2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
resent 9% of all mutations in the “Portuguese FH Study”. Our findings
emphasize that care must be taken before reporting the presence or
absence of a splice site mutation in LDLR for diagnostic purposes .
Our study also demonstrates that relatively simple, quick and inexpensive
RNA studies can evaluate putative splicing mutations that are
not always predictable by available software, thereby reducing genetic
misdiagnosis of FH patients .
P05.044
co-ocurrence of four mutations in a clinical case of Familial
mediterranean Fever
I. Navarro-Vera1 , B. Hernandez-Charro1 , M. Sanchez1 , P. Armero1 , R. Marín2 ,
P. Madero1 ;
1 2 Centro de Análisis Genéticos, Zaragoza, Spain, Hospital Puerta del Mar, Cadiz,
Spain.
Familial Mediterranean Fever (FMF) type 1 is characterized by recurrent
short episodes of fever associated with different inflammatory processes
. MEFV is the only gene currently known to be associated to
FMF . E148Q in exon 2 and several mutations in the exon 10 of MEFV
are the most common . FMF is inherited in an autosomal recessive
manner, so both parents of a proband are considered obligate carriers .
We studied a FMF clinical case and his family and surprisingly four
different mutations were found .
The family consisted in the affected proband, a sister and the parents,
all of then unaffected . The study was carried out by bidireccional
sequencing of exon 10 and targeted mutation analysis by RFLPs of
E148Q mutation in exon 2 of MEFV gene .
The E148Q was present in heterozygous state in the proband . The
sequencing analysis of the exon 10 showed the p .R653H and p .I640M
mutations and the deletion p .I692del, all of them previously described .
The study of familial samples revealed the presence of two mutations
in each and everyone: father p .I640M and p .R653H, mother E148Q
and p .I692del and sister p .I640M and p .R653H (haplotype in cis phase
from his father) .
The inheritance pattern showed that each parent carries two mutations
in cis phase, the sister inherited the wild type phase of the mother and
the mutated phase of the father and the affected son is therefore carrying
all four mutations .
This case makes us aware of the importance of considering the haplotype
phase of multiple mutations in recessive disorders .
P05.045
mEFV mutations in turkish Patients suffering From Familial
mediterranean Fever
O. Cilingir 1 , M. Ozdemir 1 , M. H. Muslumanoglu 1 , E. Tepeli 1 , G. Bademci 1 , C.
Korkmaz 2 , O. Kutlay 1 , U. Demir 1 , H. Aslan 1 , B. Durak 1 , S. Artan 1 ;
1 Eskisehir Osmangazi University, Medical Faculty, Department of Medical
Genetics, Eskisehir, Turkey, 2 Eskisehir Osmangazi University, Medical Faculty,
Department of Rheumatology, Eskisehir, Turkey.
Familial Mediterranean fever (FMF) is an autosomal recessive periodic
disorder. Over 50 mutations have been identified in the MEFV gene
responsible for FMF . For diagnosis of FMF; molecular methods and
Tel-Hashomer Clinical Criterias (THCC) can be used . AIM: To identify
distribution and frequency of the MEFV gene mutations in Turkish FMF
patients, compare two molecular different technics and correlation of
clinical-molecular diagnosis . PATIENTS-METHODS: The study was
carried out on 604 clinically diagnosed Turkish FMF patients . Mutation
screening of the MEFV gene was performed by sequencing of exon 10
in 448 patients and by FMF specific StripAssay (PCR-Reverse Hybridization)
for E148Q, P369S and F479L mutations of exons 2,3, and 5,
respectively in 256 patients . RESULTS: Of the 604 unrelated patients
investigated, 344 (56 .95%) had one or two mutations : 85 patients
(24 .71%) were homozygous; 88 (25 .58%) were compound-heterozygous;
171 (49 .71%) were heterozygote mutations . Of the mutations,
M694V (A>G), V726A, M680I (G>C), R761H accounted for 65 .99,
19 .18, 17 .44, and 4 .36 %, respectively . StripAssay was observed
completely correlate with direct sequencing in 100 patients . For determining
the correlation between mutation status, clinical diagnosis with
THCC was investigated in 90 patients . Only 33 patients were exactly
diagnosed by THCC whereas 53 patients were reported mutations by
StripAssay . CONCLUSION: Exon 10 is the most common site for FMF
mutations whereas exons 2,3 and 5 accounts for 11 .63% of the cases .
