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

Molecular and biochemical basis of disease
initiating SA or that thrombophilia itself plays a role of initial predisposition
factor in production of APA.
P0797. Mitochondrial DNA haplogroups in infertile males
M. Houshmand 1 , M. Shafa Shariat Panahi 1 , F. M.A Mohammad 2 , L. Bastaki 3 , K.
Naguib 4 , A. Olivieri 5 , B. Hooshiar Kashani 1 , V. Khalili 1 ;
1 National Institute for Genetic Engineering and Biotechnology(NIGEB), Tehran,
Islamic Republic of Iran, 2 Kuwait Medical Genetic Centre, Maternity Hospital,
Kuwait, Kuwait, 3 Faculty of Allied Health Sciences, Kuwait University,, Kuwait,
Kuwait, 4 Faculty of Allied Health Sciences, Kuwait University, Kuwait, Kuwait,
5 Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy.
A variety of mtDNA mutations responsible for human diseases have
been associated with molecular defects in the OXPHOS system. It has
been proposed that mtDNA genetic alterations can also be responsible
for sperm dysfunction. To investigate any possible association between
infertility and mtDNA haplogroups (hg), the nucleotide sequence
of the Hypervariable Segment I (HVS-I) of mtDNA was determined in
99 unrelated Kuwaiti patients with infertility and 54 normal controls with
the same ethnicity. DNA was extracted from the peripheral blood after
having obtained informed consent. The nucleotide sequence of HVS-
I (np 16,024-16,383) was directly determined. High-resolution RFLP
analysis and control-region sequencing revealed high proportion of
haplogroup J and M in normal controls (64% and 20.3%) compared to
infertile men (26.2% and 8.1%) respectively. (P= 0.002 for J and 0.03
for M) Therefore, we hypothesize that individuals classified as haplogroup
J and M demonstrate a significant decrease in risk of infertility
in Kuwaiti population.
P0798. Mutational analysis of the mitochondrial tRNA Leu(UUR) gene
in Tunisian patients with mitochondrial diseases
E. Mkaouar-Rebai 1 , A. Tlili 1 , S. Masmoudi 1 , N. Belguith 1 , I. Charfeddine 2 , M.
Mnif 3 , C. Triki 4 , F. Fakhfakh 1 ;
1 Laboratoire de génétique moléculaire humaine. Faculté de Médecine de Sfax,
Sfax, Tunisia, 2 Service d’O.R.L., C.H.U. Habib Bourguiba de Sfax, Tunisia,
Sfax, Tunisia, 3 Service d’endocrinologie, C.H.U. Habib Bourguiba de Sfax, Tunisia.,
Sfax, Tunisia, 4 Service de Neurologie, C.H.U. Habib Bourguiba de Sfax,
Tunisia., Sfax, Tunisia.
The mitochondrial tRNA Leu(UUR) gene (MTTL) is a hot spot for pathogenic
mutations that are associated with mitochondrial diseases with
various clinical features. Among these mutations, the A3243G mutation
was associated with various types of mitochondrial multisystem
disorder, such as Mitochondrial Inherited Diabetes and Deafness
(MIDD), Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and
Stroke like episodes (MELAS), Myoclonus Epilepsy with Ragged Red
Fibres (MERRF), maternally inherited Progressive External Ophthalmoplegia
(PEO), hypertrophic cardiomyopathy, and a subtype of Leigh
syndrome.
We screened 128 Tunisian patients presenting various mitochondrial
diseases (MIDD, diabetes, hearing loss, Leigh Syndrome, cardiomyopathy,
metabolic encephalopathy…) for the A3243G mutation in the
mitochondrial tRNA Leu(UUR) gene. This screening was carried out using
PCR-RFLP with the restriction endonuclease ApaI. None of the 128
patients or the 100 controls tested were found to carry the mitochondrial
A3243G mutation in the tRNA Leu(UUR) gene in homoplasmic or heteroplasmic
form. After direct sequencing of the entire mitochondrial
tRNA Leu(UUR) gene and a part of the mitochondrial NADH dehydrogenase
1, we found no mutations or polymorphisms in the MTTL1 gene
in the tested patients and controls and we confirmed the absence of
the A3243G mutation in this gene. We also found a T3396C mutations
in the mitochondrial ND1 gene in one family with nonsyndromic hearing
loss. This substitution was absent in the other patients and in 100
normal individuals. No polymorphisms or other mutations were found
in the mitochondrial tRNA Leu(UUR) gene in the tested patients.
P0799. Aberrant splicing is a common mutational mechanism in
MKS1, a key player in Meckel Gruber syndrome
V. Frank 1 , N. Ortiz Brüchle 1 , S. Mager 1 , S. G. M. Frints 2 , A. Bohring 3 , G. du
Bois 4 , I. Debatin 5 , H. Seidel 6 , N. Besbas 7 , U. Todt 8 , C. Kubisch 8 , T. Grimm 9 , F.
