PRADER -WILLI SYNDROM E IDENTIFIED BY METHYLATION SPECIFIC [617473]

PRADER -WILLI SYNDROM E IDENTIFIED BY METHYLATION SPECIFIC
MULTIPLEX LIGATION DEPENDENT PROBE AMPLIFICATION (MS –
MLPA)
Simona Loredana Vasilache ¹², Oana Marginean¹ , Adelina Micheu ²,
Claudia Banescu ³, Valeriu Moldovan ³,Carmen Duicu ¹, Ionela Maria Pascanu ²⁴
¹Department of Pediatrics , University of Medicine and Pharmacy, Targu Mures
² Department of Endocrinology, Mures County Hospital Targu Mures
³ Department of Genetics, University of Medicine and Pharmacy, Targu Mures
⁴Department of Endocrinology, University of Medicine and Pharmacy, Targu Mures

ABSTRACT
Prader -Willi syndrome (PWS) is a multisystemic complex genetic disorder caused by lack
of expression of genes on the paternally inherited chromosome 15q11.2 -q13 region . There
are three main genetic subtypes in PWS: paternal 15q11 -q13 deletion (70 % of cases),
maternal uniparent al disomy 15 (25 -30 %) and imprinting defect (1 -3 %). The challenge for
the scientific community is to differentiate more clearly between the classic PWS and the
various PWS -like phenotypes on a clinical level, for choosing the correct genetic analysis.
We present a case of a 6 years old male patient with severe hypotonia, feeding difficulties
in neonatal period, developmental delay in neur omotor acquisition, hyperphagia and
obesity (BMI: +4.66 SD) . The genetic analysis methylation specific multiplex ligation
dependent pro be amplification (MS-MLPA) revealed a rare cause of PWS, an aberrant
methylation of CpG island . It is important to mention that a precise diagnosis of PWS and
an early multidisciplinary approach are essential for efficient long -term management, for
prevent complications and improve quality of life in this patients .
Ketword s: Prader -Willi syndrome, imprin ting center, obesity
INTRODUCTION
Prader -Willi syndrome (PWS) is a
disorder characterized by neurogenetic,
neurometabolic and neurobehavioral
alteratios. It has an estimated prevalence
worldwide in the range of 1 in 10,000 to
30,000 in dividuals [1]. PWS is a genetic
condition due to paternal loss of imprinted genes on chromosome 15 and
characterized by a range of mental and
physical findings including obesity that
can be life -threatening [2]. Approximately
70% of individuals with PWS are caused
by a non -inherited deletion in the
paternally deriv ed chromosome 15q11 –
q13 region; about 25% result from

maternal disomy 1 5 and in less than 3%
of cases are individuals genomic
imprinting defects [2,3,4].
Affected infants constantly have
significant hypoto nia, feeding difficulties,
and failure to thrive, followed in early
childhood by excessive appetite with
gradual development of obesity, short
stature, intellectual disabilities and
behavioral problems [5].
In PWS a precocious multidisciplinary
approach is fundamental to prevent
complic ations, to imp rove quality of life
and prolong life expectancy.
CASE PRESENTATION
In our Department we had the opportunity
to observe the case of a child from a non –
consanguineous couple, a 6 years old
male patient, from an abnor mal
pregnancy -oligoamnios. He presented
diminished fetal activity and he was born
at 42 weeks by cesarean section, with a
birth weight of 2100 g (-2.18 SD), a birth
height of 47 cm (-1.63 SD) and APGAR
score 6/1 minute. During the neonatal
period h e presented severe hypotonia,
feeding difficulties (until 3 weeks of age
the child was fed by nasogastric tube )
and developmental delay in neuromotor
acquisition. Later t he patient presented
hyperphagia with important weight gain
and behavioral problems – temper
tantrumps and stubbornness. Karyotype from fresh blood was 46,XY. Afterwards a
molecular genetic analysis was
performed (methylation specific multiplex
ligation dependent probe ampli fication
MS-MLPA) revealing a rare cause of
PWS, an aberrant methylation of CpG
island, which belongs to SNRPN
promoter.
Thereafter , he is admi tted to our clinic;
physical examination performed reveals
dysmorphic features ( narrowed forehead,
almond shaped eyes, thinner upper lip),
delayed motor and cognitive
development, decreased stature ( height: –
1.05 SD) , excessive weight gain ( BMI:
+4.66 SD) and und erdevelopment of the
sex organs.
Associated comorbidities : congenital
microcephaly, cytomegalovirus infection
of the central nervous sy stem , severe
psychomotor and cognitive retardation,
surgically corrected cryptorchi dism .
The laborato ry investigation revealed
IGF1: 62.89 ng/ml, TSH:1.376 µUI/ml,
FT4:0.94 ng/dl and elevated liver
enzymes. The abdominal ultrasound
revealed: unilateral hydronephrosis,
hepatic steatosis and hepatomegaly . The
severe obstructive sleep apnea was
diagnosed ; the polysomnography
revealing apneea -hypopnea score (AHI) >
40. The aden oidectomy for adenoid
vegetation was performed and the post –
procedure AHI was 9.4. After

