Prevalence of Prader–Willi Syndrome among Infants with Hypotonia

Beyhan Tuysuz, MD1, Nuray Kartal, MS1, Tugba Erener-Ercan, MD2, Filiz Guclu-Geyik, PhD3, Mehmet Vural, MD2,
Yildiz Perk, MD2, Derya Erçal, MD4, and Nihan Erginel-Unaltuna, PhD3

Objective To investigate the prevalence of Prader–Willi syndrome (PWS) in infants with hypotonia between the
ages of 0 and 2 years.
Study design Karyotyping studies were performed in all infants with hypotonia. The study group was composed
of infants with hypotonia for whom the karyotyping was found to be normal. Fluorescence in situ hybridization and
methylation analysis were performed simultaneously in the study group. Molecular studies for uniparental disomy
were undertaken in the patients without deletions with an abnormal methylation pattern.
Results Sixty-five infants with hypotonia with a mean age of 8 months were enrolled. A deletion was detected in
6 patients by fluorescence in situ hybridization analysis. Only 1 patient had no deletion but had an abnormal
methylation pattern. A maternal uniparental disomy was observed in this patient. PWS was diagnosed in 10.7 %
(7/65) of the infants with hypotonia.
Conclusion The prevalence of PWS syndrome is high among infants with hypotonia. PWS should be considered
by pediatricians and neonatologists in the differential diagnosis of all newborns with hypotonia. Early diagnosis of
PWS is important for the management of these patients. (J Pediatr 2014;164:1064-7).

P
rader–Willi syndrome (PWS) is a disorder characterized by neonatal hypotonia, intellectual disability, hypogonadism,
dysmorphic facial features, and obesity. It shows great clinical variability with age.1,2 Severe hypotonia and poor suck are
typical findings in the neonatal period, and dolichocephaly, narrow bifrontal diameter, almond-shaped eyes, thin upper
lip with downturned angles of the mouth, small hands and feet, and motor delay are evident in infancy. The pathognomonic
findings of hyperphagia, obesity, and intellectual disability become evident later in childhood.1-5 Clinical diagnosis of PWS is
difficult during the neonatal period and infancy because many features of this syndrome are nonspecific and the typical clinical
features of later life are not yet present. Trifiro et al6 reviewed 21 newborns with a diagnosis of PWS to identify all clinical
markers in newborns with severe hypotonia, which could facilitate early diagnosis of the syndrome. The clinical diagnosis
of PWS in those newborns could be reached at a mean age of 7.4 months with genetic confirmation at 11 months of life.
They concluded that diagnosis by means of evaluation of dysmorphology was difficult in the neonatal period and that genetic
testing should be done in the presence of severe hypotonia.
PWS occurs as a result of the absence of expression of paternal genes in the critical chromosome region 15q11.2-13. A num-
ber of genes in this region are expressed on the paternal chromosome but imprinted on the maternal chromosome. Paternal
15q11.2-q13 deletion is responsible for 65%-75% of cases, maternal uniparental disomy (UPD) is responsible for 20%-30% of
cases, and 1%-3% of cases are sporadic or due to genomic imprinting center defects.2,7 Parent-specific DNA methylation anal-
ysis will detect more than 99% of individuals.8 Fluorescent in situ hybridization (FISH) analysis can also detect the presence of a
microdeletion in 70% of patients with PWS.2,7-9
The prevalence of PWS is reported as 1 in 10 000-30 000 live births. The real prevalence of the disease was estimated to be
more than 1 in 10 000.2,10-12 The prevalence among infants with hypotonia is unknown. Hypotonia in infants in the first year of
life is a common diagnostic and management challenge for pediatricians and neonatologists. Besides PWS, disorders with mus-
cle involvement (muscular dystrophies and myopathies), metabolic disorders such as Pompe disease, congenital glycosylation
defects, mitochondrial and peroxisomal disorders, and structural craniofacial malformations are associated with profound hy-
potonia in the neonatal and early infantile period.13-18 Identifying the underlying cause of congenital hypotonia remains diffi-
cult and may need invasive and expensive tests.
The aim of our study was to determine the prevalence of PWS in infants with hypotonia between 0 and 2 years of age by FISH
and methylation analysis.

