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

Molecular and biochemical basis of disease
the study of one heterogenetic? syndrome as BBS. Nevertheless, it is
necessary to search for new mutations to update the chip and improve
its clinical application.
Project: FIS PI060049
P0665. A digenic defect on both the chloride channels ClC-Ka
and ClC-Kb mimics a Bartter like type IV Syndrome.
N. Borsa 1 , M. Syrèn 1,2 , C. Melotti 1 , C. Radaelli 1 , A. Bettinelli 3 , D. Coviello 1 , S.
Tedeschi 1 ;
1 Fondazione IRCCS Opedale Maggiore Policlinico, Mangiagalli e Regina Elena,
Milan, Italy, 2 Dept. of Pediatrics and Neonatology, University of Milan, Milan,
Italy, 3 Dept. of Pediatrics, San Leopoldo Mandic Hospital, Merate, Lecco, Italy.
Usually mutations in the BSND gene product, a protein called barttin,
are responsible for a Bartter syndrome associated with sensorineural
deafness (Bartter type IV or BSND), an inherited tubulopathy kidney
disease. The BSND syndrome is characterized by a marked polyhydramnios,
severe renal salt wasting and deafness.
There is only one exception, previously reported, where a patient was
affected by sensorineural deafness and renal Bartter phenotype without
BSND mutations; simultaneous mutations in both CLCNKB and
CLCNKA genes (coding for ClC-Kb and ClC-Ka chloride channels respectively)
were demonstrated to be the disease-causing mutations.
We describe here a new patient who has a digenic disorder due to
a contemporary combined impairment of two closely related genes,
CLCNKA and CLCNKB, leading to a phenotype that combines a severe
renal salt wasting and sensorineural deafness. The molecular
analysis performed on the BSND, CLCNKB and CLCNKA genes revealed
the absence of mutations in the former gene and a homozygous
deletion of exons 1-6 for the CLCNKB gene and of exons 7-19 for
the CLCNKA.The simultaneous disruption of both ClC-Ka and ClC-Kb
chloride channels leads to a syndrome clinically not distinguishable
from Bartter type IV. This event should be due to the tight topology
of the highly homologous CLCNKA gene which might predispose to
an unequal crossing over leading to partial or complete deletions of
the CLCNKB gene. We hypothesize that this chimaeric resulting gene
interferes with the correct function of both the channels, and leads to a
Bartter IV-like phenotype.
P0666. Evaluation of the stress effect on expression of BEST-5
gene in cultured hepatocytes and cloning of this gene
G. Hassanshahi;
Rafsanjan University of Medical Sciences, Rafsanjan, Islamic Republic of Iran.
Liver has important roles in body metabolic regulation and for this
reason hepatocytes are used worldwide. Investigations showed that
isolation of hepatocytes causes activation of stress related genes. The
aim of this study was to study the stress related expression of BEST-5
following hepatocytes isolation and culture.The BEST5 gene is cloned
and analysed for the first time from isolated and cultured rat hepatocytes.
Very little is known about this gene and almost nothing is known
about its function. RNA was isolated from hepatocytes after 3h culture
and used for generation of PCR products corresponding to the BEST5.
cDNA generated was cloned into pCR ® 2.1 plasmid vector. Following
transformation into TOPO10 oneshot ® cells, the cells were grown in LB
agar plates containing X-Gal and ampicillin, overnight at 37°C. To confirm
that the plasmids contained inserts of the correct size, the vectors
obtained from mini-preparations were digested with the desired restriction
enzymes. Sequencing was performed for the gene. RT-PCR and
Northern blotting analysis showed that BEST5 mRNA is expressed, 3h
after isolation and culture of primary hepatocytes (3h) BEST5 mRNA
was observed until 5h of culture and then there is no detectable band
of BEST5 at further time points. Comparison of expression of the level
of mRNA of BEST5, when data statistically were analysed, there was
a significant difference between the expression of BEST5 mRNA expression
at 3h with 0h, 24h, 35h and 48h of culture (PT substitution
at IVS1 nucleotide 7 was identified in an adult woman in the
heterozygous state and in her child related to the codon 39 mutation.
