European Human Genetics Conference 2007 June 16 – 19, 2007 ...

Molecular and biochemical basis of disease
tions were 3.3%, 0.4%, and 4.8%, respectively. Five novel amino acid
substitutions (R38M, W355X, P727L, W907R, R1019X) were found in
five CD patients; four of these patients also carried another CARD15
variant. The biochemical nature of these mutations, cross-species
comparisons, and low IL8 production all favour their pathogenic role.
These mutation carriers had a complicated form of ileal or ileocolonic
disease. We were unable to identify any novel population-prevalent
CD susceptibility allele, and only the 1007fs mutation associated with
CD susceptibility. In conclusion, our data suggest that there may be yet
undetectable pathogenic mutations in the flanking or intronic regions of
the CARD15 gene. Compound heterozygosity or homozygosity for the
CARD15 mutations should be considered in complicated CD patients.
P0693. Molecular analysis of the CUL7 gene in 3 M syndrome
C. Huber 1 , A. L. Delezoide 2 , C. Baumann 3 , A. Munnich 1 , V. Cormier-Daire 1 , the
international collaborative group on 3 M syndrome;
1 Department of Medical Genetics and INSERM U781, Hopital Necker, Paris,
France, 2 Foetopathology Unit , Hopital Robert Debré, Paris, France, 3 Department
of Medical Genetics, Hopital Robert Debré, Paris, France.
3M syndrome is an autosomal recessive condition characterized by
pre- and post-natal growth retardation, facial dysmorphism, large head
circumference, normal intelligence and skeletal changes including long
slender tubular bones and tall vertebral bodies. Studying a series of 29
families, we have mapped the disease locus gene on chromosome
6p21.1, and then identified mutations in the CUL7 gene.
Following this initial study, we have collected the samples of 29 additional
3 M families and identified CUL7 mutations in 16/29 comprising
18 novel mutations. This series included one terminated pregnancy
at 33 weeks of gestation. The fetus presented with severe growth
retardation, normal head circumference, facial features, prominent
heels and long slender tubular bones suggestive of 3 M syndrome.
By direct sequencing, we first identified a CUL7 splice site mutation
(c.1215+1G>A), inherited from the father. In addition, we found a large
de novo deletion (> 3.84 Mb) encompassing the CUL7 gene. Histological
study of the femoral growth plate from this case showed an
increase in the chondrocyte density and size in the resting and proliferative
zones but no major abnormalities in the prehypertrophic and
hypertrophic zones, suggesting that CUL7 is mainly involved in the
chondrocyte proliferation but not in their differentiation.
We conclude that CUL7 is the major gene responsible for 3 M syndrome
accounting for 77.6 % of our cases (45/ 58). The absence of
any mutation in 13 patients and the exclusion of the 6p21.1 locus in 5
consanguineous families argue in favor of a genetic heterogeneity.
P0694. Mutational analysis of CYP 21 gene in patients with nonclassical
and heterozygous forms of 21-Hydroxylase deficiency
T. Bilgen 1 , G. Karaguzel 2 , S. Karauzum 1 , I. Bircan 2 , S. Akcurin 2 ;
1 Departments of Medical Biology and Genetics, Faculty of Medicine, Akdeniz
University, Antalya, Turkey, 2 Department of Pediatric Endocrinology, Faculty of
Medicine, Akdeniz University, Antalya, Turkey.
Congenital adrenal hyperplasia (CAH) is a common autosomal recessive
disorder and more than 90% of CAH cases are caused by mutations
in the CYP 21 gene. Variable degrees of 21-hydroxylase enzyme
impairment caused by different mutations are correlated with the clinical
severity of the disease. Two forms of CAH exist depending on the
clinical symptoms. The classical, severe form consists of the simple
virilizing and the salt-wasting types, whereas the non-classical (NC)
form is milder and later onset. In this study, we screened CYP 21 gene
in forty-one NC and heterozygote form of patients to detect the most
common mutations, other rare mutations, and possible novel mutations.
At first, we screened all cases for the V281L mutation by PCR-
RFLP. We detected V281L mutation in three patients, one of whom
was homozygote, and two were heterozygote for the mutation. Using
sequencing technique, we detected two different partial gene conversions
comprising the promoter and the first exon of the CYP 21 gene.
Some alterations associated with 21-hydroxylase deficiency were
detected, such as C89T, C/A655G, C2108T, and C2578T. We also
described three novel non-synonymous exonic alterations (T2555A,
A2094T, and C2700A) that could be considered as potential mutations
affecting the enzymatic activity. In addition, we identified a new genomic
change (G-47A) in the promoter region of the gene. The other
alterations detected in this study were intronic or at the 3’ UTR of the
CYP 21. This is the first study characterizing the molecular basis of NC
and heterozygous forms of CAH in Turkey.
