2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
P05.075
three novel mutations in slc40A1 gene causing
hemochromatosis type 4
V. Koubi 1 , F. Houriez 1 , C. Delacroix 2 , J. Marie 3 , S. Pissard 1,4 ;
1 laboratory of genetics, AP-HP,Hop Henri Mondor, creteil, France, 2 Hematology
Unit, AP-HP,Hop cochin, Paris 13, France, 3 Hematology Unit, AP-HP, Hotel
dieu, Paris 1, France, 4 University Paris 12, Creteil, France.
The Hemochromatosis type 4(OMIM : 606069, 2q32) is caused by
mutations in the SLC40A1 gene which encodes the ferroportin, a 10
transmenbrame domains protein’s which export iron from enterocytes
and RE cells to blood transferrin . It is supposed to be regulated through
interaction with the Hepcidin . Nowadays, 21 mutations and 7 polymorphisms
of the SLC40A1 gene are described . It is the most frequent
etiology for the “non HFE” hemochromatosis and it is inherited as an
autosomal dominant trait . We found common mutations in SLC40A1
in 13 from 93 families referred to the laboratory for a “non HFE” hemochromatosis
:G490D, G490S, G80S, V162Del and Q248H . The Study
of three families which do not displayed a typical HH 4 iron overload
(hyperferritinemia and low transferrin saturation), allowed us to discovered
3 new mutations : c . 212C >T (p .Ser71Phe) in exon 3, c .697C>
A (p .ala232Asp) in exon 6 and c .797T>C (p .Met266Thr) in exon 7 .
All patients were heterozygous for the mutation and the autosomal
dominant inheritance has been proven for p .Ser71Phe mutation since
HH type 4 and mutation were present in mother and son . Interestingly,
from those 3 mutations two are located in the loop between the TM
domain 4 and 5 (p .ala232Asp and p .Met266Thr)where are located 11
from the 21 mutations already described giving more evidences for the
role of this loops in the function or the regulation of this protein . This
work highlight the frequency of HH type 4 and brings data to better
understands the function of the protein .
P05.076
Novel sLc40A1 mutations in centre-south mediterranean
families with autosomal dominant iron overload.
F. C. Radio1 , S. Majore1 , N. Preziosi1 , A. Villa1 , M. De Muro2 , R. Villani3 , C. De
Bernardo1 , P. Grammatico1 ;
1Medical Genetics, “Sapienza University”, S.Camillo Hospital, Rome, Italy,
2 3 Emathology, University “Campus Biomedico”, Rome, Italy, Epathology,
S.Camillo Hospital, Rome, Italy.
Iron overload due to mutations in ferroportin is the most common form
of “Non HFE hemochromatosis” . Ferroportin disease or hemochromatosis
type IV has a dominant inheritance and two main clinical phenotypes
. Most cases show early increase in serum ferritin in the presence
of a low-normal transferring saturation, iron loading predominantly in
reticoloendothelial cells and, sometimes, low tolerance to the phlebotomy
program . On the contrary, some cases are similar to the typical
“classical hemochromatosis” characterized by early high transferring
saturation and prevalent parenchimal iron overload .
Different kind of mutations in the iron exporter ferroportin (SLC40A1)
result in hemochromatosis type IV . Loss-of-function mutations cause
an impairment of iron export from reticuloendothelial cells with tissue
iron accumulation but decreased availability of iron for circulating
transferrin . Instead a phenotype overlapping with ‘classical hemocrhomatosis’
is the result of gain-of function mutations in SLC40A1 .
We describe four novel missense mutations and a rare polymorphism
(p.Arg561Gly) in ferroportin (SLC40A1) found in members of five different
families of Centre-South Mediterranean showing autosomal dominant
iron overload . Both loss-of-function (p .Ala69Thr; p .Asp181Asn)
and gain-of-function (p .Arg296Gln; p .Tyr501Cys) mutations are recognizable
in these families, so that a genotype-phenotype correlation
is possible .
P05.077
Detection of large duplications within the FViii gene by mLPA
S. Rost1 , S. Loeffler1 , A. Pavlova2 , J. Oldenburg2 , C. R. Mueller1 ;
1Dept. of Human Genetics, University of Wuerzburg, Wuerzburg, Germany,
2Inst. of Hematology and Transfusion Medicine, University of Bonn, Bonn, Germany.
