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

Molecular and biochemical basis of disease
P0651. Androgen insensitivity syndrome: mutations in exon 3 of
AR gene in patients with partial and mild forms
C. Vretos, E. S. Ramos;
University of São Paulo, Ribeirao Preto, Brazil.
Androgen insensitivity syndrome (AIS) is one of the entities of absent
or deficient masculinization in individuals 46, XY. It can present
a clinical spectrum [complete (normal female phenotype), partial (P)
or frankly ambiguos, and mild (M) or normal or near-normal male], depending
on alterations of the androgen receptor gene (AR), located on
chromosome Xq11-12. The objective of the present study was to evaluate
patients referred to the University Hospital, School of Medicine
of Ribeirão Preto (USP), with a clinical diagnosis of AIS. The medical
records of 31 patients were reviewed, and 14 patients with a probable
diagnosis of AISP and eight with AISM were submitted to cytogenetic
analysis and studied by PCR-SSCP, for each of the eight exons of
the AR gene. One novel point mutation not previously reported in the
literature involving exon 3 was detected in an AISP patient (Met623Ile).
Three other point mutations previously described in the same exon
were identified: one patient with AISP (Arg607Gln), one patient with
AISM (Gly589Gln), and one patient with AISM (Tyr602Pro). Based on
these results, it was verified that it is possible to systematically employ
SSCP-PCR as a very useful screening tool of patients referred to medical
services, for the confirmation of the etiopathogeny of the disease,
the clinical management of patients and genetic counseling.
P0652. Genotyping of patients with arrhythmias
A. Bittnerová 1 , J. Kadlecová 1 , T. Novotný 2 , O. Toman 2 , J. Špinar 2 , R. Gaillyová
1 , P. Kuglík 1,3 ;
1 University Hospital Brno, Department of Medical Genetics, Brno, Czech
Republic, 2 University Hospital Brno, Department of Cardiology, Brno, Czech
Republic, 3 Masaryk University, Faculty of Science, Department of Genetics and
Molecular Biology, Brno, Czech Republic.
Electrical heart disorders are caused by problems with the electrical
system that regulates the steady, rhythmic beat of the heart. Some
arrhythmias are dangerous and cause sudden cardiac death. Other
type of the electrical heart disorder is the Long QT Syndrome (LQTS).
LQTS is associated with two cardiac muscle ion channels: voltage-gated
K + channels and voltage-gated Na + channels. To the voltage-gated
K + channels belong I Kr and I Ks channels. I Ks channels consist of KvLQT1
(KCNQ1) as an α subunit and minK (KCNE1) as a β subunit, while I Kr
channels have HERG (KCNH2) subunit and MiRP1 subunit.
Common genetic variations might modify arrhythmia susceptibility in
the general population. Polymorphisms located within the gene coding
region can directly influence the structure of its protein product,
while others located within the gene regulatory sequence can influence
the regulation of its protein expression levels. For this reason it
is important to study polymorphisms of genes associated with LQTS
(play important role in standard heart function) in sudden cardiac death
survivors.
The mutation analysis of KCNQ1 and KCNH2 was performed using
methods multiplex-PCR, multiplex SSCP (single strand conformation
polymorphism) as the screen method, and automated sequencing.
In the group of of 20 patients (sudden cardiac death survivors), we
have identified some characteristic nucleotide polymorphisms (Y662Y,
I489I, F513F, IVS13+22G>A, IVS8+40GG>CA).
Prevalence of cardiac ion channel genes polymorphisms seems to be
much more common than hypothesized thus far.
This work was supported by grants IGA MZ CR NR/9340-3, MSM
0021622415 and by the Czech Society of Cardiology.
P0653. Identification and functional characterization of three
novel mutations in desmocollin-2 gene associated with
arrhythmogenic right ventricular cardiomyopathy
G. Beffagna 1 , M. De Bortoli 1 , A. Nava 2 , M. Salamon 1 , A. Lorenzon 1 , M. Zaccolo
3 , L. Mancuso 4 , L. Sigalotti 5 , B. Bauce 2 , G. Occhi 1 , C. Basso 6 , G. Lanfranchi 1,7 ,
J. A. Towbin 8 , G. Thiene 6 , G. Danieli 1 , A. Rampazzo 1 ;
1 Department of Biology, University of Padova, Padua, Italy, 2 Department of
Cardiothoracic-Vascular Sciences, University of Padua Medical School, Padua,
Italy, 3 Venetian Institute of Molecular Medicine, Padua, Italy, 4 Venetian Institute
of Molecular Medicine, University of Padua, Padua, Italy, 5 Cancer Bioimmunotherapy
Unit, Department of Medical Oncology, Padua, Italy, 6 Institute of Pathology,
University of Padova, Padua, Italy, 7 CRIBI Biotecnology Centre, University
of Padua, Padua, Italy, 8 Department of Pediatrics, Section of Cardiology, Baylor
College of Medicine, Houston, TX, United States.
