2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
tion in exon 6 (C>T) which converts arginin into lysine . Additionally
there was an intronic mutation (C>A) which has not been reported up
to this time although predicted to have no effect on protein truncations .
In conclusion, this is the first frameshift mutation in Iranian population
and we have provided additional data of KCNH mutations in LQTS
patients. These findings will contribute to further understanding of the
function and structure of KCNH and the phenotype-genotype correlation
in hereditary LQTS .
P05.109
Gene scanning of KCNQ and KCNE by high-resolution melting
analysis
A. Bittnerová 1 , J. Kadlecová 1 , T. Novotný 2 , R. Gaillyová 1 , M. Kozák 2 , L. Křivan 2 ,
J. Špinar 2 ;
1 University Hospital Brno, Department of Medical Genetics, Brno, Czech Republic,
2 University Hospital Brno and Masaryk University, Department of Internal
Medicine and Cardiology, Brno, Czech Republic.
KCNQ1 encodes the larger subunit (KvLQT1) and KCNE1 the small
subunit (MinK) of the I Ks protein . I Ks protein is the slowly activating delayer
rectifier potassium channel. Mutations in single genes reduce the
I Ks current and cause the similar Long QT Syndrome (LQTS) phenotype
. LQTS is a cardiovascular disorder characterized by an abnormality
in repolarization, leading to a prolonged QT interval .
KCNQ1 consists of 16 exons and encodes a protein of 676 amino
acids. There have been identified 246 mutations in this gene. KCNE1
consists of just 3 exons, encoding a protein of 129 amino acids . Only
30 mutations of this gene have been found .
For the mutation scanning we have used multiplex SSCP with sensitivity
about 80 % . Now, we detect the mutations using high-resolution
melting analysis . This method is based on PCR in the presence of the
double-strand DNA binding dye, and tracking the nucleic acid melting
by monitoring the fluorescence of the sample across a defined temperature
range. Melting profiles can be used to identify the presence of
sequence variation within the amplicon . The method has almost 100%
sensitivity and specificity when used on products up to 400bp. We
have used this method for detection of 7 described mutations (S225L,
T312I, G314S, G325R, T587M, A590T and R591H) in KCNQ1 and two
SNPs (S38G and D85N) in KCNE1 with the sensitivity 100 % .
This work was supported by grants IGA MZ CR NR/9340-3, MSM
0021622415 and by the Czech Society of Cardiology .
P05.110
Identification of mutations in KLK4 genes among Iranian
patients with amelogenesis imperfecta
P. Aref, M. Bahaminpour, M. Shahrabi, M. Ghandehari Motlagh, B. Seraj, M.
Heidari;
Faculty of dentistry, Tehran, Islamic Republic of Iran.
Amelogenesis imperfecta (AI) refers to a group of inherited tooth disorders
characterized by abnormal enamel formation . The incidences of
AI vary widely; from 1 in 700 people in northern Sweden to 1 in 14,000
people in the United States In spite of the fact that many studies have
been carried out in different centers on molecular aspects of this disorder,
the genetic basis of non-syndromic forms of AI is unknown . So far,
four genes have been documented that associated with AI, AMELEX,
ENAM, KLK4 and MMP20 genes . We performed molecular genetic
studies on 10 Iranian families with different models of inheritances
according to pedigree analysis . In this study, three genes including
ENAM, KLK4, and MMP20 which account for majority of AI and autosomally
inherited were chosen for mutation detection by a polymerase
chain reaction (PCR) and single-stranded conformation polymorphism
(SSCP) . Our results from SSCP revealed genetic alterations in ENAM,
MMP20 and KLK4 genes . We found mutations in KLK4 exon 3 in 7
patients . In order to identify the type of mutation, samples were subjected
for DNA sequencing. Our findings suggested that the incidences
of KLK4 mutations in Iranian population might be higher than other
population .
P05.111
three novel mutations in the lactase gene (Lct) underlying
congenital lactase deficiency (CLD)
S. Torniainen 1 , C. Catassi 2 , T. Routi 3 , C. Gijsbers 4 , E. Savilahti 5 , I. Järvelä 1,6 ;
1 Department of Medical Genetics, University of Helsinki, Helsinki, Finland,
2 Department of Paediatrics, Universita Politecnica delle Marche, Ancona, Italy,
3 Department of Paediatrics, University of Turku, Turku, Finland, 4 The Juliana
Children’s Hospital, The Hague, The Netherlands, 5 Hospital for Children and
Adolescents, University of Helsinki, Helsinki, Finland, 6 Helsinki University Hospital,
Laboratory Service, Helsinki, Finland.
