2008 Barcelona - European Society of Human Genetics

Cytogenetics
There are several molecular abnormalities associated with BWS . The
majority of BWS patients (50-60%) have an epigenetic defect at the
DMR2 of the 11p15 region . The epigenetic defect present in this region
involves loss of methylation (LOM) of the maternally inherited copy of
KvDMR (DMR2), a CpG island localised at the intron 10 of the maternally
expressed gene KCNQ1 .
In this same intron 10, this CpG island surrounds the promoter that
drives a long transcript (KCNQ1OT1) with antisense transcription to
KCNQ1 . Normally, the maternal allele of DMR2 is methylated and
KCNQ1OT1 is silenced . If there are epigenetic alterations in this region,
loss of maternal methylation of DMR2 is accompanied by biallelic
expression of the KCNQ1OT1 transcript, usually only paternally
expressed .
We studied the methylation pattern by means of MS-MLPA technique
(Methylation-Specific MLPA). With this technique is also possible to
study the extension of the hypomethyation in the DMR2’s CpG islands
.
We studied 87 patients with BWS and among them we found that 37
presented hypomethylation at the DMR2 (42 .5%) . Moreover our results
are consistent with similar studies which demonstrated that the
hypomethylation includes the 5’end of the KCNQ1OT1 gene. Our findings
suggest a clear pattern of hypomethylation at the KvDMR with a
5’-3’ gradient of demethylation at this region .
P02.056
Laboratory progress in molecular diagnosis of Prader-Willi
syndrome and Angelman syndrome - A new strategy by
methylation-specific competitive multiplex PCR
S. Y. Lin, C. H. Lin, Y. N. Su;
National Taiwan university hospital, Taipei, Taiwan.
Introduction: Most of the Prader-Willi syndrome and Angelman syndrome
patients are caused by the deletion of chromosomal 15q11-13
or uniparental disomy of chromosome 15 . There are many diagnostic
tools available like the FISH analysis, methylation-based PCR, multiplex
PCR, High Resolution Melting analysis and commercialized MS-
MLPA . Each of them has some disadvantages and limitations such as
high costs, time-consuming and requiring DNA bisulfite-treatment. We
introduce a novel, in-house designed methylation-specific competitive
multiplex PCR, which takes three hours for the reaction, avoids bisulfite-treatment
of DNA and provides reliable results of PWS and AS.
Material and methods: 68 patients with clinical suspicion of PWS, two
with AS, and 20 unaffected individuals from the general population
were analyzed. In the MS-competitive multiplex PCR, we amplified
one SNRPN control gene, the KRITI gene to serve as internal control,
and the promoter of the SNRPN gene, which could be digested them
with HhaI enzyme . By comparing the copy number of the three regions,
we could differentiate the wild type, deletion type and UPD type
PWS within one reaction . We also evaluated other different diagnostic
technologies in clinical applications .
Results: In this article, we successfully made the molecular diagnosis
of one AS patients and 46 PWS patients by several different techniques
. In the MS-competitive multiplex PCR, the total copy numbers
were accurately calculated in all of the cases .
Conclusions: The MS-competitive multiplex PCR strategy is a good alternative
for molecular diagnosis of PWS and AS . This approach could
serve as an alternative genotyping platform for epigenetics .
P02.057
From phenotype to mosaic paternal UPD11p15 in Beckwith
Wiedemann syndrome
C. Skrypnyk 1 , M. Bembea 1 , V. Bica 2 , C. Rusu 3 , C. Jurca 1 , W. Kress 4 , A. Baumer
5 ;
1 University of Oradea, Genetics Department, Oradea, Romania, 2 Institute for
Mother and Child Care, Bucharest, Romania, 3 University of Medicine and Pharmacy,
Genetics Unit, Iasi, Romania, 4 Institute of Human Genetics, Wurzburg,
Germany, 5 Institute of Medical Genetics, Zurich, Switzerland.
Introduction: Beckwith-Wiedemann syndrome (BWS; MIM 130650) is
a model human imprinting disorder resulting from altered activity of
one or more genes in the 11p15.5 imprinted gene cluster and defined
by overgrowth, macroglosia, visceromegalia and tumor predisposition .
