2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
(1989) .Family History, Electroencephalography (EEG) recordings and
CT Scan were obtained from most patients . The study was approved
by the Iranian institutional ethics committees . DNA was obtained from
50 unrelated families with idiopathic generalized epilepsy as index
families . The 26 exons of SCNIA were Screened for deletions and duplications
by MLPA. In order to finding point mutations and SNPs each
exon individually amplified from genomic DNA in PCR reactions using
intronic primers and were analyzed by Single Strand conformation
Polymorphism gel electrophoresis (SSCP) and conformation-sensitive
gel electrophoresis(CSGE) and so the PCR products with mobility variants
were sequenced by ABI sequencer. We have identified some new
intronic variants in SCNIA and new mutations too, in patients with IGE
subtypes .
Allele and genotype frequencies in the patients and in the control
groups were compared Statisticaly
P05.096
molecular genetic analysis of SCN B gene in Russian patients
with idiopathic ventricular arrhythmias
E. Ivanova1 , H. Zaklyazminskaya2 ;
1Russian Research Center for Medical Genetics, Moscow, Russian Federation,
2Center of Molecular Genetics, Odintsovo, Russian Federation.
Cardiac channelopathies is a new class of diseases characterized by
high risk of life-threatening arrhythmias and sudden cardiac death with
apparently normal heart . Mutations in more than 10 different genes
can cause these diseases . For different disorders within this group
approximately 25-60% patients remain genotype negative . Recently, a
new gene SCN4B encoding β4-subunit of Na 1 .5 was shown in Long
v
QT Syndrome (LQTS) family . The aim of this work is to screen mutation
in SCN4B gene in patients with idiopathic ventricular arrhythmias and
to estimate the prevalence of genetic alterations in this gene in Russian
patients . The DNA samples of 48 unrelated patients (mutations in
KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 had been excluded)
with ventricular arrhythmias had been analyzed . Fourteen patients had
LQTS, 14 patients had Brugada Syndrome (BrS) and 20 patients had
Idiopathic Ventricular Tachycardia (IVT) . Molecular genetic testing was
performed by PCR-SSCP analysis with following direct sequencing of
abnormal conformers . We did not found any disease-causing mutation
in SCN4B gene . We suppose that SCN4B-associated arrhythmias are
extremely rare in Russian patients with LQTS, BrS and IVT . Polymorphism
c . C174T (p . C58C) in exon 2 was found within group of patients
with BrS . The prevalence of this SNP among the patients with BrS
and control group (100 unrelated unaffected individuals) was about
7% without significant differences. Thus, it’s unlikely that c. C174C can
significantly modify Na + -channel function . By now, routine analysis of
SCN4B gene for patients without mutations in KCNQ1, KCNH2, SC-
N5A, KCNE1 and KCNE2 seems to be inexpedient .
P05.097
CS expression in infertile men correlates with the efficiency of
the spermatogenic process
S. Bonache1 , E. Terribas1 , J. Sánchez2 , L. Bassas2 , S. Larriba1 ;
1Medical and Molecular Genetics Center-Fundació IDIBELL, BARCELONA,
Spain, 2Andrology Service-Fundació Puigvert, BARCELONA, Spain.
Germ cells use mitochondrial Krebs cycle (KC)/electron transport system
and glycolysis for the maintenance of their ATP concentration, but
each stage of spermatogenesis rely on one or other pathway for ATP
production . Interestingly, spermatocytes and spermatids RNA and protein
synthesis is highly dependent on mitochondria as the major ATP
supplier .
Citrate synthase (CS) is the first enzyme in the KC. We have assessed
the role of KC in the progression of spermatogenesis by the CS gene
expression analysis in testicular biopsies of 39 non-obstructive and
20 obstructive infertile men. Samples were classified into four groups,
from 1 (total absence of germ cells) to 4 (conserved spermatogenesis),
on the basis of the Johnsen score count .
The current method has been the real time RT-PCR [LightCycler
Instrument (Roche) and SYBR Green I fluorescence dye] and the
relative quantification strategy, using cyclophilin-A as an endogenous
control gene . The Mann-Whitney U test was used to analyse gene
expression differences between groups .
CS transcript levels positively correlated with all germ cell stages number
but a negative correlation was found with Sertoli cells supporting
the greater contribution of germ cells to CS transcription . Transcript
levels of CS were significantly decreased in spermatogenic failure-affected-samples
(p=0.01), suggesting to play a role in the efficiency of
spermatogenesis. Additionally, its significant correlation with the elongated
spermatid number may indicate that CS has an active transcription
in the latest stages of spermatogenesis with potential implications
in the metabolic activity of sperm .