Mutations in genes encoding desmosomal proteins have been reported
to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an
autosomal dominant disease characterised by progressive myocardial
atrophy with fibro-fatty replacement. We screened 54 ARVC probands
for mutations in desmocollin-2 (DSC2), the only desmocollin isoform
expressed in cardiac tissue. We identified three heterozygous mutations
(c.304G>A (p.E102K), c.631-10C>T and c.1034C>T (p.I345T)) in
three probands and in five family members. The two missense mutations
p.E102K and p.I345T map to the N-terminal region, relevant to
adhesive interactions. In individual carrying the c.631-10C>T mutation,
cDNA analysis failed to detect aberrant transcripts, and real-time quantitative
RT-PCR showed an approximately 50% reduction of DSC2 expression,
implicating haploinsufficiency as the operant mechanism in
this DSC2 mutation.
To evaluate the pathogenic potentials of the DSC2 missense mutations
detected in patients affected with ARVC, full-length wild-type and mutated
cDNAs were cloned in eukaryotic expression vectors to obtain a
fusion protein with green fluorescence protein (GFP); constructs were
transfected in neonatal rat cardiomyocytes and in HL-1 cells. Unlike
wild-type DSC2, the N-terminal mutants are predominantly localised in
the cytoplasm, thus suggesting the potential pathogenic effect of the
reported mutations.
Mutations in DSC2 gene in patients with ARVC provide further evidence
that many forms of ARVC are due to alterations in cell-cell adhesion.
P0654. Assay of determination of the frequency of different
desmosomal genes in Arrhythmogenic right ventricular
cardiomyopathy
V. Fressart 1 , E. Delacretaz 2 , F. Hidden Lucet 1 , F. Simon 1 , C. Coirault 3 , D.
Keller 4 , P. Cosnay 5 , R. Frank 1 , P. Charron 1 , B. Hainque 1 ;
1 Hôpital de la Pitié Salpétrière, Paris, France, 2 University Hospital, Bern, Switzerland,
3 Hôpital Bicêtre, Le Kremlin Bicêtre, France, 4 Hôpital de Bale, Bale,
Switzerland, 5 Hôpital Trousseau, Tours, France.
Background. Arrhythmogenic Right Ventricular Cardiomyopathy
(ARVC) is a rare disease characterized by fibrofatty replacement in
the right ventricular. It is a familial disease in 30-50%, with autosomal
dominant inheritance. Genes encoding desmosomal proteins were
identified as responsible for ARVC. Preliminary studies suggest that
plakophilin-2 (PKP2) is involved in about 11-47%, desmoglein (DSG2)
in 10% and desmoplakin (DSP) in 5-16% of ARVC. Mutation in Plakoglobin
gene (JUP) is reported in autosomal recessive Naxos disease.
Objectives. Our aim was to identify mutations in PKP2, DSG2, DSP and
JUP genes and estimate their prevalence in ARVC patients. Methods.
For 23 independent patients with classical ARVC, after DNA extraction
from blood samples, mutation screening analysis was performed
by direct sequencing of PKP2, DSG2, JUP and DSP genes. Results.
PKP2 gene: Eight causal mutations (3 frame shift, 2 small deletions, 1
insertion, 2 missense) and 3 additional missense variants were identified
in 10/22 index patients. DSG2 gene: three mutations were found
in 6 / 16 patients (1 frame shift and 2 missense mutations). Among 19
analysed patients, two missense mutations were identified in DSP and
none in JUP. Conclusions. We reported high prevalence of PKP2 and
DSG2 gene mutations among ARVC patients with respective frequency
of 45% and 37%. Rarely, we found mutations in DSP gene (1%)
and none in JUP gene in our population. These results highlight the
frequency of the PKP2 and DSG2 gene involved in ARVC and suggest
the interest of molecular analysis of this gene for clinical purpose.
P0655. ARX syndrome is due to a variable duplication size at the
critical region
O. Reish1 , T. Fullston2 , M. Regev1 , J. Gecz2 ;
1 2 Assaf Harofeh Medical Center, Zerifin, Israel, Women’s and Children’s Hospital,
University of Adelaide, Adelaide, Australia.
We describe a novel polyalanine tract expansion mutation of the ARX
gene, a 27-bp duplication of exon 2, which segregates in a newly identified
family.
The index case was ascertained due to psychomotor retardation,
hypotonia and myoclonic seizures with normal brain MRI. Additional
affected boys were reported in this family, which is otherwise compatible
with X linked inheritance. Using PCR and sequence analysis on
peripheral blood cell DNA, a 27-bp duplication of the critical region of
1