adenoidectomy g rowth hormo ne therapy
(rhGH) was initiated. rhGH doses were
progressively increased , and after 6
months of treatment an improvement is
noted in height: -0.46 SD, BMI : +3.82 SD
and height velocity : 9 cm/year .
DISCUSSIONS
The PWS has common signs and
symptoms with other diseases, which
makes it difficult to accurately diagnose.
The challenge for the clinicians is not only
to differentiate more clearly between the
classic PWS and the various PW S-like
syndrome (PWLS) on a clinical leve l but
also to provide precise genetic
explanations for these phenotypes and
provid e proper genetic counseling and
treatment. PWLS share elements of the
PWS phenotype and the gene functions
disrupted in PWLS are likely to be
involved in genetic pathways that are
significant for the development of PWS
phenotype [6].
Considering this aspects, it is important to
choose the correct genetic test to identify
patients with clas sic PWS because
Rocha et al [7] reveals in a meta -analysis
other mutation associated with PWS -like
phenotype, such us: deletion of 6q,
paracentric inversion, fragile X syndrome .
DNA methylation is considered a powerful
tool to assess paternal -only, maternal –
only, or biparental (normal) inheritance,
but can not dis tinguish between maternal
uniparental disomy 15 or imprinting
defects [5]. Compared to traditional DNA methylation, a new semi -quantitative
method, namely “methylation -specific
multiplex -ligation probe amplification”
(MS-MLPA) is recommended for
methylation detection in imprinting
disorder s (ex. Beckwith Wiedemann
syndrome , Silver Russel syndrome ) and
uniparental disomy . MS-MLPA will
determine the methylation status by using
5 methylation specific probes (4
for SNRPN gene and one for NDN gene )
but also detect the copy number changes
(deletions on chromosome 15q11),
therefore being indicated to confirm the
diagnosis o f PWS [8.9].
For th is reason , MS -MLPA analysis might
be considered as the fi rst testing when
suspecting PWS as a possible diagnosis,
especially since important genotype –
phenotype correlations will likely be
expected. [10].
Understanding the particular genetic
etiology in patients with PWS is essential
for the appropriate genetic counseling of
affected families. Estimation of
recurrence risk is dependent on the
genetic defect causing PWS. [11]
Imprinting defects are rare but significant
cause of PWS. Pat ients with an imprinting
defect have apparently normal
chromosome 15 of parental and maternal
origin, but present aberrant DNA
methylation of SNRPN promote r. When
counseling patients with a detected

imprinting defect, a recurrence risk is up
to 50%, because the mutation is likely
dominant and occurred in the paternal
grandmother's germ line [12]; those
without an IC deletion would be expected
to have a lower risk.
Maternal uniparental disomy of 15 is the
second cause of PWS an present also
aberant methylation . Frequently m aternal
UPD 15 i s de novo (recurrence <1%)
except if a Robertsonian translocation is
present in either parent .[2]
CONCLUSIONS
It is important to mention that a precise
diagnosis of PWS and an ear ly
multidisciplinary approach are essential
for efficient long -term management, for
prevent complications and improve
quality of life. The genetic basis of these
rare disorder differ but MS-MLPA is a
simple, rapid and useful method for
identification of PWS cases with aberrant
methylation status.
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