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

Molecular and biochemical basis of disease
P0633. Acute intermittent porphyria: impact of newly found
mutations on the biochemical and enzymatic protein properties
D. Ulbrichova, M. Hrdinka, J. Zeman, P. Martasek;
1st School of Medicine, Prague 2, Czech Republic.
Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant
inborn error. It results from the half-normal activity of porphobilinogen
deaminase (PBGD, EC 4.3.1.8), the third enzyme in the heme
biosynthetic pathway. AIP is manifested by life-threatening neurovisceral
attacks. Acute attack can be provoked by various factors such
as drugs, hormones, and alcohol, and is accompanied by massive
accumulation of porphyrin precursors in the urine. To date, over 300
different mutations have been found in the PBGD gene.
During systematic genetic analysis of Czech and Slovak AIP patients,
we found four novel mutations (610 C>A, 675 delA, 750 A>T, 966 insA)
and five previously reported mutations (76 C>T, 77 G>A, 518 G>A,
771+1 G>T, 973 insG) in eight unrelated families. Mutational screening
was performed by PCR, denaturing gradient gel electrophoresis
(DGGE), and DNA sequencing.
To establish the effects of these mutations on the protein structure of
PBGD, we expressed mutant constructs with the described mutations
in E. coli, and we analyzed their biochemical and enzymatic properties.
All purified enzymes carrying causative mutations had relative activities
lower then 1.0 % of the average level expressed by the normal
allele.
The identification and characterization of these novel mutations within
the PBGD gene of the newly diagnosed AIP patients provide insight
into the molecular heterogeneity of AIP. Investigation of the effects of
the present mutations on the protein structure and function provide
further understanding of the molecular basis and interaction of the molecules
in the system.
(Supported by Grants MSM0021620806, 1M6837805002, GAUK)
P0634. Neurodegeneration in X-Adrenoleukodystrophy: a
mitochondrial disease?
A. Pujol1 , S. Fourcade1 , J. López-Erauskin1 , A. Schlüter1 , E. Domènech-Estévez1
, J. Galino1 , J. Martínez-García1 , M. Portero-Otin2 , I. Ferrer1 ;
1 2 IDIBELL, L’Hospitalet de Llobregat, BArcelona, Spain, Universitat de Lleida,
Lleida, Spain.
X-linked adrenoleukodystrophy (X-ALD), is the most frequent inherited
monogenic demyelinating disease (minimal incidence 1:17,000).
X-ALD leads to death in boys or to motor disability in adults (adrenomyeloneuropathy
or AMN). The gene mutated in the disease (ABCD1)
is a peroxisomal ATP-binding transporter of very-long-chain-fatty acids
(VLCFAs), whose accumulation in plasma and tissues is the hallmark
of the disease. We have generated and characterized mouse models
for X-ALD, by classical knockout of the ABCD1 gene. These mice
exhibit a late-onset phenotype closely related to AMN patients, with
neurodegenerative features (excitotoxicity, microgliosis, axonal degeneration,
slower nerve conduction velocity and psychomotor impairment),
that begins at 15 months of age (1,2,3). The pathogenesis of
X-ALD is largely unknown, so are the mechanisms of toxicity through
VLCFAs. Using microarrays, Q-PCR and Western Blots of mouse spinal
cords, we have identified and confirmed dysregulation of oxidative
stress routes and mitochondria depletion as early events in the
pathogenesis. Indeed, markers of oxidative lesions of lipids (MDAL),
glucides (CML) and proteins (AASA) are upregulated in mutant spinal
cords. Treatment with VLCFAs of glial and neuronal cultures induces
ROS generation and decrease of mitochondria membrane potential.
Ex-vivo organotypic spinal cord slice cultures recapitulate closely the
pathogenic events seen in spinal cords, and will constitute a powerful
screening tool for therapeutic agents, and for deciphering molecular
cues underlying neurodegeneration in X-ALD.
(1) Pujol et al, Hum Mol Genet. 2002 Mar 1;11(5):499-505; (2) Pujol
et al, Hum Mol Genet. 2004 Dec1;13(23):2997-3006; (3) Ferrer et al,
Hum Mol Genet 14(23):3565-77 (2005)
P0635. Functional analysis of secretion-impaired fibrinogen
mutants rescued at low temperature: implications for treatment
of protein-misfolding disorders.
D. Vu 1 , C. Di Sanza 1 , M. Neerman-Arbez 1,2 ;
1 University Medical Centre, Department of Genetic Medicine and Development,
Geneva, Switzerland, 2 University Hospital, Division of Angiology and Haemo-
stasis, Geneva, Switzerland.
