2008 Barcelona - European Society of Human Genetics

Clinical genetics
evidence that the number of SMN2 copies acts as a phenotypic modifier.
Acute type I patients usually have one-two copies and chronic type
II and III patients usually have three-four copies . Traditionally, the linkage
analysis with C212 and C272(ag1-CA) markers was helpful in estimating
the SMN2 copies . Quantitative real time analysis (Q-RT) and
recently multiple ligation-dependent probe amplification (MLPA) have
been incorporated into laboratory diagnosis to establish the SMN2
copies with greater accuracy .
We compared the SMN2 copy number of 22 unrelated Spanish SMA
patients with SMN1 absence, correlating the three aforementioned
approaches . Using marker analysis, we determined the maximum
number of alleles by C212 or C272(ag1-CA), estimating the number of
SMN2 copies . The marker results of the respective parents were used
to confirm the copy number. In 4 cases, marker analysis predicted two
SMN2 copies and Q-RT (LightCycler) and MLPA revealed three SMN2
copies . This discordance may be the result of an uninformative marker .
In 2 other cases, marker results predicted at least three SMN2 copies
although Q-RT showed two copies and MLPA results were compatible
with three copies. This discordance may reflects the lack of probe hybridisation
in some samples and highlights the importance of matching
results of the three approaches . FIS05-2416/CIBERER/Proyect GE-
NAME .
P01.220
DNA diagnostic of Duchenne-Becker muscular dystrophy in
Belarus
S. Miasnikov 1 , I. Naumchik 2 , R. Khmel 2 , N. Rumyantseva 2 ;
1 International Sakharov Environmental University, Minsk, Belarus, 2 National
centre of research and applied medicine «Mother and child», Minsk, Belarus.
Duchenne-Becker muscular dystrophy (D/BMD) is X-linked recessive
muscle-wasting disease with incidence 1:3500 and 1:20000 of male
newborns respectively . The cause of this disorder is mutations of the
gene DMD, located on locus Xp21 . The most frequent mutation - gross
deletions in «hot spot» regions of 2-19 and 41-53 exons, which detected
in 35-75% of cases in different populations .
We have performed molecular-genetic diagnostic in cohort of 52 belarussian
D/BMD patients . All of them had typical clinical features and
high level of CPK . To detect deletions of Pm/1, 3, 4, 6, 8, 12, 13, 16, 17,
19, 32, 34, 41-45, 47-53 and 60 exons used both multiplex and routine
PCR methods . 20 of 52 (38%) patients had deletions of different size
and localization . Only 7 patients had one exon deletion . The largest
deletion we had found was deletion of 35 exons (region 8-44 exons) .
In 4 of 20 cases exon 48 was deleted . 35% of mutations localized in
proximal part of gene DMD .
Thought, multiplex PCR is rapid and suitable method to detect gross
deletion, due to low level of its occurrence in Belarus, there is need in
other methods to find both duplication and point mutation within gene
DMD .
P01.221
molecular genetic analyses of the dystrophin gene in Hungarian
Duchenne/Becker muscular dystrophy patients: Comparison of
multiplex PcR, southern blot and mLPA analyses
V. Vancso 1 , H. Piko 1 , B. Nagy 2 , Z. Ban 2 , A. Herczegfalvi 3 , V. Karcagi 1 ;
1 National Institute of Environmental Health, Dept. of Molecular Genetics and Diagnostics,
Budapest, Hungary, 2 Semmelweis Medical School, Clinic of Obstetrics
and Gynaecology, Laboratory of Genetics, Budapest, Hungary, 3 Bethesda
Children’s Hospital, Dept. of Neurology, Budapest, Hungary.
Duchenne/Becker muscular dystrophy (DMD/BMD) is a severe Xlinked
neuromuscular disease caused by mutations in the dystrophin
gene . Deletions, rarely duplications and point mutations can occur almost
anywhere in the gene, which makes the molecular diagnosis difficult.
Here we present a comprehensive study of a large portion of the
Hungarian DMD/BMD families using different molecular approaches .
Deletions in the hot spots regions were identified by multiplex PCR,
whereas rare deletions and duplications were detected by Southern
blot analysis and Multiplex Ligation-dependent Probe Amplification
(MLPA) technique in the patients . Moreover, the same techniques were
used for detecting carrier status in female relatives and in manifesting
carriers and efficiencies of the two techniques were compared.
