2009 Vienna - European Society of Human Genetics

Molecular basis of Mendelian disorders
recessive inheritance, caused by a homozygous mutation in VEGFR3.
The novel mutation is a transition from alanine-to-threonine in amino
acid 855, located in the ATP binding domain of the VEGFR3 receptor.
Assessment of receptor function showed impaired ligand-induced
internalization and ERK1/2 activity. Moreover, receptor phosphorylation
was reduced, although, less so than for a kinase-dead VEGFR3
mutation, which causes Nonne-Milroy disease. In conclusion, a hypomorphic
VEGFR3 mutation, with moderate effect on the receptor, in
a homozygous state can result in insufficient lymphatic functioning.
Thus, in addition to Nonne-Milroy disease with dominant inheritance,
VEGFR3 alterations can cause isolated recessive primary congenital
lymphoedema. These data expand our understanding of the aetiology
of congenital lymphoedema and suggest that large sale screening of
VEGFR3 in all primary lymphoedema patients is necessary.
P12.133
Novel PccA gene rearrangements causing propionic acidemia
represent 21% of the total mutant alleles
L. R. Desviat1 , R. Sánchez-Alcudia1 , B. Pérez1 , C. Pérez-Cerdá1 , R. Navarrete1
, R. Vijzelaar2 , M. Ugarte1 ;
1Centro de Biología Molecular Severo Ochoa CSIC-UAM, Madrid, Spain,
2MRC-Holland, Amsterdam, The Netherlands.
Propionic acidemia is caused by mutations in the PCCA or PCCB
genes coding for the two subunits of the propionylCoA carboxylase
enzyme. Most of the mutations detected to date in both genes are missense.
In the case of PCCA deficient patients, a high number of alleles
were uncharacterised, some of them suspected to carry an exonic deletion.
We have now employed multiplex ligation probe amplification
(MLPA) to screen for genomic rearrangements in the PCCA gene in
20 patients with incomplete genotype. Eight different deletions were
found, corresponding to a frequency of 21,3% of the overall PCCA alleles.
Two of the exonic deletions were frequent, one involving exons
3-4 and another exon 23. Long-range PCR and chromosomal walking
were performed to identify the deletion breakpoints. This revealed two
different genomic deletions, both including exons 3 and 4, present in
our sample. In all the cases studied, repetitive elements, Alu sequences
or simple repeats were found at both sides of the deletion suggesting
that these repeats are involved in its generation. The pathogenicity
of all the deletions is established based on their predicted effects or on
expression analysis of the deletion alleles. This work describes for the
first time the high frequency of large genomic deletions in the PCCA
gene, which could be due to the characteristics of the PCCA gene
structure and its abundance in intronic repetitive elements. Our data
underscore the need of using gene dosage analysis to complement
routine genetic analysis in PCCA patients.
P12.134
the effect of lysinuric protein intolerance (LPi) -causing
mutations on y+LAt-1 / 4F2hc dimerization analyzed with
acceptor photobleaching FREt microscopy
M. Tringham 1 , M. Toivonen 1 , J. Salmi 1 , P. Terho 2 , K. Huoponen 1 , O. Simell 3 , J.
Mykkänen 3 ;
1 Department of Medical Biochemistry and Genetics, University of Turku, Turku,
Finland, 2 Turku Centre for Biotechnology, University of Turku, Turku, Finland,
3 Department of Paediatrics, University of Turku, Turku, Finland.
y+LAT-1 and 4F2hc form a transporter complex for cationic amino acids
in the basolateral membrane of epithelial cells, mainly in the small
intestine and proximal kidney tubules. Mutations of y+LAT-1, 51 of
which are currently known, cause lysinuric protein intolerance (LPI,
OMIM #222700), characterized by diminished intestinal absorption of
the cationic amino acids lysine, arginine and ornithine and severe loss
of these amino acids into the urine. We previously established fluorescence
resonance energy transfer (FRET) microscopy as a tool in
studying the interactions of y+LAT-1 and 4F2hc. We now have expanded
the field into exploring the effects of three different LPI-causing mutations
(Finnish founder mutation 1181-2 A->T, G54V and 1548delC)
on the dimerization of the transporter complex. Based on the results
of this study, the mutations studied do not alter the dimer formation
but mutated y+LAT-1 interacts with 4F2hc in a manner similar to that
of the wild type. This can be observed by means of fluorescence resonance
energy transfer confocal microscopy. The unquenching of the
donor fluorescence reports of the occurrence of FRET and thus of the
interaction of y+LAT-1 and 4F2hc. Consequently, we conclude that the
interaction between the subunits is a primary phenomenon, which occurs
irrespective of the LPI-causing mutations in the light subunit.
