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

Molecular and biochemical basis of disease
P0852. Mutations in the MECP2 gene in patients of Slavonic
origin with Rett syndrome
D. Zahorakova 1 , A. Baxova 2 , J. Zeman 1 , P. Martasek 1 ;
1 Department of Pediatrics, First School of Medicine, Charles University, Prague,
Czech Republic, 2 Institute of Biology and Clinical Genetics, General University
Hospital, Prague, Czech Republic.
Rett syndrome is an X-linked dominant neurodevelopmental disorder
affecting almost exclusively females. It has an incidence of 1:10,000
female births and is characterized by apparently normal development
for the first 6-18 months of life followed by the loss of acquired motor
and language skills, autistic features, and development of stereotypic
hand movements. Rett syndrome is caused primarily by de novo mutations
in the methyl-CpG-binding protein 2 gene (MECP2). Methyl-
CpG-binding protein 2 plays important roles in the regulation of gene
expression and RNA splicing. Here we report the mutational analysis
of the MECP2 gene in 95 patients from the Czech and Slovak Republics
and the Ukraine with classical Rett syndrome. The patients, all
girls, were screened for mutations by DNA sequencing of the entire
coding sequence and the exon/intron boundaries of the MECP2 gene.
RFLP analysis was performed to confirm the mutations that cause the
creation or abolition of the restriction site. Mutation-negative cases
were subsequently examined by multiple ligation-dependent probe
amplification (MLPA) to identify large deletions. Sequencing analysis
revealed 33 different mutations in 74 patients, and MLPA analysis revealed
large deletions in two patients. The detection rate was 78%.
One mutation in exon 1 (c.48_55del8) has not been published yet. Our
results confirm the high frequency of MECP2 mutations in females with
Rett syndrome and provide data concerning the mutation heterogeneity
in the Slavonic population. The project was supported by grants
MSM0021620849 and IGA 8355-3.
P0853. Large genomic rearrangements in the MECP2 gene in
Polish patients with Rett syndrome
E. Popowska, D. Jurkiewicz, E. Ciara, M. Borucka - Mankiewicz, P. Kowalski,
D. Piekutowska - Abramczuk, M. Gajdulewicz, K. Spodar, K. Chrzanowska, A.
Tylki - Szymanska, M. Krajewska - Walasek;
The Children’s Memorial Health Institute, Warsaw, Poland.
Rett syndrome (RTT, MIM#312750) is a neurodevelopmental disorder
affecting almost exclusively females with prevalence estimated to
be 1 in 10000-15000 live births. In affected girls a period of apparently
normal development is followed by mental retardation, loss of
acquired skills, stereotypical hand movements, seizures, scoliosis and
autonomic dysfunction. The disease is caused by mutations in the Xlinked
MECP2 gene encoding methyl CpG binding protein (MeCP2).
The gene contains four exons and produces two transcripts consisting
of exons 2, 3 and 4 (MeCP2A isoform) or exons 1, 3 and 4 (MeCP2B
isoform). To date, more than 370 various MECP2 mutations have been
found. Of 140 Polish patients with classic form and variants of RTT, 57
harbor point mutations and small deletions or insertions in MECP2 exons
3 and 4. Recently, several patients with mutations involving exon
1 have been reported.
The aim of our study was to identify large genomic rearrangements
in the MECP2 gene in Polish RTT patients. Molecular analysis was
performed in forty-six patients, who had been tested negative for mutations
by SSCP and sequencing of MECP2 exons 2, 3 and 4. Patients’
genomic DNAs were screened for large deletions/duplications by multiplex
ligation-dependent probe amplification (MLPA) using SALSA
P015C kit (MRC-Holland). Large deletions including promoter region
and exon 1 were identified in three patients, and deletions covering
exon 4 and, probably, exon 3 in two patients. Besides, partial deletion
of exon 4 was observed in one RTT girl.
The study was supported by MNiI Project 2P05A12129.
P0854. Monosomy of the X chromosome in primary and
overlapping autoimmune diseases.
O. M. Mutchinick, Y. Svyryd, G. Hernández, N. Monroy, J. Sánchez;
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México,
Mexico.
