2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
ples of three groups of women: 42 known PM females from families
of FXS, 25 with POF and 17 without; and 80 females with POF, and
normal FMR1 . The following genes were analysed: FMR1; BMP15;
GDF9; INHA; NOBOX and FOXE1 polyalanine tract length . Two kits of
MLPA for the X chromosome were also studied .
Our results revealed 3 new PM carrier women without history of MR
(3/80=3.75%) which confirms that FMR1 gene is the first cause of
POF . Furthermore, we found a microdeletion by MLPA (Xq28-Xqter)
(1/80=1 .25%) in a woman with POF and a family history of POF . In
the group of previously known PM carrier women no differences were
found except for the number of CGG repeats that is higher than 100 in
the group without POF .
As a conclusion, our work demonstrates the need of performing genetic
analysis in women with POF .
P05.161
Acute intermittent porphyria: impact of mutations in the
porphobilinogen deaminase gene on the protein structure
D. Ulbrichova, J. Zeman, P. Martasek;
1st School of Medicine, Prague 2, Czech Republic.
Acute intermittent porphyria (AIP) is an autosomal dominantly inherited
disorder, classified as acute hepatic porphyria. It is characterized
by a deficiency of porphobilinogen deaminase (PBGD, EC 4.3.1.8), the
third enzyme in heme biosynthesis . Expression of the disease is highly
variable, determined in part by environmental, metabolic and hormonal
factors . Clinical expression of AIP is associated with an acute neurologic
syndrome, which includes both neuropsychiatric symptoms and
neurodegenerative changes . Autonomic neuropathy may underlie severe
abdominal pain, the most typical symptom of acute attack . Porphobilinogen
deaminase is monomeric protein with a single catalytic
active site and is organized in three domains approximately equal in
size . Several crystallographic structures of PBGD from E . coli have
been determined .
To establish the effects of newly found mutations on the protein structure
of PBGD, we expressed mutant constructs with the described mutations
in E . coli, and we analyzed their biochemical and enzymatic
properties. All purified enzymes carrying causative mutations had relative
activities dramatically decreased according to the average level
expressed by the normal allele .
E . coli and human PBGD amino acid sequences have 35% homology
and more than 70 % similarity, and considering this fact, it is possible
to extrapolate structure/function relationships for human mutations
leading to simple amino acid substitution based on comparative E .
coli/human analyses . These analyses together with the kinetic studies
of existing mutations can help predict the impact on the enzyme
function in the living organism and further improve our understanding
in this field. (Supported by Grants MSM0021620806, 1M6837805002,
GAUK 257540 54007)
P05.162
molecular characterization of Premature Ovarian Failure
associated with FmR1 premutation
N. Locatelli 1 , K. Camelin 2 , B. Bodega 1 , S. Bione 3 , D. Toniolo 4 , C. Santoro 2 , E.
Ginelli 1 , A. Marozzi 1 ;
1 Dept. of Biology and Genetics for Medical Sciences - University of Milan,
Milan, Italy, 2 Dept. of Medical Sciences - University of Piemonte Orientale, Novara,
Italy, 3 Institute of Molecular Genetics-CNR, Pavia, Italy, 4 Dibit-San Raffaele
Scientific Institute, Milan, Italy.
Premature Ovarian Failure (POF) is defined as secondary hypergonadotropic
(FSH ≥ 40 IU/l) amenorrhea occurring before the age of
40, and affects ~1% of females . Women who carry the FMR1 premutation
are at an increased risk for ovarian insufficiency. To precisely
define the range of FRAXA expansions and its incidence in POF manifestation
209 POF and 200 control women were sized for the CGG
tract. We found a significant association (10%, P < 1x10-6) between
POF and FRAXA premutation (range 60-163 repeats), and a significant
enrichment (5,7%, P = 0 .006) of POF carriers of intermediate
expansions (range 41-58 repeats) . The results obtained strengthen
the correlation between FMR1 expansion and POF . The molecular
pathogenesis this association is still unclear . As for FXTAS patients,
a toxic RNA−mediated gain of function model for POF women carrying
the FMR1 premutated allele was hypothesized . The POF condition
might be a consequence of a toxic effect of FMR1 expanded mRNA
on ovarian target tissue (e .g . oocytes or granulosa cells), leading to
increased atresia/apoptosis of follicles . To determine the possible interaction
between premutated FMR1 RNA and ovaric proteins, gel shift
assay were set up incubating the nuclear extracts of the granulosa
cell line COV434 with premutated FMR1 RNA. A specific interaction
between granulosa nuclear proteins and FMR1 premutated RNA were
observed, suggesting that rCGG- repeated binding protein are present
in granulosa cells . To identify and characterize rCGG-repeat-binding
proteins, pull-down assays will be set up .