The most common mutation among Turks is M694V (A>G) . As a result,
StripAssay for 12 common mutations might be used in routine mutation
screening analysis .
P05.046
Promoter studies in the FA genes
D. Meier, K. Neveling, D. Schindler;
Department of Human Genetics, Biocenter, Wuerzburg, Germany.
Fanconi anemia (FA) is a rare genome-instability disorder with the frequent
presence of congenital malformations and bone marrow failure .
Other characteristic features include predisposition to FA-typical malignancies
and cellular hypersensivity to DNA-interstrand crosslinking .
At least 13 genes and corresponding complementation groups are underlying
the disease . Eight of the FA proteins (FANCA, -B, -C, -E, -F,
-G, -L and -M) and other components assemble in a nuclear complex,
the FA “core complex” .
Little if any is known about the promoter regions. However, identification
and characterization of the promoters would be essential for understanding
the regulation of transcription, including intergenic regulation,
of the FA genes . Our aim is to provide an explanation for the equimolar
composition of the core complex . We used a variety of in silico methods
to predict potential promoter regions. To confirm these, we have
set up dual luciferase reporter assays. We cloned the identified regions
(~1kb) in the pGL3 basic vector that carries the reporter gene for firefly
luciferase . As co-reporter we used the pRL null vector that contains
the renilla luciferase gene . With these constructs, we transfected HeLa
and HEK293 cells to assay luciferase activity using a luminometer . In a
second approach we used mutated constructs to detect any decrease
of activity . Further characterization includes transcription factor binding
sites and conserved sequence elements . First results indicate that
there is generally strong promoter activity . Short half-life of the gene
products is consistent with high transcriptional activity of the FA genes,
and rapid regulation in response to DNA damage .
P05.047
MYCN gene mutation screening in 9 patients with Feingold
syndrome
A. Nougayrede, C. Golzio, A. Munnich, S. Lyonnet, J. Amiel, L. de Pontual;
INSERM U-781, Department of Genetics, Necker Hospital, Paris, France, Paris,
France.
MYCN is a transcription factor amplified in about 30% of neuroblastoma
. MYCN is also the disease-causing gene in Feingold syndrome,
with a dominant mode of inheritance. Feingold syndrome is defined
by microcephaly, digital anomalies (syndactyly, brachymesophalangy),
digestive atresia (oesophagus, duodenum), facial features (retrognatism,
small palpebral fissures) and occasional malformations of heart,
kidney and spleen .
We studied the coding sequence of MYCN in 9 patients and found
3 mutations in the bHLH or the Leucine Zipper domain: 2 frameshift
mutations leading to a truncated protein and a nonsense mutation .
Loss-of-function is highly likely for frameshift mutations, while a dominant-negative
due to altered DNA binding and preserved dimerisation
is hypothesised for the nonsense mutation . MYCN deletion are currently
being tested for the remaining patients .
We performed in situ hybridization on human embryos (Carnegie
stages 12, 15 and 17) to determine MYCN expression pattern during
development . At C12, MYCN is ubiquitous expressed . Later (C15
and C17), expression is restricted to mesencephalon, diencephalon,
spinal cord, limb buds, oesophagus and stomach, in accordance with
Feingold syndrome features . MYCN is also expressed in the primitive
mesonephros and the Rathcke pouch. These later findings argue for
kidney anomalies to be regarded as a feature of Feingold syndrome
and raise the hypothesis of an endocrine involvement for the short
stature observed in the syndrome .
P05.048
molecular characterization of familial hypercholesterolemia in
iranian patients
M. -. Hashemzadeh Chaleshtori 1 , E. Farrokhi 1 , F. Shayesteh 2 , M. Modarresi 2 ,
M. Shirani 1 , F. Roghani Dehkordi 3 , K. Ghatreh Samani 4 , G. Mobini 1 , M. Banitalebi
1 , K. Ashrafi 1 , M. Shahrani 1 , N. Parvin 1 ;
1 Cellular and Molecular Research Center, Shahrekord, Islamic Republic of Iran,
2 Department of Biology, School of Basic Sciences, Payam-e-noor University,
Esfahan Center, Esfahan, Islamic Republic of Iran, 3 Department of Cardiology,