Supported by FIS (PI02/0120, PI05/0759, CP03/00088, CA06/0055)
P05.098
Differential susceptibility of imprinting centers to epimutations
under DNA demethylation influence
E. A. Sazhenova1 , I. N. Lebedev1 , A. V. Eremeev2 , A. V. Svetlakov2 ;
1 2 Institute of Medical Genetics, Tomsk, Russian Federation, Center of Reproductive
Medicine, Krasnoyarsk, Russian Federation.
Genomic imprinting is an epigenetic phenomenon, which is involved in
regulation of embryonic development, placental function and neurobehavioural
processes . Changes in differential methylation of imprinting
control (IC) regions are one of the possible mechanisms leading to
aberrant gene inactivation or loss of imprinting associated with some
hereditary diseases and cancer . However, incidence of epimutations
in different imprinted loci as well as underlined mechanisms of its arising
remains elusive . Previously we have reported about differential
susceptibility to epimutations of three IC’s in spontaneous abortions
and described the loss of methylation of KCNQ1OT1 in 9 .5% of embryos
. The aim of the present research was analysis of methylation
stability of previously investigated IC’s (SNURF-SNRPN, H19/IGF2,
KCNQ1OT1) under influence of DNA demethylating agent such as 5aza-2-deoxycytidine
in vitro. Fetal fibroblasts cell cultures from 17 induced
abortions (9 .6±0 .2 weeks of pregnancy) with normal karyotype
were treated by 5-aza-2dC with 5 μg/ml for 72 h. Methylation analysis
was performed by methyl-specific or methylation-sensitive PCR. A normal
differential methylation of SNURF-SNRPN and H19/IGF2 was observed
in all cell cultures after 5-aza-2dC treatment . As to KCNQ1OT1,
all cell cultures have revealed a loss of methylation in maternal allele
. Loss of imprinting in KCNQ1OT1 was observed in our previous
research of spontaneous abortions as well as in most children born
after IVF procedures with Beckwith-Wiedemann syndrome reported in
literature . Our results indicate a different susceptibility of IC’s to epimutations,
providing evidence for KCNQ1OT1 as a «hot spot» of aberrant
epigenetic modifications in the human genome.
P05.099
Infantile neuroaxonal dystrophy: challenges for identification
and new advances in prenatal diagnosis
W. Lissens 1 , G. Buyse 2 , J. Misson 3 , L. Lagae 2 , C. Ceuterick-De Groote 4 , R. Van
Coster 5 , E. Schmedding 1 , S. Seneca 1 , L. De Meirleir 1 ;
1 University Hospital UZ Brussel, Brussels, Belgium, 2 University Hospitals K.U.
Leuven, Leuven, Belgium, 3 CHU Sart-Tilman, Liège, Belgium, 4 Born-Bunge
Institute, Antwerp, Belgium, 5 University Hospital Ghent, Ghent, Belgium.
Infantile neuroaxonal dystrophy (INAD; OMIM #256600) is a rare disease
belonging to the group of neurodegenerative disorders together
with NBIA (neurodegeneration with brain iron accumulation, PANK2
gene, OMIM #234200), KARAK syndrome and Schindler disease
(OMIM #609241) . These diseases share the pathologic feature of axonal
degeneration with distended axons throughout the central nervous
system . In previous work there has been some controversy regarding
whether INAD is a separate entity or is part of a spectrum of diseases
with panhothenate kinase-associated neurodegeneration (PANK) .
Based on molecular studies of the PANK2 gene it became clear that
INAD and PANK are genetically heterogeneous disorders . Recently,
a gene PLA2G6, encoding a phospholipase A2, has been recognized
as causing INAD . However, there is still some overlap in phenotypes
resulting from deficiencies in the PLA2G6 and PANK2 genes. We present
here the clinical features, diagnosis and molecular results of four
patients from three unrelated families in whom a definitive diagnosis
could be made by molecular analysis .
In all four patients clinical data and biochemical studies were in agreement
with a diagnosis of neuroaxonal dystrophy . Molecular studies of
the PLA2G6 were performed to confirm this diagnosis. The two male
siblings from family 1 were homozygous for a p .Val371Met substitution;
there was no apparent consanguinity in this family . A healthy carrier
female was born after prenatal diagnosis through chorionic villus
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