2008 Barcelona - European Society of Human Genetics

Clinical genetics
tion . Ferritin is the major intracellular iron store protein and consists H
(heavy) and L (light) subunits which are encoded in chromosomes 11
and 19, respectively . The iron responsive element (IRE) in the 5’ untranslated
region (5’-UTR) of L-ferritin mRNA interacts with trans-acting
iron regulatory proteins (IRPs) to regulate ferritin translation . Mutations
in the 5’UTR of the L-ferritin gene, result in reduced affinity for
IRPs and subsequently in constitutive L-ferritin synthesis which results
in congenital cataract . In the present study, we analyzed two families
with early onset bilateral cataract and marked hyperferritinemia .
Genomic DNA was isolated from peripheral blood and the IRE L-ferritin
gene was completely sequenced . Morphology of cataracts of both
families showed different phenotypes . Onset of cataract symptoms
ranged from 15 year-old to 40 year-old . No other ocular or systemic
anomalies were found to be present . Propositi underwent surgery with
satisfactory results . Levels of ferritin serum were compatible with hyperferritinemia
. DNA analysis of both families detected two different
misssense mutations in the loop region of the L-ferritin gene, one of
them previously reported, these mutations in the L-ferritin gene result
hereditary hyperferritinemia cataract syndrome .
P01.052
Analysis of the CTNS gene in spanish patients with cystinosis
disease
J. Macias, M. Rodes, J. M. Hernandez, M. J. Coll;
Institut de Bioquímica Clínica, Hospital Clínic. CIBERER, Barcelona, Spain.
Cystinosis (MIM #219800) is an autosomal recessive disorder characterized
by intra-lysosomal accumulation of cystine due to an impaired
transport of free cystine out of lysosomes .
Three phenotypical forms have been described according to the age
of onset and severity of the clinical symptoms: infantile, juvenile and
ocular nonnephropathic cystinosis .
The gene for cystinosis, CTNS, has 12 exons and encodes a 367 amino-acid
lysosomal membrane protein called cystinosin . Up to now 84
different mutations have been described .
We have analyzed the CTNS exons and intron boundaries, as well
as the promoter region in 21 unrelated Spanish cystinosis patients, in
order to find possible genotype-phenotype correlations and to enable
identification of carriers and prenatal diagnosis.In this study, 11 different
mutations were identified, 5 of which are novel. The 57-kb deletion
is the most prevalent mutation in our country (33,3% of the alleles), as
seen in other studied populations . This deletion together with other 4
mutations (previously described p .T7fsX6, p .T216fsX11, p .G308R and
p .M1T which is novel) accounted for 80% of the alleles, which facilitates
molecular diagnosis and genetic counselling in this disease .
P01.053
characteritzation of cystinuria double knockout for b 0,+ At and
rBAt
M. Espino Guarch 1 , M. Font-Llitjós 1,2 , R. Sillué 1,2 , S. Mañas 1 , J. Visa 3 , M.
Palacín 4,5 , V. Nunes 1,5 ;
1 Centro de Genética Médica y Molecular IDIBELL, L’Hospitalet de Llobregat,
Spain, 2 CIBERER-U730, Barcelona, Spain, 3 Animal Facility Service IDIBELL,
L’Hospitalet de Llobregat, Spain, 4 Parc Científic de Barcelona, Barcelona,
Spain, 5 Universitat de Barcelona, Barcelona, Spain.
Cystinuria is a common recessive disorder of renal reabsorption of cystine
and dibasic amino acids that results in urolithiasis of cystine . Cystinuria
is caused by defects in the amino acid transporter rBAT/b 0,+ AT .
Mutantions in SLC3A1 (rBAT) cause cystinuria type A, characterized
by a silent phenotype in heterozygotes (phenotype I) . Mutations in in
SLC7A9 (b 0,+ AT) cause cystinuria type B, which heterozygotes in most
cases hyperexcrete cystine and dibasic amino acids (phenotype non-
I) . The Slc7a9 null knockout mouse model (Stones) and the Slc3a1
knockin (Pebbles) develop cystinuria B and A respectively . All homozygous
mutants hyperexcrete cystine and the three dibasic amino acids,
and ~40% of them present cystine calculi in the urinary system . To
facilitate in vivo investigation of cystinuria we have generated double
knockout mice by crossing the two cystinuria mice models (Stone and
Pebbles) and we have characterized the nine genotype combinations
of the F2 in mixed background C57BL/6J-C3H .
Mices were X-ray analyzed at 2 .5 and 8 months to appraise calculi
formation . Histopatology studies and metabolic cage experiments to
collect urine to quantify amino acids excretation were also performed .