Congenital afibrinogenemia is a rare bleeding disorder characterised
by the complete absence of fibrinogen in blood. To date, 72 causative
mutations accounting for complete fibrinogen deficiency have been
identified in the three fibrinogen genes, FGA, FGB and FGG. Among
these, 9 missense or late-truncating nonsense mutations have been
shown to specifically impair secretion of fully assembled fibrinogen
molecules, implying the existence of a quality control for fibrinogen
secretion. We previously revealed opposite roles for the homologous
betaC and gammaC domains in secretion and demonstrated that secretion-impaired
fibrinogen mutants are retained in a pre-Golgi compartment.
The aim of this study was to restore the secretion of these
mutants and study the properties of the rescued fibrinogen hexamers.
We found that the secretion defect of two missense mutants but not
that of late-truncating nonsense mutants can be partially corrected by
incubating cells at low temperature (27°C) in a co-transfected COS-7
cell model. By contrast, exposure of cells to the chemical chaperones
4-phenylbutyrate (4-PBA), dimethyl sulfoxide (DMSO) and triethylamine
N-oxide (TMAO) failed to rescue the secretion of any mutant.
The mutants rescued at low temperature could be incorporated into
fibrin clots and formed factor XIII-mediated gamma-gamma dimers in
contrast to a dysfibrinogenemia mutant, used as a negative control for
these assays. However, plasmin digestion analyses revealed abnormal
patterns for the mutants compared to normal fibrinogen, suggesting
that the rescued mutants have a non-native conformation. Our data
underline the importance of careful functional investigations before
validating chemical or pharmacological chaperone-based therapies for
misfolded proteins.
P0636. CACNA1F gene mutation is behind Åland Island eye
disease
R. Jalkanen 1,2 , N. Bech-Hansen 3 , R. Tobias 3 , E. Sankila 4,5 , M. Mäntyjärvi 6 , H.
Forsius 4 , A. de la Chapelle 7 , T. Alitalo 1,2 ;
1 Biomedicum Helsinki, Department of Obstetrics and Gynecology, Helsinki
University Central Hospital, Helsinki, Finland, 2 Department of Medical Genetics,
University of Helsinki, Helsinki, Finland, 3 Departments of Medical Genetics and
Surgery, University of Calgary, Calgary, AB, Canada, 4 The Folkhälsan Institute
of Genetics, Department of Molecular Genetics and Population Genetics Unit,
Helsinki, Finland, 5 Department of Ophthalmology, Helsinki University Central
Hospital, Helsinki, Finland, 6 Department of Ophthalmology, University of Kuopio,
Kuopio, Finland, 7 Comprehensive Cancer Center, Ohio State University,
Columbus, OH, United States.
Åland Island eye disease (AIED), also known as Forsius-Eriksson
syndrome, is an X-chromosomal recessive retinal disease characterized
by a combination of fundal hypopigmentation, decreased visual
acuity due to foveal hypoplasia, nystagmus, astigmatism, protan color
vision defect, progressive myopia and defective dark adaptation. We
have previously localized AIED to the pericentromeric region of the
X-chromosome, but the causative gene is still unknown. In this study,
we screened the CACNA1F gene from genomic DNA and lymphoblast
mRNA to identify the mutation underlying the disease phenotype in
the original AIED family. A novel deletion in the CACNA1F gene was
identified, covering exon 30 and portions of the flanking introns. Expression
studies indicated that the particular exon was excluded from
the mRNA. This CACNA1F mutation co-segregated completely with
the disease phenotype in the AIED family and was not observed in 121
control chromosomes. Mutations in CACNA1F are known to cause
the incomplete form of X-linked congenital stationary night blindness
(CSNB2). Since the clinical picture of AIED is quite similar to CSNB2,
it has long been discussed, whether these disorders might be allelic or
form a single entity. CACNA1F mutations have been identified in patients
with an AIED-like phenotype, but previous studies have failed to
reveal any CACNA1F mutation in patients of the original AIED family.
Our results now show that AIED is caused by a novel deletion mutation
within CACNA1F.
P0637. Gene conversion leading to a severe Adenylate Kinase
deficiency.
C. Brunel, H. Wajcman, M. Goossens, S. Pissard;
INSERM U841, Eq 11, Lab Biochimie et Génétique, Créteil, France.
We describe a family in which a 15-year-old boy displays a non-spherocytic
chronic haemolytic anaemia associated with a mental retardation,
and a Willebrand disease. This phenotype led us to measure the
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