From the Departments of 1Pediatric Genetics and

2
Neonatology, Cerrahpasa Medical Faculty,
3
Department of Genetics, Institute for Experimental
Medicine, Istanbul University, Istanbul, Turkey; and
4
Department of Pediatric Genetics, Dokuzeylul
University, _Izmir, Turkey
FISH Fluorescent in situ hybridization
The authors declare no conflicts of interest.
PWS Prader–Willi syndrome
UPD Uniparental disomy 0022-3476/$ - see front matter. Copyright ª 2014 Elsevier Inc.
All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2014.01.039

1064

Methods
Infants with hypotonia (0-24 months of age) who had been
evaluated in the pediatric genetics clinic or who were cared
for in the neonatal intensive care unit and pediatric
emergency department of Cerrahpasa Medical Faculty,
Istanbul University were recruited. All patients who had
hypotonia had been initially studied with high-resolution
chromosome analysis. Only infants with normal karyotyping
constituted the study group. Ninety-two infants with
abnormal karyotyping were excluded from the study group.
Eighty-seven of these infants had Down syndrome, and 5
were found to have deletion syndromes. Therefore, a total
of 65 infants with hypotonia with normal karyotyping were
enrolled in the study group. Males-to-females were 1.1:1
(35 males and 30 females) in the study group. Informed
consents were obtained from the mothers. The study
protocol was approved by the Ethics Committee of Istanbul
University, Cerrahpasa Medical Faculty.
Chromosome studies were performed on peripheral blood
lymphocytes using standard methods. Metaphase chromo-
somes were analyzed by standard Giemsa banding techniques.
FISH and methylation analysis were performed simulta-
neously in the study group. The presence of a deletion was
determined using FISH. UPD was investigated in a patient
with a methylation defect without a deletion.
FISH was used to detect microdeletions with the commer-
cially available probes covering the 15q11.2 PWS critical
region, according to the manufacturer’s protocols. The
methylation pattern analysis of the PWS region was
performed to detect deletions, UPD, and imprinting center
defects, according to the standard diagnostic protocols for
Southern blot and bisulphite methylation polymerase chain
reaction. Microsatellite analysis was applied to detect UPD,
with markers mapped to segment 15q11-14.19
Results
Sixty-five infants with hypotonia with a mean age of 8 months
(3 days-24 months) were enrolled in the study. Of these
65 patients, 17 of them were in the newborn period, 33
were between the ages of 1 and 12 months, and 15 of them
were between the ages of 12 and 24 months. A microdeletion
in 15q11.2-q13 region was detected in 6 (9.2%) of the
patients by FISH analysis. We found abnormal methylation
pattern in these patients, and 1 patient had no deletion by
FISH. Subsequent molecular analysis exhibited that this
patient had a maternal UPD. In the present study, PWS
was detected in 7 (10.7%) of the 65 infants with hypotonia
of whom 2 were female and 5 were male.
The clinical findings of the 7 patients with PWS with regard
to major and minor criteria of PWS proposed by Holm et al1
are summarized in Table I. The scores of the patients with
PWS ranged between 1 and 6 points according to these
proposed criteria.1 The first patient who was a 6-month-old
male scored 5 points, the second patient (3-day-old female)
Vol. 164, No. 5  May 2014
3 points, and the third patient (8-month-old male) 6
points. Only 3 of 7 patients with the diagnosis of PWS had
a score over 5 points. Though it was seen less extensively,
patients without PWS also presented with some of the
findings included in the criteria of Holm et al (Table I).
Characteristic dysmorphic features of the patients with
PWS are summarized in Table II. Dysmorphic features of
the patients in the neonatal and early infantile period were
less prominent than those in the late infantile period.
One of the patients passed away at the age of 1 secondary to
cardiomyopathy and pneumonia, and another patient
refused follow-up. The other 5 patients were followed until
they reached 3-6 years of age. Hyperphagia and obesity
were not evident at the time of diagnosis in any of the
enrolled patients but appeared later as did the characteristic
facial features. Although short stature and behavioral
problems were observed in only 1 patient, developmental
delay was identified in all of the patients.
Discussion
PWS is one of the leading causes of neonatal hypotonia.14,15
In this study, our objective was to determine the prevalence
of PWS in neonates and infants (0-2 years) whose primary
physical finding was hypotonia. PWS was diagnosed in 7
(10.7%) of the 65 patients with hypotonia enrolled.
Gillesen Kausbek et al8 performed methylation analysis in
a group of 65 infants with hypotonia of unknown cause with
the suspicion of PWS. In 45% (29/65) of them, the diagnosis
of PWS was confirmed on genetic testing by the lack of the
paternal PWS band. The high frequency of PWS among these
infants with hypotonia was attributed to the fact that the
blood samples of these infants were in fact analyzed to
ascertain the diagnosis of PWS.
The major criteria of PWS proposed by Holm et al were
characterized as: (1) infantile central hypotonia; (2) poor
suck and feeding difficulty; (3) characteristic facial features;
(4) developmental delay; (5) hypogonadism; (6) hyperpha-
gia; and (7) rapid weight gain between 1 and 6 years of age.
Minor criteria were characterized as decreased fetal
movements and lethargy, eye abnormalities, small hands
and feet, short stature, hypopigmentation, sleep disturbance
or apnea, articulation defects, and behavioral problems. Ac-
cording to the criteria by Holm et al for subjects under 3 years
of age, 4 major criteria are needed with a total of 5 points to
make a diagnosis of PWS.1 However, during the newborn
and infantile period, of the major criteria, only hypotonia
is detected in 100% of subjects and feeding problems are
seen in 95%. Characteristic facial features, hypogonadism,
and developmental delay occur in one-half of the patients.
Hyperphagia and excessive weight gain become evident later
in life.5 Therefore, if patients are evaluated according to the
criteria by Holm et al during the neonatal or infantile period,
most of the patients with PWS would be missed.
In our series, only 3 of the 7 patients with the diagnosis of
PWS had a score over 5 points. In 2001, G€ unay-Ayg€ un et al,5
after reviewing 90 patients with the clinical diagnosis of PWS
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Table I. Evaluation of the patients according to the criteria by Holm et al1