Teksen 10 , S. Balci 11 , K. Zerres 1 , C. Bergmann 1 ;
1 Department of Human Genetics, Aachen, Germany, 2 Department of Clinical
Genetics, Maastricht, The Netherlands, 3 Department of Human Genetics,
Muenster, Germany, 4 Institute of Chromosomal Diagnostics and Genetic Counselling,
Böblingen, Germany, 5 Department of Human Genetics, Ulm, Germany,
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6 Department of Human Genetics, München, Germany, 7 Department of Pediatric
Nephrology, Ankara, Turkey, 8 Institute of Human Genetics, Cologne, Germany,
9 Department of Human Genetics, Würzburg, Germany, 10 Department of Basic
Health Sciences and Biotechnology, Ankara, Turkey, 11 Department of Clinical
Genetics, Ankara, Turkey.
Meckel-Gruber syndrome (MKS) is an autosomal recessive, usually
lethal multisystemic disorder characterized by early developmental
anomalies of the central nervous system, cystic kidney dysplasia, hepatobiliary
ductal plate malformation and postaxial polydactyly. Three
MKS loci have been mapped and recently, two genes were identified:
MKS1 on 17q22 in Caucasian kindreds and MKS3 on 8q22 in Omani
and Pakistani families, putting MKS on the growing list of ciliary disorders
(“ciliopathies”). We performed linkage analysis for MKS1-3 in
14 consanguineous and/or multiplex families of different ethnic origins
with histologic diagnosis and at least three classic MKS manifestations
in each kindred. Unexpectedly, only five families were linked to any of
the known MKS loci, clearly indicating further locus heterogeneity. All
five families showed homozygosity for MKS1 and, intriguingly, were
of non-Caucasian origin. MKS1 sequencing revealed no mutation in
two of these pedigrees, whereas different, novel splicing defects were
identified in the three other families and an additional sporadic German
patient. Given that all of our mutations and two of the in total
four known MKS1 changes cause aberrant splicing (while the other
two known mutations were frameshift mutations), we hypothesize that
splicing defects are a crucial mutational mechanism in MKS1 which
apparently is one of the main loci and key players in MKS. Our results
indicate that MKS1 mutations are not restricted to the Caucasian gene
pool and suggest further genetic heterogeneity for MKS. Overall, our
data have immediate implications for genetic counselling and testing
approaches in MKS.
P0800. Multiple ligation-dependent probe amplification (MLPA)
analysis of the dystrophin gene in Bulgarian patients with
Duchenne/Becker muscular dystrophy (DMD/BMD)
A. Todorova 1 , T. Todorov 1 , M. Lukova 1 , B. Dworniczak 2 , V. Mitev 1 ;
1 Sofia Medical University, Medical Faculty, Department of Chemistry and Bioshemistry,
Sofia, Bulgaria, 2 Institute of Human Genetics, Muenster, Germany.
MLPA is a quantitative method for detection of deletions/duplications
of one or more exons of a gene.In more than 70% of the DMD/BMD
cases the disease causing mutations are deletions or duplications in
the dystrophin gene. The MLPA method provides a cheap and powerful
tool to screen the whole dystrophin gene in two multiplex reactions.
We analyzed 24 unrelated DMD/BMD Bulgarian families without deletions
in the dystrophin gene after multiplex PCR. We used SALSA
PO34/PO35 kit specific for the dystrophin gene. In 19 families we
found deletions or duplications. We found 12 deletions distributed in
the main deletion hotspot of the gene. These deletions include exons
42-45, 44 (2 times detected), 45-47, 45-50, 46-50, 46-52, 49-50, 53-
54, 55, 58, 79. In addition, 7 different duplications were detected, all
but two starting in the 5’-end of the gene. Duplications include exons
2-10, 2-33, 8-11, 8-13, 13-40, 48-50, 51. Carrier status was clarified
in all females at risk. MLPA failed to detect deletions or duplications
in five families where the disease causing mutation could be a point
mutation or the clinical diagnosis might need reevaluation.The present
work proved that MLPA is a powerful tool in clarifying the molecular defects
along the dystrophin gene. MLPA permits to detect carrier status
of female relatives of affected boys. Moreover, this analysis is the only
one which allows DNA testing in families where the index patient is no
more available. MLPA became a method of choice for genetic analysis
of Bulgarian DMD/BMD families.
P0801. Point mutation p.Tyr997X in exon 23 of the dystrophin
gene detected by MLPA analysis in Bulgarian DMD family (case
report)
T. Todorov 1 , A. Todorova 1 , M. Lukova 1 , B. Dworniczak 2 , V. Mitev 1 ;
1 Sofia Medical University, Medical Faculty, Department of Chemistry and Bioshemistry,
Sofia, Bulgaria, 2 Institute of Human Genetics, Muenster, Germany.
We introduced and optimized for diagnostic purposes the new MLPA
method. The SALSA PO34/PO35 kit specific for deletions/duplications
detection along the dystrophin gene was applied to screen Bulgarian
DMD/BMD patients. The mathematical calculations were performed
with Excel program. In 19 Bulgarian families we found 11 different deletions
and 7 different duplications in the dystrophin gene.In one of