2009 Vienna - European Society of Human Genetics

Molecular and biochemical basis of disease
tations are duplications (6%) and point mutations (34%). Deletions
and duplications causing frame shift result in the more severe DMD,
whereas mutations maintain the reading frame cause the milder BMD.
Deletions and duplications are focused in two “hot spots”. Point mutations
are scattered within whole DMD gene and difficult to identification.
The most efficient method of deletion and duplication screening is
MLPA technique, which allows identifying 100% of this kind of mutation
and upgraded routine molecular diagnostics of this disease. Additional
advantage of MLPA is possibility of carrier status determination. To
find a point changes we use electrophoretic screening techniques as
single stranded conformers polymorphism, heteroduplex analysis and
sequencing to identifying. In our studies we analyzed 150 patients with
progressive muscular dystrophy from Great Poland and Silesia region.
DNA from 72 patients was analyzed by MLPA and remaining 78 by
PCRmultiplex. Summarising, we identify 69 cases of deletion, 14 duplication
and 3 point mutation. We did not observed deletion and amplification
of whole gene. Deletions and duplication included only one
exon occurred in 32% of patient. In central part of a gene appeared
57% of this mutation and in proximal 43%. MLPA technique improoved
significantly effeciveness of molecular diagnostics of DMD/BMD and
allows to identificate of carriers.
P16.50
the novel mutation in CLCN gene resulting in recessive form of
the myotonia congenita.
E. Ivanova1 , V. Fedotov2 , S. Kyrbatov2 , A. Polyakov1 ;
1Russian Research Center for Medical Genetics, Moscow, Russian Federation,
2Center of Genetic Consultation, Voronezh, Russian Federation.
Myotonia congenita (MC) is a hereditary muscle disorder characterized
by delayed relaxation of skeletal muscle after voluntary contraction
(myotonia). MC caused by mutations in the skeletal muscle chloride
channel gene CLCN1 (7q35). The phenotypic spectrum of myotonia
congenita is very diverse from mild myotonia detected only by clinical
examination to severe myotonia with transitory weakness (TW) and
myopathy especially in recessive cases. TW correspondents with a
transitory depression (TD) of the compound muscle action potential
(CMAP) during repetitive nerve stimulation (RNS). We analyzed 35
families with revealed myotonia by clinical examination in dominant or
recessive forms and detected 11 mutations in 14 families. Two of these
mutations detected in several unrelated families: c.1436_1449del in
exon 13 of CLCN1 gene and p.493Ala>Glu. The last mutation is more
interest. This novel missense mutation is detected in compound with
c.1436_1449del in one patient and in gomozygote in two out of three
sibs in another family. Clinical symptoms of affected sibs are moderate
myotonia of masseter, transitory weakness in hands and “warm-up”
effect, hypertrophy of calf, no exacerbation with decrease of temperature,
the myotonia of lid is absent, taking of alcohol provoke the alleviation
of symptoms. The myotonia don’t progress in time (now sibs
are 36 and 40 years old). At the same time this mild form of myotonia
caused by mutation 493Ala>Glu in homozygote combines with high
level of decrement CMAP RNS (70 and 78%). By now, routine analysis
of CLCN1 gene for patients with myotonia is going on.
P16.51
molecular diagnosis of myotonic dystrophy type i in Egyptian
patients
I. Somaia1 , H. Radwan1 , H. Hosny1 , D. Helmy2 , E. Salah2 , L. Effat1 ;
1 2 National research Centre, Cairo, Egypt, Department of Clinical pathology,
Faculty of Medicine, Ain Shams University, Cairo, Egypt.
The dominantly inherited myotonic dystrophy or myotonia dystrophica
(DM) disease belongs to a group of neurodegenerative disorders that
results from the expansion of unstable trinucleotide repeats. In these
disorders, the number of repeats increases to a critical size, affecting
the gene function and leading to disease. The DM disorder has a
peculiar and rare pattern of multisystemic clinical features, affecting
skeletal muscles, heart, eye and endocrine system. The underlying
molecular basis of the disease entails excessive expansion of a repetitive
sequence in the myotonin protein Kinase gene.
The aim of this study was to employ a molecular diagnostic technique
for the disease among suspected Egyptian cases.