P0695. No evidence for a founder effect of CYP21A1P/CYP21A2
chimeric genes causing 21-hydroxylase deficiency in Brazil
F. B. Coeli 1 , R. D. Bernardi 1 , R. R. De Carvalho 1 , R. J. Petroli 1 , F. C. Soardi 1 , G.
Guerra-Junior 2 , S. H. V. Lemos-Marini 3 , M. P. De Mello 1 ;
1 Centro de Biologia Molecular e Engenharia Genética, Campinas, Brazil, 2 Centro
de Investigação em Pediatria, Campinas, Brazil, 3 Centro Investigação em
Pediatria, Campinas, Brazil.
CYP21A2 gene which encodes 21-hydroxylase enzyme, as well as the
class III histocompatibility genes, is located between HLA-B and HLA-
DR loci. In the same loci there is a cluster of overlap genes including
RP, C4, CYP21 and TNX which is known as RCCX module. Almost
70% healthy individuals present a duplicated RCCX module arranged
in tandem (bimodular), but mono- or trimodular arrangements are also
observed in normal populations. The variability of RCCX number is
considered to be a result of unequal crossovers. Mutations on CY-
P21A2 gene cause 21-hydroxylase deficiency which is responsible for
95% Congenital Adrenal Hyperplasia, an inborn error of metabolism
that impairs cortisol synthesis in the steroidogenesis. In Brazil, after
studying the molecular basis of 21-hydroxylase deficiency in 112 unrelated
patients, a total of 44 disease-causing alleles were found to carry
a chimeric CYP21A1P/CYP21A2 gene (20%). In the present study we
describe different compositions of chimeric genes observed in mono-
, bi- and trimodular alleles (50%, 41% and 9%, respectively). Seven
different chimeric CYP21A1P/CYP21A2 haplotypes were observed in
momomodular alleles whereas five and four chimeric haplotypes were
identified in bi- and trimodular alleles, respectively. In conclusion, the
results described here indicated that there is no evidence for a founder
effect of alleles bearing chimeric genes causing 21-hydroxylase deficiency
in Brazil.
P0696. Cystic fibrosis studies: an individual spectrum of CFTR
gene mutations in Lithuania
V. Kucinskas 1,2 , K. Končiūtė 1,2 , A. Utkus 1,2 ;
1 Department of Human and Medical Genetics, Faculty of Medicine, Vilnius
University, Vilnius, Lithuania, 2 Centre for Medical Genetics, Vilnius University
Hospital Santariškių Klinikos, Vilnius, Lithuania.
Mutational testing of cystic fibrosis transmembrane conductance regulator
(CFTR) gene is in action in Lithuania since 1993. However, molecular
genetic diagnostics of cystic fibrosis (CF) in is still a considerable
problem due to mutational heterogeneity of this gene in the Lithuanian
population. 68 unrelated CF patients (136 CF chromosomes)
were tested for CFTR gene mutations combining different methods.
Initial testing for F508del using PCR with oligonucleotide primers
bridging the deletion was followed by the DGGE-based screening
of all CFTR gene exons and subsequent identification of the mutations
by direct sequencing. Duplex PCR was applied to identify the
CFTRdele2,3(21kb) mutation. The CF patients with one or two CFTR
gene mutations unidentified were tested for 33 most common mutations
using Cystic Fibrosis v.3 Genotyping Assay (Abbott, Germany).
18 different CFTR gene mutations were identified in Lithuania. Out
of them, five appear to be relatively common: F508del (61.76%),
CFTRdele2,3(21kb) (5.15%), R553X, N1303K, 3849+10kbC>T (each
2.94%). Other mutations were identified in single cases (0.74%).
Some of them are relatively common in other populations (W1282X,
G542X, 394delTT), but the majority are rare (G314R, R1066H, 3667insTCAA,
574delA, L138ins, 4006-4A>G, E217G, V754M) or even not
yet described elsewhere (4171insCCTA, 794delC). Mutations on 20
CF chromosomes (14.71%) have not yet been identified. In conclusion,
the applied PCR-based approach resulted in 18 different CFTR
gene mutations in CF patients from Lithuania with the overall identification
rate of 85.3%. F508del prevails (61.76%), but the spectrum of
CF mutations in Lithuania appears to be individual in comparison with
other European populations.
P0697. Cystic fibrosis and modifier genes in mexican population
C. N. Sanchez-Dominguez1 , M. A. Reyes-Lopez1 , R. Ortiz-Lopez2 , H. L. Gallardo
Blanco2 , A. Bustamante Saenz2 ;
1 2 Instituto Politecnico Nacional, Reynosa, Mexico, Universidad Autonoma de
Nuevo Leon, Monterrey, Mexico.
Cystic fibrosis (CF) is the most common monogenic disease in Caucasian
population, its frequency is about one in 2500 live born. The
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