2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
Second, we characterized 9 known deletions of the FOXL2 region using
array CGH. In addition, 10 new deletions identified by MLPA were
further defined by qPCR. These deletions prove to be highly heterogeneous
with regard to deletion size and breakpoint localization . They
account for 12% of molecular defects in BPES, highlighting the importance
of copy number analysis in BPES .
In conclusion, we showed that copy number changes of FOXL2 comprise
a considerable fraction of the molecular defects in BPES . Moreover,
the extensive search for CNVs and point mutations in CNCs mapping
distantly of FOXL2 in BPES patients, support the importance of long
distance gene regulation in the molecular pathogenesis of this disorder .
P05.014
molecular analysis of the ATP V B and ATP V0A genes in
distal renal tubular acidosis
I. Carboni 1 , E. Andreucci 1 , I. Pela 2 , G. Lavoratti 3 , M. Materassi 3 , D. Serafini 3 , M.
R. Caruso 4 , M. Rigoldi 5 , M. Genuardi 1 , S. Giglio 1 ;
1 Dept. of Clinical Pathophysiology, University of Florence and Medical Genetics
Unit, Meyer Children’s University Hospital, Florence, Italy, 2 Paediatric Nephrology,
Dept of Paediatrics, University Of Florence and Meyer Children’s University
Hospital, Florence, Italy, 3 Paediatric Nephrology, Dept of Paediatrics, University
of Florence and Meyer Children’s University Hospital, Florence, Italy, 4 Division
of Nephrology and Dialysis, OORR Hospital, Bergamo, Italy, 5 Metabolic Diseases
Unit, San Gerardo Hospital, Monza, Italy.
Primary distal renal tubular acidosis (dRTA) is characterized by defective
secretion of H + ions by intercalary cells of the collecting duct .
Both autosomal dominant (AD) and recessive (AR) forms have been
described . The AD form is caused by mutations of the SLC4A1 gene .
The AR form has been associated with mutations of either ATP6V1B1
- in individuals who usually display sensorineural hearing loss (SNHL)
- or ATP6V0A4 - in individuals who do not have SNHL or show hearing
loss only after the first decades of life.
We report on the first investigation of the ATP6V1B1 and ATP6V0A4
genes in dRTA patients in Italy. Mutations were identified in 8/8 patients
analyzed .
The ATP6V0A4 Arg807Gln mutation was found at the homozygous
state in a patient with severe early-onset SNHL, that is generally not
observed in subjects with ATP6V0A4 defects . Only another case with
the same genotype, who had a comparably severe phenotype, is reported
in the literature .
A monoallelic de novo ATP6V1B1 mutation (Arg394Gln) was observed
in one patient with a typical dRTA renal phenotype without deafness .
This mutation has been previously described in two cases, who also
were apparently simple heterozygotes .
We also detected three previously unreported mutations, two in AT-
P6V0A4 and one in ATP6V1B1 .
Our results shed further light on phenotype-genotype correlations in
dRTA. In particular, the association of a specific monoallelic ATP6V1B1
mutation with a dRTA phenotype without hearing loss suggests that
other genetic mechanisms, in addition to autosomal recessive inheritance,
may be associated with ATP6V1B1 alterations .
P05.015
An EYA gene mutation in intron 8 [c.867+5G>A] causes
alternative RNA splicing and is a recurrent mutation causing
Branchial-oto-renal syndrome
T. L. Stockley1,2 , R. Mendoza-Londono1,2 , E. J. Propst1,2 , S. Sodhi1 , L. Dupuis1 ,
B. C. Papsin1,2 ;
1 2 The Hospital for Sick Children, Toronto, ON, Canada, The University of Toronto,
Toronto, ON, Canada.
