2008 Barcelona - European Society of Human Genetics

Clinical genetics
mal HPRT coding sequence . Determined by mean of Real-Time PCR
technology, these patients showed markedly decreased HPRT mRNA
expression . Finally, we have analysed genomic regulatory sequences
from HPRT gene in these patients but no mutation was found .
Conclusions: This is the first report of a patient with Lesch-Nyhan syndrome
due to a defect in HPRT gene expression regulation .
P01.060
clinical, biochemical, and molecular diagnosis of L-2hydroxyglutaric
aciduria; Report of three iranian families with
six affected cases
Y. Shafeghati 1,2 , G. Vakili 3 , M. Noruzinia 4 , R. Colombo PhD. 5 ;
1 Genetics Research Center, Tehran, Islamic Republic of Iran, 2 Medical genetics
department, Sarem women’s hospital, Tehran, Islamic Republic of Iran, 3 private
general practitioner, Tehran, Islamic Republic of Iran, 4 2- Medical Genetics
Department, Sarem Women’s Hospital, Tehran, Islamic Republic of Iran, 5 3-
Laboratory of Human Molecular Biology and Genetics Catholic University of the
Sacred Heart, Milan, Italy.
Background: L-2-Hydroxyglutaric aciduria is a novel autosomal recessive
neurometabolic disorder . First described by “Duran” in 1980 about
100 cases have been reported . It is characterized by slowly progressive
neurological dysfunction with cerebellar ataxia, pyramidal sign,
intellectual decline, seizure etc . . MRI scanning is highly characteristic
and screening for organic acid(L-2-Hydroxyglutaric acid) in the urine,
serum, and CSF is diagnostic .
Materials & Methods: We have investigated three Iranian families with
six affected children aged (4 to 16 years) who were suspected for this
rare disorder, by urine organic acid assay and MRI scanning . In two
families we analyzed the Duranin gene which is responsible for hydroxy-glutarate
dehydrogenase .
Results: Affected cases were evaluated because of clinical findings.
Urine levels of L-2-Hydroxyglutaric acid were strongly increased . MRI
scanning of the brain showed hyper intense signal on T2 weighted
images of the sub-cortical white matter, and basal ganglia in all of the
patients. We have identified the mutation in one of the families. It was
a large deletion encompassing at least exons 7 and 8, the process is
ongoing yet . In this family, because the mother became pregnant we
did PND, unfortunately the fetus was affected in homozygote state .
Conclusions: Because of its inheritance pattern(autosomal recessive),
and high rate of consanguineous marriages in Iran, the prevalence of
this disorder might be higher, between mentally handicapped patients,
especially those with macrocephaly . So we should consider this rare
entity in our differential diagnosis .
P01.061
Lysosomal membrane permeabilization triggers cathepsin D
secretion and cell death in lysosomal storage diseases
D. L. Medina, R. de Pablo, V. Bouché, A. Ballabio;
Telethon Institute of Genetics and Medicine, Naples, Italy.
Lysosomal storage diseases (LSDs) are a group of inherited metabolic
disorders due to the defective activity of lysosomal hydrolases
and characterized by accumulation of undegraded substrates in the
lysosomes, leading to peripheral organ cell damage, neurodegeneration,
and premature death in most cases (Saftig et al, 2005) . Here we
have used our mouse model for Multiple Sulfatase Deficiency (MSD)
(Settembre et al, 2007), to study the intracellular signals triggered by
intralysosomal storage. Using fluorescent weak bases to examine the
acidic lysosomal compartment from isolated splenocytes and mouse
embryonic fibroblasts, we have found an increased lysosomal membrane
permeabilization (LMP) in MSD cells compared with their wildtype
counterparts . These results suggest a loss of the intralysosomal
acidic pH that can be mimicked in wild-type cells by the addition of the
specific inhibitor of vesicular proton pump, Bafilomycin A1. Lysosomal
destabilization in MSD cells correlates with several hallmarks of apoptosis
such as lysosomal secretion of the aspartate protease cathepsin
D, loss of mitochondrial membrane potential, release of cytochrome
C, activation of caspase-3, and annexin V staining . Interestingly, LMP
seems to be a common sign of other LSDs as suggested by the increased
LMP observed in splenocytes from a mouse model of Hunter
disease . Together, these evidences support the role of LMP and the
release of lysosomal proteases in the initiation and execution of apoptosis
in pathological situations such as LSDs .