Background: Congenital lactase deficiency (CLD) is a severe gastrointestinal
disorder of newborns that is inherited as an autosomal recessive
trait and is enriched in the isolated Finnish population . The
diagnosis is based on clinical symptoms and low lactase activity in
intestinal biopsy specimens . Five mutations in the lactase gene (LCT)
have so far been identified to underlie CLD.
Methods: To search for new mutations underlying CLD we assayed
disaccharidase activities in intestinal biopsy specimens and screened
the coding region of LCT gene by direct sequencing from one Italian,
two Finnish and two Turkish patients with clinical symptoms compatible
to CLD .
Results: Three novel mutations in the LCT gene were identified. A single
nucleotide substitution leading to an amino acid change S688P in
exon 7 and a premature stop codon E1612X in exon 12 of the LCT were
present in the patient of Italian origin . A novel substitution of R1587H
in exon 12 was found in a heterozygous form from one Finnish patient .
Both Finnish patients were heterozygous for the Finnish founder mutation
Y1370X . Analyses of another Finnish patient are still ongoing . The
previously reported missense mutation G1363S in exon 9 was found
in a homozygous state in two siblings of Turkish origin . Comparison
of clinical phenotype and the location and/or type of a mutation in the
LCT gene shows that all mutations lead to a similar phenotype .
Conclusions: A total of eight mutations are known in CLD . This is the
first report of mutations in non-Finnish patients.
P05.112
Functional characterization of point mutations in the LDLR gene
found in Portuguese patients with clinical diagnosis of familial
hypercholesterolaemia.
S. Silva1 , D. Patel2 , A. K. Soutar2 , M. Bourbon1 ;
1 2 Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal, MRC-Clinical
Sciences Centre, London, United Kingdom.
The LDL receptor is a surface glycoprotein that mediates binding and
uptake of cholesterol-rich lipoproteins from plasma, in particular LDL .
Mutations in the LDLR cause familial hypercholesterolaemia (FH),
which results in defective catabolism of LDL leading to premature atherosclerosis
and CHD .
Five missense mutations found in the LDLR gene during the “Portuguese
FH Study”, responsible for protein variants V408L, W469R,
S627P, P664S and V838M, were studied in order to assess their
pathogenicity .
The different LDLR mutants were generated by site-directed mutagenesis
and expressed in CHO-ldlA7 cells lacking endogenous expression
of LDLR . To determine the effects of mutations on LDLR function
we measured saturable binding plus internalization and degradation of
125I-labelled LDL at 37ºC and estimated mature LDLR at cell surface
by immunoblotting .
All mutant constructs resulted in production of a detectable mature
protein in CHO-A7 cells, except variant W469R which accumulated
the precursor form . Variants W469R and V408L were severely impaired
in their ability to mediate uptake and degradation of 125I-LDL and showed reduced amounts of LDLR at cell surface . Variant S627P
retained ~40% and P664S ~60% of normal LDLR activity . V838M variant
showed essentially the same activity as the wild-type LDLR .
Results suggest that four of the variants studied are mutations causing
disease in patients carrying those alterations and V838M is a rare
non-pathogenic variant . The severe effect that V408L mutation has in
LDLR function does not correlate with the patient’s phenotype, suggesting
other genetic or/and environmental factors may be involved in
phenotype modulation .
P05.113
Genetic testing for Leber congenital Amaurosis (LcA): a 3-year
experience
F. Coppieters 1 , S. De Jaegere 1 , T. de Ravel 2 , I. Casteels 3 , F. Meire 4 , N. Van
Regemorter 5 , A. De Paepe 1 , P. J. Coucke 1 , B. P. Leroy 1,6 , E. De Baere 1 ;
1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium,
2 Center for Human Genetics, Leuven University Hospitals, Leuven, Belgium,
3 Department of Ophthalmology, Leuven University Hospitals, Leuven, Belgium,