The genetic compound codon 39/IVS1-7 was found in a fifteen years
old boy with the thalassemia intermedia phenotype.
The new mutation here described seem to be completely in superposition
with the haematological phenotype associated to the IVS1-6
mutation in carrier state. It is assumable that this mild phenotype can
be related to the progressive distance between the mutation and the
consensus sequence of the first intron donor site of the β gene, relieving
the negative consequence of the efficiency of the splicing mechanism.
P0668. A Novel Cryptic Splice Site in IVSI-110 β-thalassemia
S. Mansoori Derakhshan 1,2,3 , H. Wardan 1 , M. Kleanthous 4 , S. Christou 5 , J.
Vadolas 1 ;
1 Cell and Gene Therapy Group, The Murdoch Childrens Research Institute,
Royal Children’s Hospital, Melbourne, Australia, 2 Paediatrics department, Melbourne
University, Melbourne, Australia, 3 Tabriz University of Medical Science,
Tabriz, Islamic Republic of Iran, 4 The Cyprus Institute of Neurology and Genetics,
Nicosia, Cyprus, 5 The Thalassaemia Center, Nicosia, Cyprus.
The IVSI-110 β-thalassemia mutation was first described in 1981 and
is one of the most common β-globin splicing mutations found in patients
of Greek or Cypriot origins. This mutation is the result of a G to A
substitution at position 110 in the first intron. This mutation has previously
been reported to generate an aberrant 3’ acceptor splice site,
which is preferentially recognised by the spliceosome over the normal
3’ acceptor splice site. The IVSI-110 mutation leads to a 90% reduction
in normal β-globin chain synthesis causing transfusion-dependent
disease in homozygous patients. Our group recently reported the creation
and characterisation of ‘humanised’ transgenic mice containing
the IVSI-110 human β-globin locus. Using human β-globin-specific RT-
PCR, we noted two human β-globin-specific aberrant spliced products.
We observed quantitative differences in the aberrant β-globin specific
RT-PCR spliced products in peripheral blood and bone marrow-derived
cells from ‘humanised’ IVSI-110 transgenic mice and IVSI-110
human patient-derived peripheral blood cells. We attribute the relative
differences in the level of aberrant splice products to mRNA instability.
In this study, we confirm by cloning of RT-PCR products and
DNA sequencing the identity of the novel activated cryptic 3’ acceptor
site in mice and also in humans containing the IVSI-110 mutation. We
conclude, that the identification of novel cryptic splice site indicates
that IVSI-110 splicing is more complex than previously reported, and is
worthy of further investigation.
P0669. Combination of Hb Knossos [Cod 27 (G-T)] and IVSII-745
(C-G) in a Turkish Patient with Beta-Thalassemia Major
I. Keser 1 , E. Manguoglu 1 , O. Kayisli 1 , A. Yesilipek 2 , G. Luleci 1 ;
1 Akdeniz University, School of Medicine, Department of Medical Genetics, Antalya,
Turkey, 2 Akdeniz University, School of Medicine, Department of Pediatrics,
Antalya, Turkey.
Beta-thalassemia is the most common disease among hemoglobinopathies
in Antalya, Turkey, as well as worldwide. Mutations found in
Turkish beta-thalassemia patients constitute a heterogeneous group,
consisting mostly of point mutations. Only in very rare cases, deletions
or insertions cause affected or carrier phenotypes. Hb Knossos (beta
27 (B9) Ala-Ser) is a rare variant with a normal HbA2 level. In this
study, we aimed to investigate the effect of compound heterozygosity
for Hb Knossos [Cod 27 (G-T)] and IVSII-745 (C-G). To our knowledge,
this is the first report of such a combination related with beta-thalassemia
major phenotype in a Turkish family, where Reverse Dot Blot
Hybridisation (RDBH) and DNA sequencing analysis were used. Heterozygous
inheritance of the mutation results in mild beta-thalassemia