2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
activation.We investigated the function of the two PHD finger domains
and the COOH terminal portion of AIRE by using several mutated constructs
transfected in mammalian cells and a luciferase reporter assay .
The results predict that the second PHD as well as the COOH terminal
regions have marked transactivational properties . Our studies indicate
a prevalent role of the second PHD since the C446Y mutation, which
alters the PHD2 sequence led to a complete loss of the transactivation
activity .
The COOH terminal region contains the fourth LXXLL and the PXX-
PXP motifs which play a critical role in mediating the transactivation
capacity of the AIRE protein . On the other hand, the
crucial role of the PXXPXP sequence has already been defined by the
finding of the disease-causing mutation in the sequence of APECED
patients. Our study provides a definition of the role of the PHD fingers
in transactivation and identifies a new transactivation domain of the
AIRE protein localized in the COOH terminal region .
P05.010
Homozygosity mapping with sNP arrays as a useful technology
for diagnosis in complex diseases as Bardet-Biedl syndrome in
consanguineous families
I. Pereiro1 , M. Piñeiro1 , D. Valverde1 , D. Nishimura2 ;
1 2 University of Vigo. Spain, Vigo, Spain, Department of Paediatrics, Howard
Hughes Medical Institute, University of Iowa, Iowa, IA, United States.
Bardet-Biedl syndrome (BBS, MIM 209900) is a rare pleiotropic human
genetic disorder that has as primary phenotypic features: early-onset
retinitis pigmentosa, obesity, renal abnormalities, limb abnormalities
and also a variable degree of cognitive impairment .
This disorder is genetically heterogeneous with twelve genes identified
(BBS1-BBS12) . BBS also shows considerable inter- and intra-familial
variation of the phenotype . To date mutation screening has resulted
in the identification of approximately 70% of the causative mutations,
indicating that additional BBS genes have to be identified.
At the moment is very hard and time consuming to search for mutations
in each of the genes involved, as some of them have been implicated
in a very low percentage . In this study we employed high-density SNP
genotyping for homozygosity mapping in the identification of gene mutations
to simplify this task in consanguineous families .
Nine consanguineous families were analyzed, and in five of them linkage
to a known BBS loci was detected . Sequencing of the BBS gene
that localizes in the locus where linkage was detected, revealed 5 new
mutations (G2X, BBS3; L454P and G250R, BBS6; P108 and del 2pb
in 372, BBS12) . Cosegregation of the mutation in the family corroborates
the pattern of autosomic recessive inheritance .
For the rest of the families linkage indicated several novel candidate
BBS gene loci .
P05.011
Hypomorphic mutations in syndromic encephalocoele genes are
associated with Bardet-Biedl syndrome
A. Diaz-Font 1 , C. C. Leitch 2 , N. A. Zaghloul 2 , E. E. Davis 2 , C. Stoetzel 3 , S. Rix 1 ,
M. Al-Fadhel 4 , R. A. Lewis 5 , W. Eyaid 4 , E. Banin 6 , H. Dollfus 3 , P. L. Beales 1 , J. L.
Badano 2,7 , N. Katsanis 2 ;
1 Institute of Child Health, London, United Kingdom, 2 McKusick-Nathans Institute
of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore,
MD, United States, 3 Laboratoire de Génétique Médicale, Faculté de Médecine
de Strasbourg, Université Louis Pasteur, Strasbourg, France, 4 Department of
Pediatrics, King Fahad Hospital, Riyadh, Saudi Arabia, 5 Departments of Ophthalmology,
Molecular and Human Genetics, Pediatrics, and Medicine, Baylor
College of Medicine, Houston, TX, United States, 6 Department of Ophthalmology,
Hadassah-Hebrew University Hospital, Jerusalem, Israel, 7 Institut Pasteur
de Montevideo, Montevideo, Uruguay.
Meckel-Gruber syndrome (MKS) is a genetically heterogeneous, neonatal
lethal malformation and the most common form of syndromic
neural tube defects (NTDs) . To date, several MKS genes have been
identified, whose protein products affect ciliary function 1-5 . Here we
show that mutations in MKS1, MKS3 and NPHP both cause Bardet-
Biedl syndrome (BBS) and also have a potential epistatic effect on mutations
in known BBS loci . Five of six families with MKS1 and BBS mutations
manifested seizures, a feature that is not a typical component
of either syndrome. Functional studies in zebrafish showed that mks1
is necessary for gastrulation movements and that it interacts genetically
with known bbs genes . These observations are not restricted to
MKS1 . We also found two families with missense or splice mutations
in MKS3, one of which also bears a homozygous nonsense mutation
in NPHP6 that likely truncates the extreme C-terminus of the protein .