Subjects and Methods: Nine probands complaining of clinical and neurophysiological
features suggestive of myotonic dystrophy diagnosis
were studied. Molecular studies were performed on the nine probands
and their available family members. Genomic leukocytic DNA samples
were extracted using a salting out technique. A polymerase chain reaction
primer pair was used to amplify the designated fragment followed
by estimation of the CTG repeat length. A modified nucleotide mixture
was used for amplification of the expanded premutated and fully mutated
CTG alleles.
Results and discussion: Five families out of the nine showed abnormal
repeat expansion. The PCR products were directly detected on 8%
ethidium bromide stained-polyacrylamide gel. The utilization of molecular
methods in DM management would not only lead to definitive
molecular diagnosis and proper genetic counseling for affected families
but may also aid in future prenatal diagnosis.
P16.52
GtG repeat polymorphism in myotonic dystrophy patients
F. Koc 1 , D. Erdogan 2 , S. Kocaturk Sel 3 , A. N. Nazli Basak 4 ;
1 Cukurova University School of Medicine, Department of Neurology, Adana,
Turkey, 2 Bogazici University, Department of Molecular Biology and Genetics,
Istanbul, Turkey, 3 Cukurova University, Department of Medical Biology and
Genetics, Adana, Turkey, 4 Bogazici University, Department of Molecular Biology
and Genetics, İstanbul, Adana, Turkey.
Myotonic dystrophy (DM) is a neuromuscular disorder with autosomal
dominant inheritance pattern. It affects various organs including skeletal
muscle, heart, brain, eye, endocrine, and gastrointestinal systems.
It is characterized with progressive muscle weakness and wasting and
difficulty in muscles relaxation after contraction (myotonia). It is caused
by an excessive number of CTG repeats at chromosome 19q13.2-13.3
region.
It has four subtypes. These are classified as; premutation, mild, classical
and congenital. These subtypes are named according to changes
in repeat numbers. In a normal person CTG repeat number can go up
to 35. Trinucleotide repeat number between 35 and 50 is not attributed
to a disease condition though they are not stabile and are inherited
from one generation to the next.
Low trinucleotide repeat numbers can be readily amplified with PCR.
High repeat numbers, however, are hard to amplify. In this study in
order to amplify alleles with high CTG trinucleotide repeat numbers, “
Triple PCR” was used.
Study group was consisted of 13 unrelated DM1 patients admitted to
the Department of Neurology in Çukurova University School of Medicine.
After the proband was diagnosed as DM1, other family members
were assessed and the diagnosis was confirmed genetically. By the
data obtained from the probands, the pedigrees were constructed.
Thirty nine members including 20 men and 19 women of 13 unrelated
DM families were analyzed for CTG trinucleotide repeat polymorphism
to diagnose the DM1 patients on a molecular basis.
Method of Triple PCR had positive results in all DM1 patients and proven
to be a reliable diagnostic tool.
P16.53
Paternal repeat length instability of myotonic dystrophy type 1
pre- and protomutations
M. M. Gerrits1 , C. E. M. de Die - Smulders1,2 , C. G. Faber1 , M. J. Blok1,2 , H. J.
M. Smeets1,2 ;
1 2 Academic Hospital Maastricht, Maastricht, The Netherlands, Research Institute
GROW, Maastricht University, Maastricht, The Netherlands.
Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder,
and the most common form of muscular dystrophy in adults. The molecular
basis of DM1 lies in the instability of a CTG repeat in the 3’
UTR of the DM1 protein kinase gene. Small expanded DM1 alleles
are usually unstable and often increase in number in successive generations.
Here we evaluated the risk on expansion of an additional
22 pre- (37-50 CTG repeats) and 41 protomutation (50-80 repeats) in
relation to sex and repeat length in DM1 transmitting parents for 63
DM1 parent-child pairs (33 males, 30 females). CTG-repeat lengths
in the parents and children were determined by PCR and Genescan
analysis. For the transmitting males, 23/33 (70%) repeats expanded to
a full mutation upon transmission, whereas 5/33 (15%) repeats were
stable and 5/33 (15%) slightly increased. For the transmitting females,
these values were 5/30 (17%), 17/30 (57%) and 6/30 (20%), respectively.
When the results were subdivided by repeat length, only one
(1/8, 13%) transmitting male gave rise to a full mutation in the offspring
in the premutation range, and 9/11 (82%), 6/6 (100%) and 7/8 (86%)