P01.062
mutational spectrum and phenotype-genotype correlation in
spanish methylmalonic Acidemia with Homocystinuria, cblc
type
B. Pérez, A. Jorge-Finnigan, B. Merinero, L. R Desviat, F. Leal, E. Richard, M.
Ugarte;
Centro de Biología Molecular Severo Ochoa. CIBERER, Madrid, Spain.
Methylmalonic acidemia with homocystinuria, cblC type is the most
frequent genetic disorder of vitamin B 12 metabolism . We present the
mutational spectrum of 22 patients, classified belonging to the cblC
complementation group by biochemical and cellular approaches, to
provide insight on the phenotype-genotype correlations and on the
search for new therapeutical targets . The mutational spectrum included
five previously described mutations (M1L, R91fs, R132X, R153X,
R161X) and one new splicing change (IVS1nt2T>G) . The most frequent
change was the known mutation R91fs caused by a single
nucleotide duplication in position 271 which accounted for 82% of
the mutant alleles characterized and 77% of the cblC in homozygous
fashion . Up to date only some family studies have been performed to
rule out the presence of a big deletion . The frequency of c .271dupA
is higher than described in other studied populations such as the Italian
and the Portuguese cases. These results allow the confirmation
of the disease in our population by specific mutational analysis. The
other changes were rare mutations identified in one or two alleles (2
to 4%) . All the mutations found produce a premature truncation codon .
The majority of the patients exhibited a neonatal severe presentation .
Although most patients responded biochemically to B12 they exhibited
a fatal outcome . Further studies of the physiopathology of the disease
to provide insight about the severity of the symptoms despite of B12
responsiveness as well as the in vitro use of drugs to read-through the
PTC mutations will be discussed .
P01.063
Identification of 4 novel mutations in the NPC gene in spanish
patients with Niemann-Pick type c disease
L. Rodríguez Pascau 1,2,3 , G. Sánchez Ollé 1,2,3 , J. Macias 4,3 , M. Coll 4,3 , L. Vilageliu
1,2,3 , D. Grinberg 1,2,3 ;
1 Universitat de Barcelona, Barcelona, Spain, 2 IBUB, Barcelona, Spain, 3 CI-
BERER, Barcelona, Spain, 4 Institut de Bioquímica Clínica, Hospital Clínic,
Barcelona, Spain.
Niemann-Pick type C (NPC) is a rare autosomal-recessive lysosomal
storage disorder characterised by severe progressive neurological deterioration
. The disease is caused by mutations in the NPC1 or NPC2
genes . Approximately 95% of patients bear mutations in the NPC1
gene, which encodes a late endosomal integral membrane glycoprotein,
and about 5% of the patients show mutations in the NPC2 gene,
encoding a small soluble lysosomal protein with cholesterol-binding
properties . Both genes are involved in cholesterol and glycolipid trafficking
and transport, so NPC patients show accumulation of unesterified
cholesterol and glycosphingolipids in the lysosomal/late endosomal
system .
A mutational analysis was carried out in 5 non-related individuals affected
by this disorder . Genomic and cDNA from the patients were amplified
and sequenced. All the NPC1 mutant alleles were identified; 4 of
them have not been described before and were not found in 100 control
alleles . The new changes were: one missense mutation: p .F1079S
(c .3236T>C); one nonsense mutation: p .E1089X (c .3265G>T) and two
intronic changes that affect the splicing process: c .2604+5G>A in the
donnor splice site of intron 17 which promotes skipping of exon 17 and
c .1554-1009G>A that is located in intron 9 and results in the incorporation
of 194 pb of intron 9 in the cDNA as a new exon . Some of the
mutant alleles caused degradation of the mRNA by the nonsense-mediate
RNA decay mechanism . To identify these mutations, cycloheximide
treatment of cultured fibroblast was required.
The financial support from Fundación Niemann-Pick España and from
CIBERER (INTRA/07/720 .1) is acknowledged .