Preliminary results indicate that double knockout mice has more se-
vere phenotype than single knockouts: single mutants which are heterozygote
for the other subunit and double mutants show less viability,
higher stone rate and a more severe urinary system damage .
Supported by MEC (SAF2003-08940-01/02 and BFU2006-14600-
C02-01/02/BMC), The European Union (EUGINDAT; LSHM-CT-
2003-502852), Generalitat de Catalunya (2006 SGR00018 and 2005
SGR00947) .
P01.054
Single exon deletions in the PAH gene in Polish PKU patients
M. Bik-Multanowski, J. J. Pietrzyk;
Chair of Pediatrics, Jagiellonian University, Krakow, Poland.
The majority of molecular defects causing phenylketonuria (PKU) are
missense mutations of the phenylalanine hydroxylase gene (PAH),
which are detectable with use of classical molecular techniques basing
on single exon amplification, heteroduplex analysis and sequencing.
The cumulative mutation detection rate in various groups of PKU-patients
reaches 90-99% . However, the above procedure fails to detect
single exon deletions .
The aim of this study was to establish simple, non-laborious PCRbased
methods for detection of three PKU-causing single exon deletions
and to assess their frequency in Polish PKU-patients .
methods: An attempt to detect Ex5del955, Ex5del4232ins268 and
EX3del4765 deletions was undertaken with use of published data on
the mutated sequence of the PAH gene . DNA samples from 25 PKU
Polish patients were analyzed, in whom only one or no PKU-causing
mutations (a single case) were identified despite extensive analysis
performed with classical methods (in the remaining 245 patients, in
whom DNA samples were tested, both mutations were found) .
Results: PCR protocols for amplification of mutated alleles were
successfully established . Following PCR primers were used: Exon-
5del955for-gcacattggaatccacagcaagg,
Exon5del955rev-gtctctagactctaggagtccccag, Exon5del4232forgttcccattctgtcagttgcctg,
Exon5del4232rev-ggaggatctgtctccgctctc, Exon3del4765nestedfor-gcattgtccaagtacctagcctgg
(nested PCR), Exon-
3del4765for-acaggcacacaccaccatgc and Exon3del4765rev-gccactatgggattgggtgacc
. In the tested samples, six cases of Ex5del4232ins268
deletion were found as well as one case of each of the other deletions,
which were interestingly detected in the same patient’s sample .
conclusion: Ex5del955, Ex5del4232ins268 and EX3del4765 deletions
in the PAH gene can be detected with use of simple PCR-based
methods and are present in around 2%-3% of Polish PKU-patients .
Supported by government grant for scientific research
(N40708032/3085)
P01.055
Detection of mutations in two families with Ethylmalonic
encephalopathy using real-time PcR
V. Tiranti 1 , R. Mineri 1 , T. Georgiou 2 , G. Stylianidou 3 , A. Drousiotou 2 ;
1 Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the
Study of Children’s Mitochondrial Disorders, National Neurological Institute
“Carlo Besta”, Milan, Italy, 2 Department of Biochemical Genetics, The Cyprus
Institute of Neurology and Genetics, Nicosia, Cyprus, 3 Department of Paediatric
Neurology, Archbishop Makarios III Hospital, Nicosia, Cyprus.
Ethylmalonic encephalopathy (EE, OMIM # 602473) was diagnosed
in three children from two unrelated Cypriot families . They presented
with neurodevelopmental delay, prominent pyramidal and extrapyrimidal
signs, recurrent petechiae, orthostatic acrocyanosis and abnormal
MRI images . They had persistent lactic acidemia, markedly elevated
urinary excretion of ethylmalonic acid, 2-methylsuccinate, isobutyrylglycine
and isovalerylglycine, and moderately raised butyrylcarnitine
in blood . In one patient muscle cytochrome C oxidase activity was
greatly reduced. The diagnosis was confirmed by westernblot analysis
in fibroblasts which showed the absence of the ETHE1 protein. Molecular
analysis of the ETHE1 gene was carried out using both PCR
and sequencing. The proband of the first family was a compound heterozygote
for a deletion in exon 4 and a missense mutation in exon
5, L185R . In order to establish the presence of the deletion in a heterozygous
state, we carried out quantitative real-time PCR on DNA
extracted from both the patient and his healthy parents . The proband
from the second family was homozygous for the deletion in exon 4 and
in this case quantitative real-time PCR demonstrated that the parents
were both heterozygotes for the deletion . The exon 4 deletion and the