% (14/18) 1.0000 (2)
Birth weight (g) (%) 2521 (p3) 2693 (3,p,10) 0.3616 (3)
Birth length (cm) (%) 44,42 (,,p3) 47,87 (p10) 0.0298 (3)
Hypotonia 100% (8/8) 96% (24/25) 1.0000 (2)
Feeding problems 100% (8/8) 96% (24/25) 1.0000 (2)
Developmental delay 87,5% (7/8) 100% (26/26) 0.2353 (2)
Weight .p90 50% (4/8) 73,08% (19/26) 0.3884 (2)
Height ,p50 42,86% (3/7) 50% (12/24) 1.0000 (2)
OFC ,p50 37,5% (3/8) 68,18% (15/22) 0.2098 (2)
OFC 50,p,97 62,5% (5/8) 31,82% (7/22) 0.2098 (2)
Hypertelorism 62,5% (5/8) 40% (8/20) 0.4097 (2)
Almond shaped eyes 62,5% (5/8) 52,38% (11/21) 0.6968 (2)
Strabismus 71,42% (5/7) 45,45% (10/22) 0.3898 (2)
Narrow bifrontal diameter 50% (4/8) 86,36% (19/22) 0.0596 (2)
Small hands and feet 71,42% (5/7) 81,82% (18/22) 0.6119 (2)
Learning disabilities 100% (1/1) 100% (16/16) 1.0000 (2)
Seizures 16,67% (1/6) 54,54% (12/22) 0.1727 (2)
Behavioral problems 80% (4/5) 78,94% (15/19) 1.0000 (2)
Hyperphagia 50% (4/8) 72% (18/25) 0.3970 (2)
High pain threshold 40% (2/5) 54,54% (6/11) 1.0000 (2)
Skin picking 100% (2/2) 86,66% (13/15) 1.0000 (2)
Decreased vomiting 100% (2/2) 100% (10/10) 1.0000 (2)
a Statistical analysis with a level of confidence of a 5 0,05 to assume a statistically significance difference. (1) Mann-
Whitney test; (2) Fisher test; (3) Student’s t-test.
in babies were the main characteristics considered by the physicians
in asking for PWS genetic tests.
Some individuals with the PWS-like phenotype but with normal
methylation results have been described in the medical literature,
and hypotonia, obesity, mental retardation, and sometimes
hyperphagia are the main observed characteristics of such
cases. Additional investigation can disclose some other anomalies,
like the Fragile X syndrome (de Vries et al., 1993), interstitial
6q deletion (Villa et al., 1995; Stein et al., 1996),
UPD14 (Berends et al., 1999), some chromosome X aberrations
(Monaghan et al., 1998; Stratakis, 1998; Tümer et al., 1998),
and 1p deletions (Eugster et al., 1997). Obesity and mental retardation
can occur in many other mendelian inherited disorders,
such as the Cohen syndrome, Bardet-Biedl syndrome, and
others (see review in Gunay-Aygun et al., 1997b).
Besides obesity and mental retardation in children, a history
of neonatal hypotonia and swallowing difficulties should be
present to suggest PWS diagnosis. In babies, hypotonia, hypogonadism,
feeding problems, and decreased vomiting suggest
the diagnosis of the syndrome. The methylation analysis,
a noninvasive exam, allows one to make the PWS diagnosis,
and invasive exams, like muscle biopsy and electroneuromiography,
which are usually performed in hypotonic babies, are not
necessary. Consequently, with early PWS diagnosis, dietary

PWS SYNDROME
management to avoid obesity and physiotherapy to improve
muscle tone and avoid scoliosis, can be introduced earlier. PWS
diagnosis in babies and children can avoid obesity-related health
problems, which can cause early death. Parents and siblings of
deletion and UPD PWS patients have low genetic risks (around
1%), unless some type of chromosome aberration is disclosed
in the patient. Clinical and behavioral PWS studies in populations
of different genetic backgrounds are important to delineate
the phenotypic and behavioral variability present in this
syndrome.
ACKNOWLEDGMENTS
We thank Dr. Robert D. Nicholls for kindly provided the
SNRPN probe for methylation assay. This work is supported by
FAPESP and PRONEX.
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Address reprint requests to:
Dr. C. Fridman
Departamento de Biologia
Instituto de Biociências, USP
Caixa Postal 11.461
CEP: 05422-970, São Paulo, SP, Brazil
E-mail: cfridman@ib.usp.br
Received for publication December 2, 1999; accepted July 7,
2000.