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

Molecular and biochemical basis of disease
activity. In Latvian population the R408W mutation is associated with
RFLP haplotype 2, the VNTR-3 allele, and the 238bp-STR allele with
frequency 77% from all mutant chromosomes. Almost 56% of patients
are homozygous for the R408W, 40% are compound heterozygous
and 3,3% have no R408W mutation. According to the pre-treatment
Phe blood level and Phe tolerance, 90,2% (55/61) patients are classified
as having a classical PKU phenotype, 4,9% (3/61) patients - moderate,
and 4,9% (3/61) patients - mild.
Patients with moderate and mild PKU phenotype are compaund heterozygotes,
five of them have R408W in one allele and G272X, A104D
and E178G in other allele, two patients have an unidentified other
allele and one patient has no R408W mutation. Final results are in
progress.
P0839. PLP1 overexpression in PMD and PMLD patients: interest
of fibroblasts analysis
M. Bonnet-Dupeyron 1,2 , P. Combes 1 , L. Ouchchane 3 , G. Giraud 1 , O. Boespflug-
Tanguy 1,4 , C. Vaurs-Barriere 1 ;
1 INSERM, UMR384, Clermont-Ferrand, France, 2 CHU de Clermont-Ferrand,
Cytogénétique Médicale, Clermont-Ferrand, France, 3 CHU de Clermont-
Ferrand, Département de Santé Publique, Unité de Biostatistique, Télématique
et Traitement d’Image, Clermont-Ferrand, France, 4 CHU de Clermont-Ferrand,
Génétique Médicale, Clermont-Ferrand, France.
The myelin proteolipid protein 1 (PLP1), which encodes for the main
proteins of the CNS myelin, is implicated in the X-linked disorders of
CNS myelination, Pelizaeus-Merzbacher Disease (PMD) and Spastic
Paraplegia 2 (SPG2). Large duplications including the whole gene account
for 60% of gene alterations. Transgenic mice with additional Plp
gene copies are identically hypomyelinated and have demonstrated
that Plp overexpression may act in a dominant negative effect on myelinating
oligodendrocytes that enter into apoptosis.
By real time quantitative RT-PCR, we quantified PLP/DM20 mRNA
levels in nerve biopsies (n=6) and primary cultured fibroblasts (n=14),
from PMD patients carrying a PLP1 gene duplication. An overexpression
of the PLP/DM20 transcripts was observed in all samples compared
to controls, suggesting that PLP1 expression in fibroblasts and
in PNS may reflect PLP1 expression level in the CNS. Therefore, fibroblasts
from 17 male PMD/SPG2 patients without identified PLP1
or GJA12 abnormality (PMD Like, PMLD), were analyzed. Among
them, 8 present with a PLP/DM20 mRNAs overexpression, implicating
a PLP1 dysregulation due to mechanism(s) alternative to the gene
duplication. All have a mild form of dysmyelination. Neither genomic
mutations nor rearrangements have been found in the PLP1 promoter
and in the ASE cis regulating elements. A mutation was observed for
one patient in the 3’UTR. Its functional relevance on the mRNAs stability
is under evaluation.
In conclusion, fibroblasts represent a useful tool to quantify the PLP1
gene expression level and have allowed us to demonstrate that the
PLP1 gene may remain a candidate gene for PMLD patients.
P0840. Splicing abnormalities associated with different types of
PLP1 mutations.
M. Bonnet-Dupeyron1 , P. Combes1 , O. Boespflug-Tanguy1,2 , C. Vaurs-Barriere1
;
1 2 INSERM UMR384, Clermont-Ferrand, France, CHU de Clermont-ferrand,
Génétique Médicale, Clermont-Ferrand, France.
The myelin proteolipid protein 1 (PLP1) gene encodes, by alternative
splicing of its exon 3, for the main proteins of the CNS myelin: PLP
and DM20. Duplications of the gene and point mutations are implicated
in the X-linked disorder of CNS myelination: Pelizaeus-Merzbacher
Disease and Spastic Paraplegia 2. To assess the functional
consequences of different types of PLP1 mutations (7 exonic punctual
mutations, 3 deletions of 1 nucleotide, 3 consensus splicing site and 1
nucleotide substitution in intron 3 of unknown significance), PLP/DM20
transcripts were sequenced from peripheral nerves and/or primary cultured
fibroblasts.
