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 Ercal, MD4, and Nihan Erginel-Unaltuna, PhD3

Objective To investigate the prevalence of Prader–Willi syndrome (PWS) in infants with hypotonia between theages of 0 and 2 years.Study design Karyotyping studies were performed in all infants with hypotonia. The study group was composedof infants with hypotonia for whom the karyotyping was found to be normal. Fluorescence in situ hybridization andmethylation analysis were performed simultaneously in the study group. Molecular studies for uniparental disomywere 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 in6 patients by fluorescence in situ hybridization analysis. Only 1 patient had no deletion but had an abnormalmethylation 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 consideredby pediatricians and neonatologists in the differential diagnosis of all newborns with hypotonia. Early diagnosis ofPWS is important for the management of these patients. (J Pediatr 2014;164:1064-7).

Prader–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 aretypical 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 pathognomonicfindings of hyperphagia, obesity, and intellectual disability become evident later in childhood.1-5 Clinical diagnosis of PWS isdifficult during the neonatal period and infancy because many features of this syndrome are nonspecific and the typical clinicalfeatures of later life are not yet present. Trifiro et al6 reviewed 21 newborns with a diagnosis of PWS to identify all clinicalmarkers in newborns with severe hypotonia, which could facilitate early diagnosis of the syndrome. The clinical diagnosisof 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 genetictesting 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. Paternal15q11.2-q13 deletion is responsible for 65%-75% of cases, maternal uniparental disomy (UPD) is responsible for 20%-30% ofcases, 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 amicrodeletion 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 bemore than 1 in 10 000.2,10-12 The prevalence among infants with hypotonia is unknown. Hypotonia in infants in the first year oflife 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 glycosylationdefects, 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 FISHand methylation analysis.

From the Departments of 1Pediatric Genetics and2Neonatology, Cerrahpasa Medical Faculty,3Department of Genetics, Institute for ExperimentalMedicine, Istanbul University, Istanbul, Turkey; and4Department of Pediatric Genetics, DokuzeylulUniversity, _Izmir, Turkey

The authors declare no conflicts of interest.

0022-3476/$ - see front matter. Copyright ª 2014 Elsevier Inc.

All rights reserved. http://dx.doi.org/10.1016/j.jpeds.2014.01.039

FISH Fluorescent in situ hybridization

PWS Prader–Willi syndrome

UPD Uniparental disomy

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Vol. 164, No. 5 � May 2014

Methods

Infants with hypotonia (0-24 months of age) who had beenevaluated in the pediatric genetics clinic or who were caredfor in the neonatal intensive care unit and pediatricemergency department of Cerrahpasa Medical Faculty,Istanbul University were recruited. All patients who hadhypotonia had been initially studied with high-resolutionchromosome analysis. Only infants with normal karyotypingconstituted the study group. Ninety-two infants withabnormal karyotyping were excluded from the study group.Eighty-seven of these infants had Down syndrome, and 5were found to have deletion syndromes. Therefore, a totalof 65 infants with hypotonia with normal karyotyping wereenrolled in the study group. Males-to-females were 1.1:1(35 males and 30 females) in the study group. Informedconsents were obtained from the mothers. The studyprotocol was approved by the Ethics Committee of IstanbulUniversity, Cerrahpasa Medical Faculty.

Chromosome studies were performed on peripheral bloodlymphocytes using standard methods. Metaphase chromo-somes were analyzed by standardGiemsa banding techniques.

FISH and methylation analysis were performed simulta-neously in the study group. The presence of a deletion wasdetermined using FISH. UPD was investigated in a patientwith a methylation defect without a deletion.

FISH was used to detect microdeletions with the commer-cially available probes covering the 15q11.2 PWS criticalregion, according to the manufacturer’s protocols. Themethylation pattern analysis of the PWS region wasperformed to detect deletions, UPD, and imprinting centerdefects, according to the standard diagnostic protocols forSouthern blot and bisulphite methylation polymerase chainreaction. Microsatellite analysis was applied to detect UPD,with markers mapped to segment 15q11-14.19

Results

Sixty-five infants with hypotonia with amean age of 8months(3 days-24 months) were enrolled in the study. Of these65 patients, 17 of them were in the newborn period, 33were between the ages of 1 and 12 months, and 15 of themwere between the ages of 12 and 24 months. A microdeletionin 15q11.2-q13 region was detected in 6 (9.2%) of thepatients by FISH analysis. We found abnormal methylationpattern in these patients, and 1 patient had no deletion byFISH. Subsequent molecular analysis exhibited that thispatient had a maternal UPD. In the present study, PWSwas detected in 7 (10.7%) of the 65 infants with hypotoniaof whom 2 were female and 5 were male.