The significant female predominance of autoimmune diseases (AID)
has been associated to acquired X-chromosome monosomy (XCM)
and foetal cell microchimerism (MC). However, this characteristic was
not investigated in overlapping AID like Reynolds syndrome (RS). Rationale:
RS show higher rate of XCM than single AID. To address this
22
hypothesis we studied women with RS (12), CREST syndrome (13),
Primary Biliary Cirrhosis (PBC) (12) and a control group paired by sex
and age (24).
Methods: after signed consent, information on male pregnancies,
transfusions, abortions and older brothers in the sibship of index
cases, 5 ml of whole blood was obtained for lymphocytes cultures,
FISH studies in interphase nuclei (IN) and DNA extraction for Q-PCR
of SRY gene sequences. CEP-X spectrum green probe was used for
MX analysis, and CEP-15 spectrum orange as control. Samples were
blindly assessed and 500 IN were analysed in each subject.
Results: Median ages in the different groups were quite similar (59-
61). Proportion of MX IN were 14% (13.1-14.8); 11.5% (10.5-12.1);
11.3% (10.7-12.3) and 7.2% (6.8-7.7) for RS, CREST, PBC and controls
respectively. Chi-square comparisons of the amount of MX cells,
showed significant statistical differences (SSD) between each AID and
controls, between RS and CREST, and RS and CBP. Q-PCR of SRY
sequences discarded MQ as a confounding factor.
Conclusions: Our results confirm that MX is significantly increased in
AID respect healthy controls, that MQ is not an associated cause and
that probably an additive effect could explain the SSD between RS and
the other two AID.
P0855. A real time PCR assay to detect causative mutations of
the ryanodine receptor gene
I. Valášková 1 , E. Flodrová 1 , R. Gaillyová 1 , Š. Prášilová 1 , P. Kuglík 1,2 ;
1 University Hospital Brno, Department of Medical Genetics, Brno, Czech Republic,
2 Masaryk University, Faculty of Science, Department of Genetics and
Molecular Biology, Brno, Czech Republic.
The ryanodine receptor (RYR1) is an essential component of the calcium
homeostasis of the muscle in mammals. Defects in the RYR1
gene in humans (chromosome 19q13.1) are associated with malignant
hyperthermia (MH), life-threatening and frequently fatal disorder
triggered by commonly used anesthetics. Susceptibility to MH (MHS),
dominantly inherited predisposition to MH, is diagnosed by using an
invasive diagnostics test on excised muscle bundles, the in vitro contracture
test (IVCT). This test is based on the differential contractile
response of normal and MH muscle to caffeine and halothane.
Molecular genetic screening plays an integral role in the diagnosis of
MHS. Genetic data provide additional diagnostic information or contribute
information independent of IVCT. Genetic screening is very difficult
due to low incidence of each mutation and the vatness of the
RYR1. Until now, 23 RYR1 mutations causing MH have been listed by
European MH Group. A detection MH causative RYR1 mutations can
be used in predictive genetic testing. We describe an assay that allows
analysis of RYR1 genotypes using real time PCR. We provide a test
based on melting point analysis of fluorscently labelled probes after
high speed PCR amplification. After nucleic acid extraction from the
white blood cells, or muscle cells RYR1 mutation can be detected in
less than 1hour. This assay is cheaper and faster than traditional one
used to date. Our RYR1 mutation analysis protocol currently screens
for the most common RYR1 mutations in Czech population. Our aim is
to develop analysis test for all published MH causative mutations.
P0856. Characterization of COX16, a novel human COX
assembly gene
L. Salviati1 , E. Trevisson1 , A. Casarin1 , M. Doimo1 , S. Sacconi2 ;
1 2 Dept. of Pediatrics, Padova, Italy, Dept. of Neurology, University of Nice,
France.
COX16 is a gene essential for cytochrome c oxidase (COX) biosynthesis
in yeast.. We have now cloned and characterised its human
homologue.
The human gene was identified in the expressed sequence tags (EST)
database using a cyberscreening method.. Human COX16 is comprised
of 4 exons on chromosome 14 and encodes a 106 aa protein
with a N-terminal putative transmembrane domain and a C-terminal
hydrophilic domain, with no homology to any known family of proteins.
The gene is expressed ubiquitously with highest levels in muscle,
heart, and liver. We found multiple transcription initiation sites, the
most upstream of which is located 100 bp from the ATG.
Cox16 protein is located in mitochondria, presumably in the mitochondrial
inner membrane.
Silencing of the COX16 transcript by siRNA caused a significant reduction
of COX activity after 48 hours in transfected cells.