P12.135
A Proteus syndrome case with PtEN gene mutation
B. Imko-Walczuk 1 , M. Podralska 2 , W. Cichy 3 , A. Plawski 2 ;
1 University of Medical Sciences, Bydgoszcz, Poland, 2 Institute of Human Genetics,
Poznan, Poland, 3 University of Medical Sciences, Poznan, Poland.
Proteus syndrome is severe disorder of symmetric and disproportionate
overgrowth of body parts. The first manifestations of disease are
observed at birth and progress significantly with age. Proteus disease
belongs to hamartomatous syndromes and is characterized by multifocal
overgrowth of tissue, especially ectodermal and mesodermal
tissue. We present a case of 12-year-old boy treats in A. Jurasz University
Hospital in Bydgoszcz, Poland. Patient was born by natural
childbirth at 33 week of gestation. Birth weight was 3200 g. The Apgar
scale was scored on 9 points. The family history was negative. Since
births time it has been observed overgrowth and deformation of lower
right limb and left foot. Follow-up examination showed extensive dorsal
angioma. Based on these findings at 36 months of age the Klippel-Trenaunay
syndrome was diagnosed. Because of progression of
skin, vascular and soft tissular changes the diagnosis was changed
for Proteus syndrome. At the moment patient fulfills diagnostic criteria
of Proteus syndrome. Our patient presents main sings of Proteus
syndrome like asymmetric overgrowth lower limbs, macrodactyly, protuberant
foots, scoliosis, seen skin lesions and vascular malformation.
Progression of soft tissue hyperplasia in right limb and foot deformity
is watched
The entire coding sequence of PTEN gene was sequenced by direct
PCR product sequencing. The deletion of one nucleotide was identified
at the end of intron 7. The mutation was IVS7-3delT. The deletion
occurred at splice junction.
The study was supported by the Polish Ministry of Science and Higher
Education projects no. 2PO5E02630 and N401014435
P12.136
Multiplex ligation-dependent probe amplification refines
molecular diagnosis in pseudoxanthoma elasticum
L. M. Costrop 1 , O. M. Vanakker 1 , P. Coucke 1 , L. Martin 2 , N. Chassaing 2 , I.
Pasquali- Ronchetti 3 , A. De Paepe 1 ;
1 Center for Medical Genetics, Ghent, Belgium, 2 Department of Dermatology,
Angers, France, 3 Department of Biomedical Sciences, Modena, Italy.
Background: Pseudoxanthoma elasticum (PXE) is a recessive disorder
characterized by oculocutaneous and cardiovascular manifestations,
due to mineralization of elastic fibres. The disorder is caused
by mutations in the ABCC6 gene, encoding an ATP-dependent transmembrane
transporter. By conventional methods, more than 200 missense
and nonsense mutations have been described, and the current
ABCC6 mutation detection rate accounts for 94% of disease alleles
in our cohort. Remaining disease alleles can be partly explained by
middle-sized deletions, which can be missed by direct sequencing. We
aimed to optimize ABCC6 analysis by screening for such deletions using
multiplex ligation-dependent probe amplification (MLPA).
Methods: We performed MLPA in a cohort of 35 out of 331 biopsy
proven PXE patients, in whom only one or no mutations were detected
after screening for the frequent multi-exon 23-29 deletion and direct
sequencing of the ABCC6 coding region and exon/intron boundaries.
Results: Ten deletions were observed comprising five different multiexon
deletions and five novel single-exon deletions.
Conclusion: For this cohort, we demonstrated that approximately 25%
(10/41) of currently undetected ABCC6 disease alleles can be identified
using MLPA, yielding mutation detection rates of over 95 % in our
PXE population. Our results demonstrate that the majority of PXE mutations
are situated in the ABCC6 gene and indicate that the involvement
of another gene locus in classic PXE is unlikely. These results
improve the efficacy of familial screening and genetic counseling in
PXE. Therefore, we propose MLPA as a valuable additional screening
method in the molecular analysis of the ABCC6 gene.