Haemophilia A is caused by a variety of different mutations in the FVIII
gene: missense and nonsense mutations, small and large deletions
and insertions, as well as large inversions . So far, only one duplication
of a whole exon of the FVIII gene, exon 13, has been published by an
Italian working group . Duplications comprising whole exons are dif-
ficult to detect by the usually applied methods for mutation screening
in X-chromosomal linked disorders .
In about 80 haemophilia A patients (from a total of approx . 2000) we
could not identify any mutation by long range PCR, DGGE and additional
sequencing of all 26 exons and flanking intronic regions of the
FVIII gene . These patients were investigated by multiplex ligation-dependent
probe amplification (MLPA, MRC-Holland) for large duplications
. We detected eight exon-spanning duplications of different length
in nine haemophilia A patients . In two patients we found duplications
of only one exon: exon 13 and 14, respectively . The other duplications
affected more than one exon: exons 1-5, exons 5-25, exons 23-25,
exons 2-25, exons 14-21 and exons 7-11, respectively . All duplications
were confirmed by DHPLC analysis (denaturing high performance liquid
chromatography) .
In conclusion, we found large duplications in the FVIII gene to be causative
mutations for haemophilia A in about 10% of all pre-screened
patients in which we could not detect any mutation by the conventionally
used methods .
MLPA is a convenient method for detection of large duplications and
also deletions of whole exons particularly in X-chromosomal linked disorders
as haemophilia A .
P05.078
Frequency of factor Viii intron-1 inversion among hemophilia A
patients from Republic of macedonia and Republic of Bulgaria
E. Sukarova Stefanovska 1 , M. Bojadzievska 1 , V. Dejanova 2 , P. Tchakarova 3 ,
G. Petkov 3 , G. D. Efremov 1 ;
1 Research Center for Genetic Engineering and Biotechnology, Macedonian
Academy of Sciences and Arts, Skopje, The former Yugoslav Republic of
Macedonia, 2 Center for Transfusiology, Medical Faculty, Skopje, The former
Yugoslav Republic of Macedonia, 3 Pediatric Clinic, Medical Faculty, Stara Zagora,
Bulgaria.
Hemophilia A (HA) an X-linked bleeding disorder, is characterized by
a wide allelic heterogeneity, as numerous mutations are spread out
through the whole exonic, intronic and regulatory regions of the factor
VIII gene . The most recurrent molecular defect is the inversion involving
the intron-22, accounting for >40% of the patients with severe disease
. Recently, Bagnall et al . (Blood, 2002) reported an inversion of intron-1
as a further recurrent mutation . The prevalence of this mutation
is between 0 .6 and 5%, according to reports from different groups .
The aim or this study was to determine the frequency of intron-1 inversion
among 57 severe hemophilia A patients from Republic of Macedonia
and 40 patients from Republic of Bulgaria . DNA samples from
all 97 patients were analyzed for the presence of intron-22 inversion .
Patients negative for the intron-22 inversion, were screened for the
intron-1 inversion. Two PCR reactions flanking each int1h repeat (intronic
and extragenic) were used .
We have identified the presence of intron-1 inversion in four out of
57 (7 .0%) severe HA patients from Republic of Macedonia . None of
these patients exhibited inhibitor development . Analysis of the factor
VIII gene haplotypes demonstrated that the intron-1 breaking inversion
has occurred independently in three (out of four) patients . No intron-1
inversion was detected among HA patients from Bulgaria .
Our data highlight the importance of analysis of the intron-1 inversion
in the severe HA cases from Republic of Macedonia . This will benefit
both genetic counseling and the study of the relationship between
genotype and inhibitor development .
P05.079
molecular analysis of a novel deletion at Exon 4 of factor iX gene
in a hemophilia B patient
L. Kokabee1,2 , E. Kamali1 , S. Zeinali1 , S. Jamali1 , M. Karimipoor1 ;
1 2 Pasteur Inistitute of Iran, Tehran, Islamic Republic of Iran, khatam university,
Tehran, Islamic Republic of Iran.
Hemophilia B (HB) is an X-linked bleeding disorder caused by the functional
deficiency of blood coagulation factor IX.The disease is due to
heterogeneous mutations in the factor IX gene (F9), located at Xq27 .1 .
It spans about 34 kb of genomic DNA .
Molecular analysis of F9 gene in a severe Iranian HB patient and carrier
testing for the family referred from Kashan hemophilia center .
After obtaining informed consent, genomic DNA was extracted from
the peripheral blood of the patient and his mother and sister by standard
methods. PCR amplification and single strand conformation poly-