2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
P05.071
characterization of the Dfna5 promoter region
K. Vrijens, L. Van Laer, G. Van Camp;
Center Medical Genetics, University Antwerp, Antwerp, Belgium.
DFNA5 mutations cause a non-syndromic, progressive, sensorineural
hearing loss in humans. Four different mutations have been identified
at the genomic level, all leading to exon 8 skipping at the mRNA level .
As a consequence, it was hypothesised that DFNA5-related hearing
loss is associated with a gain-of-function, and that only skipping of
exon 8 leads to disease . With the objective to clarify the molecular basis
of this gene’s regulation, we have characterized the mouse Dfna5
promoter region .
Initially, in silico analyses of the mouse Dfna5 promoter region were
performed followed by 5’-RACE experiments using mouse cochlear
cDNA. The latter enabled us to identify the cochlear Dfna5 transcription
initiation site (TIS) in vitro . Subsequently, constructs were generated
for transfection experiments in HEK293 cells. After confirmation of the
core promoter region in a 400bp construct surrounding the suspected
TIS, constructs of increasing length were generated to identify regulatory
elements . Both an enhancer and a silencer element could be
identified in the region upstream of the TIS. Next, transfection experiments
performed with the organ of Corti cell line OC-k3 demonstrated
that the suspected core promoter also drives expression in inner ear
cells . Furthermore the enhancer and silencer elements act similarly in
OC-k3 cells . Transfections using the 400bp construct in reverse orientation
were performed as negative control . However, this construct
also revealed promoter activity, suggesting the presence of an antisense
regulatory element . Finally, transcription factor binding sites in
the Dfna5 regulatory region where identified using several computer
modelling programs .
P05.072
mutational screening of GJB2 non-coding regions in Portuguese
hearing loss patients
T. D. Matos 1 , H. Simões-Teixeira 1,2 , H. Caria 1,3 , D. P. Kelsell 4 , G. Fialho 1 ;
1 Center of Genetics and Molecular Biology, University of Lisbon, Lisbon, Portugal,
2 Unidad de Genética Molecular, Hospital Ramón y Cajal, Madrid, Spain,
3 Higher College of Health, Polytechnic Institute of Setúbal, Setúbal, Portugal,
4 Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts
and The London School of Medicine and Dentistry, Queen Mary, University of
London, London, United Kingdom.
Many hearing loss mutations in the GJB2 gene have been described
in the last decade, being most of them located within the coding region
. During this period, only a few mutational studies were performed
on the non-coding regions of the gene . Such studies focused on the
first exon, the donor splice site and, occasionally, the promoter region.
None, to our knowledge, has ever included the whole 3’ UTR . Two
pathogenic mutations have so far been reported in GJB2 donor splice
site, and recently we have found a novel pathogenic mutation, -3438
C>T, occurring in the GJB2 basal promoter . These mutations, or novel
ones, in the GJB2 non-coding regions may therefore be involved in
other unelucidated cases of hearing loss .
In this study, we analysed by sequencing the GJB2 promoter, exon 1,
donor splice site and 3’UTR of about 100 unrelated Portuguese patients
previously screened for GJB2 coding region mutations .
An interesting finding was the identification of one homozygote for the
-493del10 deletion upstream GJB2 basal promoter, without any accompanying
GJB2 coding mutation. The significance of this mutation
is yet unclear . However, in a previous study, over 6% of carriers, but
no -493del10 homozygotes, were found in a control sample of 630
individuals, which might suggest this could be a pathogenic recessive
mutation . Results regarding other non-coding variants are being assessed
.
These data, and the previous reports on pathogenic non-coding GJB2
mutations, justify routine screening of these regions in order to improve
molecular diagnostic and genetic counseling of hearing loss patients .
P05.073
Genetic etiology and spectrum of mutations in GJB2 and
sLc26A4 genes
D. Raskova, D. Stejskal, P. Seeman, M. Putzova, E. Hlavova, O. Bendova, J.
Stoilova, R. Pourova, I. Vackova;
GENNET, Praha, Czech Republic.