These data extend the genetic stratification of ciliopathies and suggest
that BBS and MKS, although clinically distinct, are allelic forms of the
same molecular disorder .
P05.012
Bartter syndrome: think of CLCNKB
E. Kamsteeg, J. Schoots, N. V. A. M. Knoers, H. Scheffer, L. H. Hoefsloot;
Radboud University Medical Centre, Nijmegen, The Netherlands.
Bartter syndrome (BS) is an autosomal recessive disorder characterized
by renal salt wasting and hypokalaemic metabolic alkalosis . The
primary defect is a reduced NaCl reabsorption in the thick ascending
limbs of Henle’s loop . Four types of recessive BS exist: congenital
without (I/II) or with sensorineuronal deafness (IV) and the milder form
(III) . Mutations in SLC12A1, KCNJ1, CLCNKB and BSND give rise to
BSI-IV, respectively . Here, we have analyzed CLCNKB in a group of
KCNJ1-mutation negative BS patients (n=60) .
More than half of the published mutations in BSIII are (partial)
CLCNKB deletions, caused by unequal cross between the homologous
CLCNKA and CLCNKB . Therefore, we have performed multiplex
ligation-dependent probe amplification analysis of CLCNKB . Deletion
of the entire gene has been observed homozygously in four and heterozygously
in 2 patients . Three patients have a heterozygous deletion
of the 5’ end of the gene (promoter region through exon 8) . Additionally,
sequence analysis revealed a second mutation in the five patients
with a heterozygous deletion . In the remaining patients, we have found
seven patients with two mutations (both in the homozygous or compound
heterozygous state), but also 7 patients with only one mutation .
It is unclear whether the second mutation is present elsewhere in the
CLCNKB gene, or whether one of the other genes involved in BS is
mutated .
Together, BS type III has been confirmed in 16 of the 60 BS patients.
Since we have found mutations in KCNJ1 in 10 patients, we suggest
that BSIII is more prevalent than BSII .
P05.013
Exploring the contribution of conserved Non-coding sequences
(cNcs) to Blepharophimosis syndrome (BPEs)
B. N. D’haene 1 , C. Attanasio 2 , M. Friedli 2 , D. Beysen 1 , B. Lorenz 3 , P. Lapunzina
4 , B. Lowry 5 , M. Pugeat 6 , T. de Ravel 7 , W. Reardon 8 , G. Pierquin 9 , A. Reiner 10 ,
R. Fisher 11 , S. Del Polo 12 , B. Menten 1 , K. Buysse 1 , F. Pattyn 1 , F. Speleman 1 , A.
De Paepe 1 , S. E. Antonarakis 2 , E. De Baere 1 ;
1 Center for Medical Genetics, Ghent University, Ghent, Belgium, 2 Department
of Genetic Medicine and Development, University of Geneva Medical School,
Geneva, Switzerland, 3 Regensburg University Medical Center, Department
of Pediatric Ophthalmology, Regensburg, Germany, 4 Department of Medical
and Molecular Genetics, Pathology and Paediatric Endocrinology, Hospital
Universitario La Paz, Madrid, Spain, 5 Department of Medical Genetics, Alberta
Children’s Hospital, Calgary, AB, Canada, 6 Department of Endocrinology, East
Pole of Lyon Hospitals, and INSERM Unit 0322, Lyon, France, 7 Center for
Human Genetics, Leuven University Hospitals, Leuven, Belgium, 8 National
Centre for Medical Genetics, Our Lady‘s Hospital for Sick Children, Crumlin,
Dublin, Ireland, 9 Department of Human Genetics, Hôpital du Sart Tilman, Liege,
Belgium, 10 Institute of Human Genetics, International Centre for Life, University
of Newcastle, Newcastle, United Kingdom, 11 St James’s University Hospital,
Leeds, United Kingdom, 12 Department of Paediatrics, Hospital 12 de Octubre,
Madrid, Spain.
Blepharophimosis syndrome (BPES) is a development disorder caused
by FOXL2 mutations, total gene deletions or extragenic deletions . In
12% of patients however, the molecular defect remains unknown . We
hypothesise that copy-number variations (CNVs) or point mutations in
cis-regulatory regions mapping within the minimal extragenic deletion
region could affect FOXL2 transcription and cause disease .
In a panel of 33 molecularly unresolved BPES patients, one novel extragenic
deletion upstream of FOXL2 was found by array CGH . This
deletion overlaps with the previously defined shortest region of overlap
(SRO) of upstream extragenic deletions, and was confirmed by qPCR.
In addition, qPCR of 25 CNCs located in this SRO revealed putative
CNVs in 9 patients, of which the significance is being evaluated. Moreover,
sequencing of the 25 CNCs revealed 4 putative pathogenic variants,
which are further being investigated by luciferase assays .
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