European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Genomics, technology, bioinformatics
P1278. Multiplex Ligation-dependent Probe Amplification
(MLPA), a new assay for the molecular diagnosis of Duchenne
and Becker muscular dystrophies
L. Adala 1 , M. Gribaa 1 , H. Elghezal 1 , J. Bouguila 2 , H. Ajina 3 , A. Saad 1 ;
1 Cytogenetics, Molecular genetics and human Reproduction Biology laboratory
Farhat HACHED Hospital, Sousse, Tunisia, 2 Pediatric department Farhat
HACHED Hospital, Sousse, Tunisia, 3 Pediatric department Sahloul Hospital,
Sousse, Tunisia.
Duchenne muscular dystrophy (DMD) is one of the most common and
severe inherited neuromuscular diseases, affecting 1 in 3500 live born
males.
Both of the DMD and BMD (Becker muscular dystrophy), a milder and
less prevalent form of the disease, are X-linked recessive disorders
caused by mutations of the dystrophin gene located at Xp21.
Mutation detection in the DMD gene defective in these diseases is a
problematical trial complicated by the large size of the gene, which
consists of 79 exons and 8 promoters spread over 2,2 Mb of genomic
DNA. Hence, a molecular diagnosis should be proposed to the families
in order to detect the carrier women and to suggest an antenatal
diagnosis.
On this aim, we developed a protocol which is based on the semiquantitative
technique of diagnosis; the MLPA (Multiplex Ligation-dependent
Probe Amplification) and the linkage analysis by tripled primers
PCR (Reaction of polymerization in chain). These 2 techniques allowed
us to study 13 cases including 7 clinically suspected DMD/BMD
patients, 4 antenatal diagnoses and 2 girls suspected to be carrier.
The results showed that 5 of these cases have dystrophin’s anomalies
(38,4%); in four of them it is a deletion and only one is carrying
duplication.
The maternal profile was studied in 9 cases. Three of them showed
heterozygous deletions and only one, showed duplication.
This protocol allowed us the identification of homozygous and heterozygous
deletions and duplications of dystrophin gene in only one
step.
P1279. Multiplex Ligation-dependent Probe Amplification (MLPA)
- an interlaboratory collaborative validation study
T. Janssens, A. Corveleyn, F. Le Calvez, E. Dequeker, G. Matthijs;
Center for Human Genetics, EuroGentest, Leuven, Belgium.
The EU Network of Excellence EuroGentest aims to improve and harmonize
the overall quality of genetic services throughout Europe. The
thorough validation of new methods is one of many contributions to
accomplish this, and Multiplex Ligation-dependent Probe Amplification
(MLPA) is the first technology to be validated in this context.
MLPA is a relatively new method to determine the copy number of
nucleic acid sequences by hybridisation of specific MLPA probe pairs
that bind to adjacent sites and are joined by a ligation reaction before
PCR.
A plethora of popular MLPA kits for different genetic diseases are available
from the manufacturer. But even if their performance was already
established in different molecular diagnostic laboratories, so far no
major nor interlaboratory collaborative programs have been undertaken
to fully validate MLPA.
Therefore, a validation study was set up, including 13 international
diagnostic laboratories. Questionnaires were answered by the participants
to evaluate the variability concerning MLPA protocols and
analysis tools, with the remarkable outcome that the majority had
adapted the original protocol. Furthermore, the P002 BRCA1 MLPA kit
(MRC-Holland) was used on 10 wildtype DNA samples and 3 deletion
or duplication samples to evaluate the performance and the precision
of the method itself. Finally, a data set of 89 MLPA files for DMD was
analysed by different software tools for the evaluation of performance
and internal quality control.
We will present an analytical and diagnostic validation report and a
generic standard operating procedure (SOP), and propose guidelines
for diagnostic laboratories to successfully implement MLPA.
P1280. MS-MLPA (Methylation Specific Multiplex Ligationdependent
Probe Amplification) in the diagnosis of Angeman
Syndrome
M. Gribaa 1 , S. Kacem 1 , H. Elghezal 1 , M. Yaacoub 2 , N. Gaddour 3 , A. Saad 1 ;
1 Service de Cytogénétique et de Biologie de la Reproduction. Hôpital Farhat
Hached, Sousse, Tunisia, 2 Service de Pédiatrie. Hôpital Farhat Hached, Sous-
se, Tunisia, 3 Service de Psychiatrie. Hôpital Fattouma Bourguiba, Monastir,
Tunisia.
