2009 Vienna - European Society of Human Genetics

Molecular and biochemical basis of disease
P16.40
Post mortem Duchenne muscular dystrophy DNA diagnostics
from archival milk teeth
C. Stein 1 , W. M. Schmidt 2 , D. Jovanovic 2 , H. Rehder 3 , R. E. Bittner 2 ;
1 Department of Forensic Medicine, Medical University of Vienna, Vienna, Austria,
2 Neuromuscular Research Department, Center of Anatomy & Cell Biology,
Medical University of Vienna, Vienna, Austria, 3 Department of Medical Genetics,
Medical University of Vienna, Vienna, Austria.
The X-linked Duchenne muscular dystrophy (DMD) remains an untreatable
lethal disease causing the premature death of affected patients
in their early twenties. DMD is caused by mutations in the highly
complex, 79 exons spanning DMD gene. Since the characterization of
the complete DMD coding sequence in the late 1980ies, thousands of
different mutations scattered throughout the gene have been reported.
If a DMD-patient has died prior to this era, the molecular basis for his
disease remained unresolved, which hampers the accurate and direct
genetic counseling of his family members, such as the establishment
of carrier-diagnosis in female relatives.
Here, we report on the successful post mortem analysis of the DMD
gene in two different cases. In both cases, we could get hold of preserved
milk teeth of the deceased patients and successfully isolated
genomic DNA thereof. Subsequently, we subjected these DNA-samples
isolated from milk teeth to multiplex ligation-dependent probe
amplification (MLPA)-based screening for whole exon deletions or
duplications within the DMD gene. In both cases we could detect the
pathogenic mutation, i.e. an out-of-frame deletion of exon 21 in one
case and an out-of-frame consecutive deletion of exons 46-48 in the
other.
In both cases reported here, the DNA samples extracted from teeth
(which had been kept for more than 20 years and more than 40 years,
respectively) were suitable for MLPA and PCR procedures and were
the biologic source for establishing the causative mutation within the
largest gene known, the DMD gene, even decades after the patient’s
death.
P16.41
comparative clinical and molecular analysis of DmD gene
deletions and duplications
E. Neagu 1 , G. Girbea 1 , A. Constantinescu 1 , C. Constantinescu 1 , D. Iancu 1 , G.
Talpes 1 , E. Manole 2 , E. Ionica 3 , N. Butoianu 4 , D. Plesca 5 , A. Todoran 6 , L. Barbarii
1 ;
1 National Institute of Legal Medicine “Mina Minovici”, Bucharest, Romania,
2 Neuropathology Clinic - Colentina Hospital, Bucharest, Romania, 3 Multiuser
Research Unit – University of Bucharest, Bucharest, Romania, 4 Neuropediatric
Clinic - “Al.Obregia” Hospital, Bucharest, Romania, 5 Pediatric Hospital “V.
Gomoiu, Bucharest, Romania, 6 Medical University Targu Mures, Bucharest,
Romania.
Deletions and duplications in dystrophin gene are known to cause
approximately 65% of Duchenne and Becker muscular dystrophies
(DMD/BMD). To detect these major DMD gene mutations multiplex ligation-dependent
probe amplification (MLPA) was applied on samples
from 107 unrelated patients with clinical diagnostic or clinical suspicion
of DMD/BMD. In our study, by comparing the major clinical and
mutational features (location, extent, predicted effect on gene reading
frame) of the 11 duplications lot and the 55 deletions lot, we have tried
to asses the usefulness of the molecular analysis for the prediction of
the clinical form severity and natural history of DMD/BMD in order to
improve their clinical management.
P16.42
mutation-associated exon skipping pinpoint localization of
exonic splicing regulatory elements in the DmD gene
S. Le Guedard-Mereuze1 , D. Thorel2 , D. Méchin2 , C. Saquet2 , J. Miro3 , P. Khau
van Kien2 , M. Claustres2,1,3 , S. Tuffery-Giraud1,3 ;
1Inserm U827 - Laboratoire de Génétique Moléculaire, Montpellier, France,
2CHU Montpellier - Laboratoire de Génétique Moléculaire, Montpellier, France,
3Université Montpellier 1 - Faculté de Médecine, Montpellier, France.