P05.163
Novel type of mutation in Pseudohypoparathyroidism type ia
E. Fernández-Rebollo 1 , R. Barrio 2 , G. Pérez-Nanclares 1 , A. Carcavilla 2 , I. Garin
1 , L. Castaño 1 , G. Pérez de Nanclares 1 ;
1 Endocrinology and Diabetes Research Group, Hospital de Cruces, Barakaldo,
Spain, 2 Pediatric Endocrinology, Hospital Ramón y Cajal, Madrid, Spain.
Individuals with AHO and resistance to PTH, TSH, and often, additional
hormones are referred to as having PHP-Ia . These patients carry
heterozygous inactivating mutations, including translation initiation
mutations, amino acid substitutions, nonsense mutations, splice site
mutations and small insertions or deletions, in one of the 13 GNAS exons
encoding the α-subunit of the stimulatory G-protein (Gsα). GNAS
has been describeb as one of the most complex gene loci as several
different transcript variants have been detected .
Objective: We sought to identify the molecular defect in a patient apparently
suffering from PHP-Ia .
Methods and results: The GNAS gene of a 5-yr-old boy with brachydactily,
mental retardation, pseudohypoparathyroidism, and congenital
hypothyroidism was investigated . Sequencing analysis of DNA extracted
from peripheral blood revealed a heterozygous mutation in exon 2,
consisting in an inversion of de novo origin and maternal inheritance,
that was explained by the analysis of the SNP located in exon 5 .
Molecular studies of cDNA prepared from blood RNA demonstrated
that both the normal and the mutant variants were stable . The anomalous
RNAs were generated by new splice-sites in intron 1, that produced
a protein of only 59 aminoacids due to the creation of a premature
stop codon in the processed intron 1 .
Conclusion: This report demonstrates for the first time an inversion at
the GNAS gene responsible of pseudohypoparathyroidism type Ia .
P05.164
Deficient inhibitors of calcification act as common final pathway
in PXE and the PXE-like syndrome
O. M. Vanakker 1 , L. Martin 2 , L. J. Schurgers 3 , P. J. Coucke 1 , C. Vermeer 3 , I.
Pasquali-Ronchetti 4 , A. De Paepe 1 ;
1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium, 2 Department
of Dermatology, Angers University Hospital, Angers, France, 3 Department
of Biochemistry (VitaK), University of Maastricht, Maastricht, The Netherlands,
4 Biomedical Sciences Department, University of Modena and Reggio
Emilia, Modena, Italy.
Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder,
characterized by oculocutaneous and cardiovascular manifestations,
due to mineralization and fragmentation of elastic fibres. The
causal ABCC6 gene encodes a transmembrane transporter; however,
the pathogenetic link with the elastic fiber abnormalities remains unknown
.
We recently identified a novel PXE-like syndrome - resembling PXE -,
associated with a vitamin K (VK)-dependent clotting factor deficiency.
We identified causal mutations in GGCX, encoding a gamma-carboxylase
. Together with phosphorylation by the Golgi casein kinase,
carboxylation represents an important posttranslational modification
necessary for activation of VK-dependent proteins, such as matrix gla
protein (MGP) and osteocalcin (OC), known to be calcification inhibitors
. Hence, this disorder provides novel possibilities to unravel the
pathogenetic events causing PXE .
In PXE-like patients, immunohistochemistry on skin tissues and serum
ELISA tests revealed increased levels of uncarboxylated (inactive)
MGP and OC, as a result of the GGCX mutations . In PXE patients,
similar results were obtained . An important difference in PXE patients
was however an increase of the phosphorylated fraction of inactive
MGP, whereas the phosporylated/unphosphorylated MGP ratio in
PXE-like samples was normal . This suggests that in PXE not only the
carboxylase activity is downregulated but also an upregulation of the