Approximately 20% of BWS cases have uniparental disomy (UPD) of
chromosome 11 . Material and methods: We report 4 patients, three
boys and one girl, clinically diagnosed with BWS . Genomic DNA was
extracted from peripheral blood lymphocytes and PCR methylation
tests for 11p15.5 loci were performed to confirm the diagnosis. Results:
All patients accomplished the consensus clinical diagnosis criteria and
had ispislateral hemihyperplasia . Transient neonatal hypoglycemia
was recorded in two cases, three cases had cardiac involvement . The
cases where confirmed by DNA methylation for loci LT1 and H19, microsatellite
analysis was performed for one case and showed 11p15 .5
mosaic paternal UPD . Conclusions: The consensus criteria enabled a
correct and precocious clinical diagnosis in the first two months of life.
The DNA tests confirmed the cases and showed that interchromatid
somatic recombination was the causal mechanism for one case . Considering
that all cases had a similar phenotype we assumed the same
casual mechanism and established a correct tumor screening .
P02.058
Methylation Specific Multiplex Ligation-dependent Probe
Amplification (MS-MLPA) in routine diagnostics of Prader-Willi
and Angelman syndromes
A. Szpecht-Potocka1 , M. Gos1,2 , J. Bal1 , T. Mazurczak1 ;
1 2 Institute of Mother and Child, Warsaw, Poland, MSCM Cancer Center and
Institute of Oncology, Warsaw, Poland.
Methylation Specific Multiplex Ligation-dependent Probe Amplification
(MS-MLPA) is a novel technique that allows for simultaneous detection
of copy number variation and CpG methylation . The usage of probes
specific for 15q11-q13 locus makes it useful as a diagnostic tool in the
diagnosis of Prader-Willi and Angelman syndromes (PWS/AS) . The
aim of our study was the implementation of MS-MLPA technique as a
standard diagnostic procedure in PWS/AS patients .
The Salsa MS-MLPA Prader Willi / Angelman Kit (ME028) was used
according to MRC-HOLLAND suggested protocol . The MS-MLPA peak
analysis, normalization and calculation of dosage ratio were performed
with the SoftGenetics LLC GeneMarker ver 1 .6 software . For the pilot
study, we have chosen 20 cases with established PWS or AS molecular
diagnosis based on PCR-based DNA methylation analysis of
SNRPN locus (MS-PCR) and the STR analysis of deletion or UPD
presence .
The results of the analysis of the copy number variation and the methylation
status in 15q11-q13 locus with MS-MLPA confirmed the previous
findings obtained for the pilot patients. We have also performed
the copy number variation analysis in additional 16 AS and 24 PWS
patients with abnormal methylation status detected by MS-PCR . The
MS-MLPA technique allowed to exclude from the further molecular
analysis patients with the deletion without the necessity parents testing
. Therefore, it seems that the MS-MLPA is a useful method in the
molecular diagnosis of PWS/AS syndromes .
The study was supported by grant PBZ-KBN-122/P05/01-4 .
P02.059
New hypotheses in PWS/AS research: a multidisciplinary
approach of rare diseases in Romania
M. Puiu 1 , M. Serban 1 , N. Cucu 2 , G. Anton 3 , D. Dan 4 , C. Popoiu 1 , C. Rusu 5 , V.
Pop 6 , C. Badiu 7 ;
1 University of Medicine and Pharmacy, Timisoara, Romania, 2 University of
Biology, Bucharest, Romania, 3 National Institute of Virusology, Bucharest, Romania,
4 APWR, Zalau, Romania, 5 University of Medicine and Pharmacy, Iasi,
Romania, 6 University of Medicine and Pharmacy, Cluj, Romania, 7 Institute of
Endocrinology Parhon, Bucharest, Romania.
SPW and SA are two clinical, metabolic and neurological different syndromes
with 1 case for 15000 new born . The molecular mechanisms
identified imply large deletions, uniparental disomia (DUP), intragenic
and epigenetic modifications in the processes of imprinting and only
rare balanced translocations . The actual studies have extended the
area of epigenetic modifications, involving chromatin dynamic structure
through covalent modifications of their components. A group of
researchers from medical centers of Romania together with APWR
suggests studying new etiological hypotheses of PWS/AS .
Since a small number of cases with SPW/SA do not integrate with
the etiologies described by now our project have a great opportunity
to discover new and interesting aspects . The project aims to follow in
these patients aspects connected to environment, diet, pollution and
way of life influence trying to solve the weak points of the correct imprinting
process .
Knowing the epigenetic aspects from SPW/SA will allow a more pre-