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

Molecular and biochemical basis of disease
role of each nucleotide change within the NF1 coding sequence, since
a significant proportion of NF1 exon 7 mutations affects pre-mRNA
splicing, by disrupting exonic splicing motifs and modifying the delicate
balance between aberrantly and correctly spliced transcripts.
P0820. The role of Nonsence-mediated RNA decay (NMD)
mechanism in modifying clinical course and mode of inheritance
of cardiac arrhythmias
E. V. Zaklyazminskaya, A. V. Polyakov, M. Kurnikova;
Russian Research Center for Medical Genetics, Moscow, Russian Federation.
Background: Nonsense-mediated mRNA decay (NMD) is an mRNA
quality-control mechanism that degrades aberrant mRNAs containing
premature translation termination codons (PTCs). The PTC mutations
in cardiac ion channels genes can realize through haploinsufficiency
due to elimination of the mutant mRNA. Thus, these mutations lead
to the presence only normal ion channel proteins on surface of the
cardiomyocyte but in depressed amount.
Methods: Blood samples were collected from 85 unrelated Russian
families with Long QT syndrome (LQTS) and from 14 Brugada syndrome
(BrS) patients. Genomic DNA and total RNA were extracted
from blood using standard methods. Mutation screening was performed
using PCR-SSCP method and direct sequencing. Results: We identified
4 PTC mutations in 18 LQT1 patients (fifteen heterozygous and 3
homozygous mutations), 1 PTC mutation in 5 related LQT2 patients
and 3 PTC mutations in 3 BrS patients. All heterozygous carriers of
PTC mutations in KCNQ1 had favorable clinical course of disease. Homozygous
carriers of PTC mutations in KCNQ1 had extremely malignant
forms. Heterozygous patients with PTC mutations in KCNH2 and
SCN5A had characterized by moderate clinical sings but SCD were
recorded in these families. Conclusions: All heterozygous carriers of
PTC mutations in KCNQ1 had mild clinical course of disease. Such
mutations in KCNH2 and SCN5A were not so propitious. Homozygous
LQT1 probands with two “favorable” mutations had unexpectedly fatal
manifestations. We speculate that haploinsufficiency of IKr- and INachannel
subunits have more serious consequences for repolarization
than the same for IKs.
This work was partly supported by Russian President’s grants NSh-
5736.2006.7; RFFI-06-04-08363-ofi
P0821. Biochemical and structural characterization of Noonan
syndrome-causing mutations affecting SHP-2’s phosphotyrosylbinding
pockets
S. Martinelli1 , P. Torreri1 , G. Bocchinfuso2 , L. Stella2 , E. Flex1 , A. Grottesi3 , G.
Chillemi3 , M. Ceccarini1 , A. Palleschi2 , B. D. Gelb4 , T. C. Petrucci1 , M. Tartaglia1 ;
1 2 Istituto Superiore di Sanità, Rome, Italy, Università “Tor Vergata”, Rome, Italy,
3 4 CASPUR, Rome, Italy, Mount Sinai School of Medicine, New York, NY, United
States.
Missense mutations in PTPN11 cause Noonan syndrome (NS), a genetically
heterogeneous developmental disorder with a pleiomorphic
phenotype. PTPN11 encodes SHP-2, an SH2 domain-containing protein
tyrosine phosphatase that relays signals from activated cell-surface
receptors to RAS. NS-causing mutations promote SHP-2’s gain of
function by either destabilizing its catalytically inactive conformation or
increasing the affinity and/or specificity of the SH2 domains for phosphotyrosyl
ligands. While the identity of substitution does not seem to
be critical in some cases, suggesting a crucial role in SHP-2’s function
for the residue being replaced, an invariant amino acid change is frequently
observed, indicating a specific role for the introduced residue.
Here, we characterized functionally and structurally two invariant NScausing
mutations, T42A and E139D, affecting residues placed in the
N- and C-SH2 pockets which mediate SHP-2 binding to phosphotyrosyl-containing
signaling partners. By analyzing in vitro biochemical
behavior (basal and ligand-stimulated phosphatase activity, and
ligand-binding properties assayed by surface plasmon resonance) of
all possible substitutions arising from a single base change affecting
codons 42 and 139, we show that T42A and E139D SHP-2 proteins
are the only mutants exhibiting a significant increase in ligand-induced
phosphatase activity and enhanced phophopeptide binding affinity.
Molecular dynamics simulations performed on selected mutants provide
structural insights of the effects generated by individual mutations
on protein function. In conclusion, this study provides functional explanation
for the invariant occurrence of the T42A and E139D mutaions in
NS as well as the molecular mechanism of their pathogenicity.