Mutation-associated exon skipping has been recognized in an increasing
number of genes as a novel form of splicing mutation. Not only
nonsense mutations, but also missense and even translationally silent
mutations can lead to alternative splicing events. In the DMD gene,
the identification of a mutation introducing a premature stop codon in
patients with a milder (Becker-like) phenotype than expected (Duch-
enne-like) is a pointer of such mechanisms. The rescue of the phenotype
results from the partial elimination of the truncating mutation from
dystrophin mRNA by skipping of an in-frame exon. A large fraction of
these mutations are supposed to exert their effects by disrupting the
activity of Exonic Splicing Enhancers (ESEs), but repression of splicing
may also be due to the creation of an Exonic Splicing Silencer (ESS).
Using cDNA-based mutation detection analysis, we have identified 9
novel mutations in exons 19, 29, 38, 39, 59 and 66 of the DMD gene
that lead to different extents of misspliced transcripts in Duchenne
(DMD) and Becker (BMD) patients. To further elucidate the underlying
mechanisms, disruption of cis-regulatory splicing sequences was assessed
by using the Human Splicing Finder software (http://www.umd.
be/HSF/). Seven out of the 9 mutations were found to abrogate one
or several ESE(s). An ESE-dependent splicing assay has been set up
to confirm the in silico predictions. This study illustrates how diseasecausing
mutations can contribute to shed light on new modulatory elements
important for splicing in the DMD gene, which may be important
for the exon-skipping therapeutic strategy.
P16.43
Analysis of the dystrophin gene in Duchenne muscular
dystrophy patients from Bashkortostan Republic of Russia
I. Khidiyatova1 , I. Gilyazova1 , I. Khidiyatova2 , R. Magzhanov2 , E. Khusnutdinova1
;
1 2 Institute of Biochemistry and Genetics, Ufa, Russian Federation, Bashkir
State Medical University, Ufa, Russian Federation.
Duchenne muscular dystrophy is caused by mutations in the dystrophin
gene (DMD) consisting of 79 exons. It is known that 25%-75%
of all DMD gene mutations in different populations are huge deletions
covering one and more exons and located in 2 hot points - 5’ (6-19
exons) and 3’ (40-53 exons) regions. More than 6% of mutations are
large duplications, the rest are point mutations.
Using the multiplex PCR we studied mutations spectrum of twenty exons
(3, 4, 6, 8, 13, 17, 19, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
60) and promoter region of DMD gene in patients from 60 unrelated
families, living in Bashkortostan Republic (the territory of the Southern
Urals). Exon deletions of the gene were revealed in 31.75% of families
- 30% in 5’-region, 70% - in 3’-region. SSCP-analysis and consequent
sequencing of DNA samples without large deletions revealed 2 new
point mutations not described before and 1 polymorphism. In 3 patients
from 2 families of Russian ethnic origin we found c.401_404delCCAA
(p.Thr134ThrfsX7) in the 6th exon; it’s frequency is 3.17% among unrelated
patients. Mutation c.6626delA (p.Lys2210ArgfsX11) (1.6%) was
revealed in 46th exon in 1 patient in Tatar ethnic origin. We revealed
mutation c.7728T>C (Asn2575Asn) in the 53th exon, the functional significant
of which is unknown. Thus, specific mutation spectrum of DMD
gene was found in patients from Bashkortostan Republic that is very
important for analysis of structural and functional features of the gene
and for optimal DNA-diagnostics approaches development.
P16.44
mRNA-based analysis of point mutations in DmD gene in a
selected cohort of spanish patients
J. Juan Mateu1,2 , M. Rodriguez1,2 , M. Moragues3 , L. Gozalez-Quereda1,2 , M. J.
Barcelo1,2 , J. Colomer4 , A. Nascimento4 , E. Tizzano1,2 , P. Gallano1,2 ;
1 2 Genetics Dept Hospital Sant Pau, Barcelona, Spain, CIBERER, Spain,
3 4 CIBERNED, Barcelona, Spain, Neurology Dept Hospital Sant Joan De Déu,
Barcelona, Spain.
The most common form of Duchenne and Becker muscular dystrophies
causing mutations are large intragenic deletions and duplications
that account for 60 to 70% of all cases. The remaining cases are
due to small mutations consisting in nonsense mutations, missense
mutations, splicing mutations, frameshift small deletions or insertions
and midintronic insertions. The detection of these small mutations in
routine diagnosis have long been a difficult issue due to the complexity
and large size of the DMD gene. In order to identify these mutations we
have chosen a previously described method based on whole sequencing
of mRNA from muscle biopsy.
To validate the method, we studied a selected cohort of nine unrelated
patients with clinical and immunohistochemical data compatible
with distrophinopathy and without deletions or duplications in the DMD
gene. A total of new seven mutations were found, six consisting in different
nonsense mutations and the remaining consisting in a acceptor