Angelman Syndrome (AS) is a complex neurobehavioral disorder associated
with loss of function of differentially expressed imprinted gene
at the region 15q11-q13. It is a rare disorder. It occurs in one of 20
000 live births. It is due either to a microdeletion in the 15q11 locus
(65-75%), or to uniparental disomy (5%), or to UBE3a gene mutations
(10%), or to an anomaly of printing center (5%). In the 10% remainders
cases, the cause still unknown.
The molecular study of the patients appear with a great importance
with an aim of being able to conclude about the parental origin and,
therefore, about the disease.
In this work, we developed an experimental protocol able to ensure
the molecular detection of this pathology. It is based on the MS-MLPA
technique (Methylation Specific Multiplex Ligation-dependent Probe
Amplification), which is able to detect abnormal methylation of imprinted
genes as well as deletion occurring at 15q11-q13 locus. The study
of STRs markers of chromosome 15 allowed us to distinguish between
uniparental disomy and microdeletion when abnormal methylation was
detected.
These two complementary techniques allowed us to study 8 suspected
AS patients. Only one of them showed a deletion in the 15q11-q13
region. This anomaly was confirmed with FISH (Fluorescent In Situ
Hybridysation).
The combination of these two techniques, in the diagnosis of AS,
represents a very good strategy to find more than 85% of causative
anomalies. It can easily be used in the majority of laboratories because
of its low price and simplicity.
P1281. Evidence for a degenerating X chromosome in the rodent
species Ellobius lutescens
A. Süß1 , J. Högel1 , I. Bakloushinskaya2 , W. Just1 ;
1 2 Inst. of Human Genetics, Ulm, Germany, Koltzov Inst. of Developmental Biology
Russ. Acad. Sci, Moscow, Russian Federation.
The mole vole Ellobius lutescens is an exceptional mammal with an
odd karyotype of 2n = 17,X with a single X chromosome in both sexes.
We postulate that the single X of E. lutescens will degenerate in consequence
of its lacking partner for recombination. In analogy to the fate
of the Y chromosome of male mammals we suggest an accumulation
and fixation of non-deleterious mutations that will finally lead to the
inactivation of X-chromosomal genes. This proceeding accumulation
of mutations may cause the extinction of the species E. lutescens.
To test this hypothesis we compared sequences of essential (Zfx, Atrx,
Flna) and non-essential (Opn1mw, Nr0b1, Xist, Mecp2) X-chromosomal
genes to orthologous sequences of other mammals; as control
for the mutation rate of recombining chromosomes we analysed
regions of the autosomal genes Sfrs3 (essential) and App (non-essential).
We compared the ratio of accumulated mutations in essential
versus non-essential X-chromosomal genes to the ratio of autosomal
essential versus non-essential genes.
The results confirmed our hypothesis of a degenerating X chromosome.
A comparison of the sequence conservation of the studied essential
X-chromosomal genes between E. lutescens and E. fuscocapillus
showed a highly significant accumulation of mutations in non-essential
X-chromosomal genes (p = 1.95 · 10-9 ) whereas the analysis of
autosomal genes did not show significant accumulation of mutations (p
= 0.73). So we expect a progressive decay of the single X chromosome
and as a consequence the extinction of the species E. lutescens.
P1282. Identification of primate-specific non-coding RNAs at
human chromosome 15q11.2
C. Cerrato Rivera1 , M. Ogorelkova1 , X. Estivill1,2 ;
1 2 Centre de Regulació Genòmica, Barcelona, Spain, Spanish Nacional Genotyping
Center, Barcelona, Spain.
Non-coding RNAs play important roles in many cellular processes
rather than serving as templates for protein synthesis. Computational
and experimental approaches have predicted thousands of ncRNAs in
human genome, but most of them are yet unknown and only a small
fraction have been partially characterised in terms of function or expression
pattern. We observed that the chromosomal region 15q11.2
share characteristics with other regions containing ncRNAs: repetitive
elements in tandem, imprinted locus, and signals of instability. We
have performed a computational analysis of the target one-megabase
1