Hereditary hearing impairment clinic was established at our institute
in October 2003 . 226 families have been investigated until October
2007 . Genetic etiology of hearing impairment was found at 70,35 %
of families (53,46% with autosomal recessive inheritance,15,10% with
autosomal dominant inheritance and 13,83% with genetic syndromes,
chromosomal aberrations and another genetic diseases with deafness,
at 17,61 % families the mode of inheritance could not be determined) .
The rest (29,65%) were families with idiopathic hearing loss .
The mutations in the GJB2 gene (Cx26) were investigated at 321 patients
by sequencing of entire coding region of GJB2 . In patients carrying
only one pathogenic mutation the IVS 1+1 G to A mutation in
the non-coding region was further tested . We found 4 mutations not
reported before (Ala149Thr, Ile140Ser, c .683+3 C to A, Gly130Val) .
At least one pathogenic mutation was found at 157 (48,91%) patients .
Both pathogenic mutations were detected at 65 (20 .25%) patients .
No pathogenic GJB2 mutation was detected at 134 (41,74%) patients
and 30 (9,35%) patients are carriers of various polymorphisms or mutations
not reported before . The mutation 35delG is by far the most
common of all pathogenic mutations and with mutations Trp24Stop,
313del14 and -3170 G to A accounted in total for 95% of all causal
mutated alleles in all patients .
Molecular genetic analysis of SLC26A4 gene was introduced in Czech
Republic in 2006 . 23,08% patients with congenital deafness, goiter or
enlarged vestibular aqueduct (EVA) have both pathogenic mutations
of SLC26A4 gene .
P05.074
A systems Biology Approach to Hearing: combining Genomic,
Proteomic and microRNA characterization
K. B. Avraham 1 , T. Elkan 1 , A. Dror 1 , R. Elkon 1 , T. Satoh 2 , G. Toren 1 , R. Hertzano
1 , M. Irmler 3 , J. Beckers 3 , E. Hornstein 4 , D. M. Fekete 2 , L. M. Friedman 1 ;
1 Department of Human Molecular Genetics & Biochemistry, Sackler School
of Medicine, Tel Aviv University, Tel Aviv, Israel, 2 Biological Sciences, Purdue
University, West Lafayette, IN, United States, 3 GSF-National Research Center
for Environment and Health, GmbH, Neuherberg, Germany, 4 Department of Human
Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Systems biology involves studying the interaction and interplay of
many levels of biological information . We have combined transcriptomic
and proteomic analyses of cochlear and vestibular sensory epithelia
in order to identify networks of genes and proteins essential for
the development and function of these inner ear organs . We further
identified microRNAs (miRNAs) that are uniquely expressed in the auditory
and vestibular sensory epithelia using bioinformatics tools and
experimental approaches, including microarray profiling and in situ
hybridization .
Expression profiling of vestibular and cochlear sensory epithelia using
Affymetrix microarrays and proteomics analysis using the Q-TOF
mass spectrometer with ITRAQ labeling has led to the identification of
genes and protein networks which function differently in the cochlear
and vestibular systems . A network analysis was applied to determine
if a set of proteins of interest are physically connected, assuming that
physical interaction between proteins points to some common function/pathway/complex
. Two major sub-networks emerged from the
integrated transcriptomic-proteomic clusters, indicating multiple interactions
between proteins expressed in the cochlear and vestibular
systems .
miRNAs are recognized as important regulators of gene expression at
the post-transcriptional level and mutations in miRNAs can lead to disease
. Combining our transcriptomic and proteomic data with miRNA
identification in the inner ear has led us to make predictions regarding
putative targets, which are being experimentally validated . For a number
of miRNAs, morpholino experiments in zebrafish demonstrated abnormalities
in inner ear development and/or structure, demonstrating
the importance of these miRNAs in the inner ear . These miRNAs are
candidates for causing deafness .