The clinical findings of the 7 patients with PWSwith regardto major and minor criteria of PWS proposed by Holm et al1

are summarized in Table I. The scores of the patients withPWS ranged between 1 and 6 points according to theseproposed criteria.1 The first patient who was a 6-month-oldmale scored 5 points, the second patient (3-day-old female)

3 points, and the third patient (8-month-old male) 6points. Only 3 of 7 patients with the diagnosis of PWS hada score over 5 points. Though it was seen less extensively,patients without PWS also presented with some of thefindings 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 ofthe patients in the neonatal and early infantile period wereless 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 patientrefused follow-up. The other 5 patients were followed untilthey reached 3-6 years of age. Hyperphagia and obesitywere not evident at the time of diagnosis in any of theenrolled patients but appeared later as did the characteristicfacial features. Although short stature and behavioralproblems were observed in only 1 patient, developmentaldelay 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 prevalenceof PWS in neonates and infants (0-2 years) whose primaryphysical 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 withthe suspicion of PWS. In 45% (29/65) of them, the diagnosisof PWS was confirmed on genetic testing by the lack of thepaternal PWS band. The high frequency of PWS among theseinfants with hypotonia was attributed to the fact that theblood samples of these infants were in fact analyzed toascertain the diagnosis of PWS.The major criteria of PWS proposed by Holm et al were

characterized as: (1) infantile central hypotonia; (2) poorsuck 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 fetalmovements and lethargy, eye abnormalities, small handsand feet, short stature, hypopigmentation, sleep disturbanceor apnea, articulation defects, and behavioral problems. Ac-cording to the criteria by Holm et al for subjects under 3 yearsof age, 4 major criteria are needed with a total of 5 points tomake a diagnosis of PWS.1 However, during the newbornand infantile period, of the major criteria, only hypotoniais detected in 100% of subjects and feeding problems areseen in 95%. Characteristic facial features, hypogonadism,and developmental delay occur in one-half of the patients.Hyperphagia and excessive weight gain become evident laterin life.5 Therefore, if patients are evaluated according to thecriteria 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

Patients without PWSDeletion (+) UPD

Patient number 1 2 3 4 5 6 7 58Age 5 m 3 d 7 m 2.5 m 45 d 11 m 11 m 1-24 m

Major criteriaNeonatal hypotonia 1 1 1 1 1 1 1 58/58Feeding problems in infancy 1 1 1 14/58Facial features 1 1 1 1 1 1 1 14/58Hypogonadism 1 1 1 1 12/58Developmental delay 1 1 1 1 10/58HyperphagiaExcessive weight gain

Minor criteriaDecreased fetal activity 0.5 4/58Behavior problemsSleep disturbance/sleep apneaShort stature 0.5 2/58Hypopigmentation 0.5 0.5 0.5 4/58Small hands and/or feet 0.5 0.5 0.5 0.5 0.5 4/58Narrow hands/straightUlnar bordersEye abnormalitiesThick viscous salivaArticulation defectsSkin-picking

Score 5 3 6 4 3 4 5

Major criteria 1 point. Minor criteria ½ point.

THE JOURNAL OF PEDIATRICS � www.jpeds.com Vol. 164, No. 5

retrospectively with regard to the criteria of Holm et al,proposed that neonatal hypotonia and feeding problems ininfancy, helped diagnose the syndrome during the first yearsof life, whereas others, such as excessive eating, were usefulfindings during early childhood.

When patients were evaluated with regard to the character-istic facial features such as dolichocephaly, narrow bifrontaldiameter, almond-shaped eyes, small appearing mouthwith thin upper lip, and down-turned corners of the mouth,we observed that facial dysmorfism was evident after theneonatal period (Table II). Small hands and feet were alsogenerally recognized after the neonatal period.

Classical features such as hyperphagia, obesity, anddevelopmental delay appeared after the infantile period in

Table II. Distribution of dysmorphic findings amongpatients with PWS

Deletion (+)UPD(+)

Patient number 1 2 3 4 5 6 7Age 6 m 3 d 7 m 2.5 m 45 d 11 m 11 mDolichocephaly + + � � � + +Narrow face or bifrontaldiameter

+ � + � � + +

Almond-shaped eyes + � + + + + +Down-turned corners ofthe mouth

+ � � + � + +

Small mouth with thinupper lip

+ � � + � + +

Microretrognathia + + + + + + +Small hands and/or feet + � + � � + +Hypopigmentation + � � + � + +

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our patients. This observation supports our suggestion thattesting for PWS should be performed in all infants withhypotonia to prevent the delay in diagnosis.Short stature was detected in only 1 of our patients. In

PWS, between the ages of 3 and 13 years, the 50th percentilefor height is roughly identical with the third percentile inhealthy controls and the pubertal growth spurt is also atten-uated. Short stature may be apparent in childhood and isalmost always present by the second decade in the absenceof growth hormone replacement.20 In most recent studies,infants treated with growth hormone therapy showedimprovements not only in stature but also in acquisition ofthe gross motor skills and language and cognitive scores.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 andinfantile period includes neonatal sepsis, hypoxic ischemicencephalopathy, muscle diseases such as myastheniasyndromes, congenital myopathies, and congenital myotonicdystrophy, craniofacial malformations, peroxisomal andmitochondrial disorders, and congenital glycosylationdefects. These diseases have similar clinical findings to thoseseen in PWS, and invasive or expensive tests like musclebiopsy or molecular analysis might be required for thedifferential diagnosis. One concern is that patients mightdie during this extensive diagnostic work up period beforethese tests are completed. The death of the patient beforeany diagnosis is reached creates a challenge for the physicianin terms of prenatal diagnosis and genetic counseling.PWS should be considered by pediatricians and neona-

tologists in the differential diagnosis of all newborns with

Tuysuz et al

May 2014 ORIGINAL ARTICLES

hypotonia to eliminate the unnecessary burden of invasivetests on the patient for the diagnosis of other possible causesof hypotonia. Early diagnosis of PWS is also important forthe early intervention of disease specific problems and toprevent 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,

CerrahpasaMedical Faculty, Istanbul University, 80630 Istanbul, Turkey. E-mail:

beyhantuysuz@yahoo.com

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