21
P0822. Cancer or not cancer : differential impact of mutations
in the XPD helicase in xeroderma pigmentosum and
trichothiosdystrophy ?
T. Magnaldo, Valérie Bergoglio, Lydia Riou, Odile Chevalier-Lagente;
CNRS, Villejuif, France.
Genetic alteration of the repair of UV-induced DNA lesions (cyclobutane
pyrimidine dimers, CPD, and 6-4 photoproducts, 6-4 PP) by nucleotide
excision (NER) may result in photo genodermatoses, such
as the cancer prone xeroderma pigmentosum (XP) syndrome or, the
cancer free trichothiodystrophie (TTD) syndrome. We aimed at comparing
the impact of mutations in the XPD gene resulting in either the
XP-D (R683W) or the TTD/XP-D (R112H) syndrome. We cultured primary
fibroblasts and keratinocytes from small skin biopsies of XP-D
and of TTD/XP-D patients. Comparative study of DNA repair kinetics
demonstrated faster and better DNA repair capacity of TTD/XP-D
cells compared to XP-D cells and hence, a shorter P53 stabilisation in
the former. In order to reproduce the 3D cutaneous architecture, we
elaborated organotypic TTD/XP-D and XP-D skin cultures. Response
to UVB (290_320 nm) irradiation in term of DNA repair and apoptosis,
was also better in TTD/XP-D XP-D than in XP-D organotypic skins.
We also assessed expression of markers of epidermal homeostatis
and revealled alteration or extinction of some epidermal differentiation
markers such as filaggrin in XP-D, but not in TTD/XP_D organotypic
skin cultures.
Altogether these results indicated that the R112H XPD mutation resulting
in the cancer-free TTD/XP-D syndrome are associated to a better
vital prognostic of cutaneous cells compared to the XPD R683W mutation
characteristic of XP-D cancer-prone syndrome.
P0823. Investigation of plasma carnitine ester profiles in a family
with homozygous and heterozygous OCTN2 deficiency
G. C. Talián 1 , K. Komlósi 1 , L. Magyari 1 , É. Nemes 2 , R. Káposzta 2 , G. Mogyorósy
2 , K. Méhes 1 , B. Melegh 1 ;
1 Department of Medical Genetics and Child Development, University of Pécs,
Pécs, Hungary, 2 Department of Pediatrics, Medical and Health Science Center,
University of Debrecen, Debrecen, Hungary.
OCTN2 is the high affinity transporter protein for carnitine uptake into
the cells. In the gene coding for this protein, slc22a5, a homozygous
844C deletion causing a V295X nonsense mutation was found in a
three-year old male Roma patient with hepatopathy and cardiomyopathy.
This kind of mutation has already been described in another Hungarian
Roma subject with primary systemic carnitine deficiency. We
measured 20 short-, medium- and long-chain carnitine esters by ESI
tandem mass spectrometry from plasma samples of the patient, his
consanguineous parents (first cousins) and siblings. The free carnitine
and all circulating carnitine esters were severely decreased in the proband.
The three heterozygous pediatric siblings in the family (2 males
and 1 female) showed also reduced carnitines levels between the normal
controls and the proband. The parents, who were also heterozygotes,
exhibited a highly similar pattern. Oral supplementation with
50 mg/kg/day dose of L-carnitine normalized the hepatomegaly, elevated
transaminases and the previous pathologic cardiac ultrasound
parameters of the proband. In the plasma samples 2 and 13 months
after the onset of carnitine treatment the free carnitine and many of
the carnitine esters increased to about half-normal level in response
to the therapy; however, some individual esters remained still much
below the controls. The data presented here show severely affected
carnitine ester metabolism besides the dramatic decrease of the free
carnitine in primary systemic carnitine deficiency caused by deleterious
slc22a5 mutations. The possible functional consequences of the
reduced carnitine esters associated with the heterozygous genotype
require further investigations.
P0824. Mutational analysis of OCA patients in Denmark
K. Grønskov, J. Ek, A. Sand, T. Rosenberg, K. Brøndum-Nielsen;
Kennedy Institute - National Eye Clinic, Glostrup, Denmark.
Oculocutaneous albinism (OCA) is a genetic heterogeneous disorder
caused by hypopigmentation of the eyes, hair and skin. The hypopigmentation
results from defects in melanin production. Lack of melanin
in the eyes causes misrouting of the optic nerve fibers, resulting in
nystagmus, foveal hypoplasia, strabismus, photophobia and greatly
decreased visual acuity. Hypopigmentation of the skin results in enhanced
sensitivity to light and increased risk of skin cancers.