Transcripts analysis has shown that the intron 3 mutation leads to the
intron retention, demonstrating its implication in the phenotype and
suggesting the potential existence of an intron splicing regulatory element
in the surrounding sequence. Unexpectedly, one of the 7 exonic
punctual mutations, initially thought to be a missense, was demonstrated
to impair the splicing by creating a new donor site exclusively
used, at least in the fibroblasts, instead of the classical site. In addition,
220
transcripts analysis has allowed to assess that mutations in the consensus
splicing sites are deleterious for PLP1 gene expression and to
identify the different cryptic sites that are alternatively used.
This study demonstrates that splicing abnormalities can result from
various types of mutations. Therefore, such approaches are useful to
better evaluate the genotype-phenotype correlations and to identify intronic
or exonic splicing regulatory elements.
P0841. Polycystin-2 regulates cellular proliferation in a p21/
Cdk2-independent manner
K. N. Felekkis 1 , P. Koupepidou 1 , E. Kastanos 1 , N. Gretz 2 , C. Bai 3 , L. Tsiokas 3 ,
C. Deltas 1 ;
1 University of Cyprus, Nicosia, Cyprus, 2 Medical Research Center, Klinikum
Mannheim, University of Heidelberg, Mannheim, Germany, 3 Department of
Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma, OK,
United States.
Autosomal dominant polycystic kidney disease (ADPKD) is a common
inherited disorder, characterized by progressive cyst formation
and loss of kidney function. ADPKD is principally linked to two genes,
PKD1 and PKD2. The pathogenesis is currently thought to involve
dysregulated epithelial cell proliferation and differentiation, alteration
in membrane proteins polarity and abnormal fluid accumulation. The
molecular mechanism of cystogenesis has not been fully elucidated.
A recent report implicated polycystin-2 in the regulation of epithelial
cell proliferation. Specifically, the authors suggested that PC-2 overexpression
suppresses cell proliferation through inhibition of the p21/
Cdk2 pathway. To better understand the role of PC-2 in epithelial cell
proliferation, we utilized various cellular models generated by stable
expression of mutated (R742X and 1-702) and wild-type PKD2. In
contrast to the previous data, over-expression of wild-type or mutated
polycystin-2 in two different cell-lines does not affect cellular growth.
On the contrary, electrophysiology experiments demonstrated that
overexpression of wild-type PKD2 increases both inwards and outwards
K+ currents in these cells. Interestingly, primary renal epithelial
cells from transgenic rats generated by expression of the 1-702 PKD2
have elevated levels of the proliferation marker, PCNA. However, in
both primary cells and stable cell lines, wild -type or mutated PC-2 do
not alter p21 levels and Cdk2 activity. Collectively, these data suggest
that in our models, PC-2 regulates epithelial cell proliferation in a p21/
Cdk2-independent manner and that PC-2 inactivation by itself is not
sufficient for abnormal cell proliferation. Experiments are underway to
verify these data by genome-wide expression and pathway analysis.
P0842. Digenic/Triallelic inheritance in polycystic kidney disease
suggests a new mutational mechanism with mutations in a
transcription factor and its regulated gene
N. Ortiz Brüchle1 , V. Frank1 , K. Häffner2 , K. Zerres1 , C. Bergmann1 ;
1 2 Department of Human Genetics, Aachen, Germany, Department of Pediatrics,
Freiburg, Germany.
The transcription factor HNF1ß has been recently shown to be crucial
for kidney development, as demonstrated by in vivo experiments
where Xenopus embryos microinjected with mutant HNF1ß RNA transcripts
had impaired pronephros development. HNF1ß binds specifically
as homo- or heterodimer in concert with HNF1α to the proximal
PKHD1 promoter and stimulates transcription of the gene responsible
for autosomal recessive polycystic kidney disease (ARPKD). Recent
studies revealed that mutations of the HNF1 binding site, expression
of a dominant-negative HNF1ß mutant and mice with renal-specific
inactivation of Hnf1ß exhibit a drastic defect in PKHD1 expression.
Here we present a patient with polycystic kidney disease heterozygote
for the novel PKHD1 1-bp deletion c.1151delA (p.P383fs), while
a second PKHD1 change was not identified. HNF1ß mutation analysis
revealed the novel, non-conservative missense mutation c.244G>A
(p.D82N) that affects an evolutionarily highly conserved residue and
considerably disturbs the protein’s DNA binding domain. Further arguments
of its pathogenic character are the conservation of p.D82 in
its counterpart HNF1α and that it was not present among 500 tested
chromosomes. Reporter gene analysis is currently underway to further
corroborate its functional significance. Beyond the first description of
digenic and/or triallelic inheritance in polycystic kidney disease, our
study is of general interest as it demonstrates an intriguing new regulatory
mutational mechanism with mutations in a transcription factor and
its activated gene.