Here we report the genetic results of 121 affected males and 95 female
relatives. The DMD/BMD disease was confirmed in 77 males using
multiplex PCR . With Southern blot analyses and later on, by MLPA
rare exon deletions were detected in 7 male patients, whereas duplications
were observed in 5 cases . Thus, the overall deletion frequency
was 69% in the Hungarian DMD/BMD patients . Out of the 95 female
samples analysed by Southern blot and MLPA techniques, 41 female
relatives proved to be carriers, including two duplication events and
three manifesting carriers .
With the help of this reliable new method a large portion of the Hungarian
DMD/BMD patients and their female relatives were exactly genotyped
and given a precise genetic counselling . Moreover, this opens
the perspective for participation in future therapeutic interventions,
also for the Hungarian patients .
P01.222
three mutations in the DYSF gene in a LGmD2B patient
L. González-Quereda 1 , N. de Luna 2 , J. Juan 3 , M. Rodríguez 3 , E. Gallardo 4 , E.
Tizzano 3 , I. Illa 4 , M. Baiget 3 , P. Gallano 3 ;
1 Ciberer, Genetics, Hospital Sant Pau, Barcelona, Spain, 2 Ciberned , Neurology,
Hospital Sant Pau, Barcelona, Spain, 3 Genetics , Hospital Sant Pau, Barcelona,
Spain, 4 Neurology, Hospital Sant Pau, Barcelona, Spain.
Mutations in the dysferlin gene (DYSF) give rice to different muscular
dystrophy phenotypes with autosomal recessive inheritance including
Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy
(MM) and Distal Anterior Compartment Myopathy (DAT) . The DYSF
gene, which maps to chromosome 2p13, has 55 exons and codifies a
protein of about 237 kDa . Given that dysferlin is expressed in peripheral
blood monocytes in addition to skeletal muscle, we performed the
screening of mutations in the DYSF gene by sequencing monocytes
cDNA . This strategy improves the isolation of the mRNA as a source
that is less invasive than the muscle biopsy .
In parallel, we analysed dysferlin expression by immunohystochemistry
in muscle biopsies and by Western blot in peripheral blood monocytes
.
We report here an sporadic case of LGMD2B, presenting a reduced
staining in inmunohistochemistry and Western Blot analyses using
anti-dysferlin antibodies .
The mutational screening revealed the presence of three mutations:
1) A nonsense mutation located in exon 34: c.3805 G>t;
p.Glu1269X.
2) A missense mutation located in exon 44: c.4820 t>c ;
p.ile1607thr.
3) A splice site mutation located in intron 21: c.2055+1 G>A, a mutation
not described to date .
The independent sequencing of the two allelles will enable us to determine
the distribution of the mutations . The distribution of the three
mutations could account for the reduced expression of dysferlin in
muscle biopsy and monocytes in contrast to the total absence in dysferlinopathy
patients .
P01.223
Genotype and phenotype studies of myotonic dystrophy type 1
(Dm1) in Hungarian patients
A. Herczegfalvi 1 , H. Piko 2 , H. Merkli 3 , R. Horvath 4 , V. Karcagi 2 ;
1 Bethesda Children’s Hospital, Dept. of Neurology, Budapest, Hungary, 2 National
Institute of Environmental Health, Dept. of Molecular Genetics and
Diagnostics, Budapest, Hungary, 3 Medical University of Pecs, Dept. Clinic for
Neurology, Pecs, Hungary, 4 Jahn Ferenc Hospital, Dept. of Neurology, Budapest,
Hungary.
Dystrophia myotonica type 1 (DM1) is a diffuse systemic disorder and
is inherited as an autosomal dominant trait with a variable penetrance .
An unstable expansion of (CTG)n repeats in the 3’ untranslated region
encoding a member of the protein kinase family in 19q13 .3 is
the causative mutation for myotonic dystrophy . Healthy individuals harbour
5-37 CTG repeats, whereas in affected individuals repeat expansion
varies between 37 and 4000 . To examine the correlation between
clinical expression and CTG trinucleotide repeat length, Southern blot
analyses using probe p5B1 .4 were carried out in families clinically diagnosed
with myotonic dystrophy . So far, 61 patients and 15 family
members from 47 families were analysed and in 34 cases the mutation
in DMPK gene was confirmed. The expanded CTG repeats were
transmitted maternally as well as paternally . In the maternally transmitted
cases the expanded fragment lengths were always larger than
in the paternally transmitted ones . Moreover, a clear correlation was
established between phenotype severity and the length of the CTG ex-