Branchio-oto-renal (BOR) syndrome is a heterogeneous autosomal
dominant disorder characterized by branchial arch abnormalities,
hearing loss and renal abnormalities . Variable expression of clinical
features is found within and between affected families . Mutations in
the EYA1 gene are reported to account for 40-70% of reported BOR
syndrome cases . We have developed a strategy for molecular testing
of the EYA1 gene causing BOR syndrome consisting of 1) sequencing
of the complete coding region and flanking intronic regions and 2) multiple
ligation probe amplification (MLPA) analysis. Using this strategy
EYA1 mutations were identified in 82% (14/17) of a cohort of paediatric
BOR syndrome probands. Forty-five percent (5/11) of BOR syndrome
probands in our cohort had de novo EYA1 mutations, suggesting that
the incidence of de novo cases in BOR syndrome is higher than 10%
previously reported . We also describe a previously unreported recurrent
EYA1 gene mutation c .867+5G>A found in 21% (3/14) of EYA1
mutation-positive BOR probands in our patient cohort . RNA analysis
indicates that the c .867+5G>A mutation affects splicing of the EYA1
gene, and produces an aberrant mRNA transcript skipping exon 8 and
leading to a premature termination signal in exon 9 . The aberrant transcript
lacking exon 8 was present at approximately 50% level of wildtype
EYA1 mRNA in fibroblasts, and suggests that certain transcripts
of EYA1 escape nonsense-mediated decay and encode truncated EYA
protein capable of dominant-negative interactions causing disease .
P05.016
BscL2 and its possible function in adipogenesis
D. Jud, T. Schwarzbraun, C. Windpassinger, K. Wagner;
Institute of Human Genetics, Graz, Austria.
Congenital generalized lipodystrophy, first described by Berardinelli
(1954) and Seip (1959), is a rare autosomal recessive disorder characterized
by near complete absence of adipose tissue from birth or
early infancy . Two distinct forms can be distinguished: CGL1 and
CGL2, whereas CGL1 is caused by mutations in AGPAT2 and CGL2
by mutations in BSCL2 . Whilst mutations in AGPAT2, a member of the
acyltransferase family, likely cause lipodystrophy by reducing triglyceride
synthesis in adipose tissue, the pathogenic effects of mutations
in BSCL2 on a molecular level are not yet fully understood . Further
clinical features of both subtypes include severe insulin resistance,
acanthosis nigricans, muscular hypertrophy, hepatomegaly, diabetes
mellitus and hypertriglyceridemia . However patients with CGL2 show a
more severe phenotype with lack of both metabolically active and mechanical
adipose tissue, a higher prevalence of mild mental retardation
and hypertrophic cardiomyopathy .
To elucidate the function of BSCL2 in adipogenesis we generated
BSCL2 knock-out flies. These knock-out strains showed altered architecture
and distribution of lipid droplets in adipose tissue . Therefore
our data suggest that BSCL2 is involved in lipid droplet formation and/
or maintenance in Drosophila. To confirm these data in mammals we
created a mouse knock-out model for BSCL2 . We chose the Cre-loxP
system to obtain conditional knock-out mice in order to investigate the
function of BSCL2 in relevant tissues specially during adipocyte development
. We will apply molecular genetic, histological, biochemical and
cell culture methods to further determine the enigmatic role of BSCL2
in adipogenesis and in the pathophysiology of CGL2 .
P05.017
The functional significance of the C1 inhibitor gene promoter
mutations
L. Grodecka 1 , H. Grombirikova 1 , B. Ravcukova 1 , J. Litzman 2 , T. Freiberger 1 ;
1 Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation,
Brno, Czech Republic, 2 Institute of Clinical Immunology and Allergology,
Faculty of Medicine of Masaryk University, St. Anne’s University Hospital,
Brno, Czech Republic.
C1 inhibitor (C1INH) is a key negative regulator of complement activation
. Decreased level of functional C1INH causes hereditary angioedema
which is usually transmitted as an autosomal dominant trait .
Till now, three promoter mutations have been detected in the C1 inhibitor
gene (C1NH), one of them (-45 C>A) having been newly identified
in our laboratory. Mutations -40 C>G and -45 C>A both appeared
to cosegregate with other mutations in coding region, while the third
mutation -103 C>T did not . Interestingly, in patients suffering from
hereditary angioedema, mutation -103 C>T was found exclusively in
homozygous state while heterozygous individuals were healthy . The
objective of our work was to assess functional importance of the three
promoter mutations on the C1NH gene expression . Results of luciferase
reporter gene assay showed that two of the mutations, -40 C>G
and -45 C>A, have negligible effect on C1INH expression . Surprisingly,
mutation -103 C>T increased luciferase gene expression by 59
% in comparison to wild type promoter construct . Yet plasma levels of
C1 inhibitor in patients with this mutation were significantly decreased.
The reason for this discrepancy is not obvious . However, a negative
correlation between amount of mRNA and plasma levels of C1INH has
already been described in healthy controls . Such a correlation suggests
that a negative feedback regulation of C1INH expression may
exist .
This study was supported by grant of IGA MZ CR No . NR 9192-3 .