Patients with PWS
Deletion (+) UPD Patients without PWS
Patient number 1 2 3 4 5 6 7 58
Age 5m 3d 7m 2.5 m 45 d 11 m 11 m 1-24 m
Major criteria
Neonatal hypotonia 1 1 1 1 1 1 1 58/58
Feeding problems in infancy 1 1 1 14/58
Facial features 1 1 1 1 1 1 1 14/58
Hypogonadism 1 1 1 1 12/58
Developmental delay 1 1 1 1 10/58
Hyperphagia
Excessive weight gain
Minor criteria
Decreased fetal activity 0.5 4/58
Behavior problems
Sleep disturbance/sleep apnea
Short stature 0.5 2/58
Hypopigmentation 0.5 0.5 0.5 4/58
Small hands and/or feet 0.5 0.5 0.5 0.5 0.5 4/58
Narrow hands/straight
Ulnar borders
Eye abnormalities
Thick viscous saliva
Articulation defects
Skin-picking
Score 5 3 6 4 3 4 5

Major criteria 1 point. Minor criteria ½ point.

retrospectively with regard to the criteria of Holm et al, our patients. This observation supports our suggestion that
proposed that neonatal hypotonia and feeding problems in testing for PWS should be performed in all infants with
infancy, helped diagnose the syndrome during the first years hypotonia to prevent the delay in diagnosis.
of life, whereas others, such as excessive eating, were useful Short stature was detected in only 1 of our patients. In
findings during early childhood. PWS, between the ages of 3 and 13 years, the 50th percentile
When patients were evaluated with regard to the character- for height is roughly identical with the third percentile in
istic facial features such as dolichocephaly, narrow bifrontal healthy controls and the pubertal growth spurt is also atten-
diameter, almond-shaped eyes, small appearing mouth uated. Short stature may be apparent in childhood and is
with thin upper lip, and down-turned corners of the mouth, almost always present by the second decade in the absence
we observed that facial dysmorfism was evident after the of growth hormone replacement.20 In most recent studies,
neonatal period (Table II). Small hands and feet were also infants treated with growth hormone therapy showed
generally recognized after the neonatal period. improvements not only in stature but also in acquisition of
Classical features such as hyperphagia, obesity, and the gross motor skills and language and cognitive scores.
developmental delay appeared after the infantile period in Therefore, consensus guidelines suggest starting this treat-
ment at 6 months and certainly before 2 years of age.21-23
The differential diagnosis of hypotonia in the newborn and
Table II. Distribution of dysmorphic findings among infantile period includes neonatal sepsis, hypoxic ischemic
patients with PWS encephalopathy, muscle diseases such as myasthenia
UPD
syndromes, congenital myopathies, and congenital myotonic
Deletion (+) (+) dystrophy, craniofacial malformations, peroxisomal and
Patient number 1 2 3 4 5 6 7 mitochondrial disorders, and congenital glycosylation
Age 6m 3d 7m 2.5 m 45 d 11 m 11 m defects. These diseases have similar clinical findings to those
Dolichocephaly + + + + seen in PWS, and invasive or expensive tests like muscle
Narrow face or bifrontal + + + +
diameter biopsy or molecular analysis might be required for the
Almond-shaped eyes + + + + + + differential diagnosis. One concern is that patients might
Down-turned corners of + + + + die during this extensive diagnostic work up period before
the mouth
Small mouth with thin + + + + these tests are completed. The death of the patient before
upper lip any diagnosis is reached creates a challenge for the physician
Microretrognathia + + + + + + + in terms of prenatal diagnosis and genetic counseling.
Small hands and/or feet + + + +
Hypopigmentation + + + + PWS should be considered by pediatricians and neona-
tologists in the differential diagnosis of all newborns with
1066 Tuysuz et al
May 2014
hypotonia to eliminate the unnecessary burden of invasive
tests on the patient for the diagnosis of other possible causes
of hypotonia. Early diagnosis of PWS is also important for
the early intervention of disease specific problems and to
prevent complications and to ameliorate the quality of life. n
Submitted for publication Aug 14, 2013; last revision received Dec 23, 2013;
accepted Jan 17, 2014.
Reprint requests: Beyhan Tuysuz, MD, Department of Pediatric Genetics,
Cerrahpaşa Medical Faculty, Istanbul University, 80630 Istanbul, Turkey. E-mail:
beyhantuysuz@yahoo.com
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