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

Molecular and biochemical basis of disease
London, London, United Kingdom, 3 South West Thames, Regional Genetics
Unit, St Georges Hospital, London, United Kingdom.
Lymphoedema is a chronic tissue swelling usually of the lower extremities
caused by abnormal lymph drainage. There are 3 main clinical subtypes
of primary lymphoedema - Milroy disease (MD), Lymphoedema
Distichiasis (LD) and Meige disease, all of these conditions exhibit autosomal
dominant inheritance. MD patients present with lymphoedema
at birth or in early infancy. The penetrance of MD is estimated to be
about 88%. LD presents with distichiasis (aberrant eyelashes arising
on the inner eyelid) and lymphoedema in late childhood or puberty and
is about 90% penetrant.
To date two genes have been identified; VEGFR3 (or FLT4), associated
with MD and FOXC2, associated with LD. Mutations reported in
the VEGFR3 gene are primarily missense and have to date only been
found in exons 17-26 which encodes the highly conserved tyrosine
kinase domain. Diagnostic testing has been developed for the 10 exons
in the VEGFR3 gene using dHPLC on the Transgenomic WAVE
Analyser and any variants observed are sequenced in both directions.
The FOXC2 gene consists of a single 1.5kb exon encoding a forkhead
transcription factor. Bi-directional sequencing of 4 overlapping
fragments of FOXC2 is employed as a screening technique. To date
FOXC2 mutations found in the laboratory consisted only of small insertions
and deletions. However, missense and stop mutations have
been previously reported. Screening of both these genes is offered as
a clinical molecular diagnostic service.
P0784. Genetic screening for patients with non-obstructive
infertility from Ukraine
O. Fesai;
Institute of Molecular Biology and Genetics National Academy of Science of
Ukraine, Kiev, Ukraine.
Microdeletions of the long arm of the human Y-chromosome are associated
with spermatogenic failure and have been used to define three
regions of Yq (AZFa, AZFb, AZFc) that are recurrently deleted in infertile
males. It was supposed that apart from cystic fibrosis, mutations
in the cystic fibrosis transmembrane conductance regulator (CFTR)
gene are involved in male infertility. Mutations in the CFTR gene cause
congenital bilateral absence of the vas deferens (CBAVD) in approximately
1% of the infertile males.
We have screened Y-chromosome microdeletions, mutations and poly-
T sequence of CFTR gene in 680 patiens (azoospermia and oligospermia)
and in 621 healthy valantiers by PCR-based methods. For each
patient, multiplex PCR analyses were performed on DNA isolated from
leukocytes derived from peripheral blood to screen 18 sY-sequences
on Yq. Cytogenetic investigation was performed according to standard
methods (GTG-banding). No chromosomal anomalies were found in
men.
Microdeletions were detected in 2,79% infertile men. During patients
research we have made a conclusions: a) damage of genes from AZFb
region results in impairments of last stage spermatogenesis; b) deletions
in AZFc region are critical for early stage spermatogenesis.
The frequency of CFTR gene mutations carriers detected in 630 patients
was statisticaly significant higer than in control group. 5T allele
of CFTR gene associated with CBAVD was detected in 1,93% of patients.
The obtained data produce the evidence of the possible involment
of CFTR protein in spermatogenesis. The genetic consultation
and preimplantation analysis were recommended for the couples with
identified Y-chromosome deletions and CFTR gene mutations.
P0785. Expression profiling of human normal testis by
microarray technology
F. Raicu 1 , V. Gatta 2 , A. Ferlin 3 , C. Foresta 3 , G. Palka 2 , L. Stuppia 2 ;
1 Department of Clinical Sciences and Imaging, G. d’Annunzio University
Foundation, Chieti-Pescara, Italy, 2 Department of Biomedical Sciences, G.
d’Annunzio University Foundation, Chieti-Pescara, Italy, 3 Department of Medical
and Surgical Sciences, University of Padova, Padova, Italy.
Spermatogenesis is a complex process that demands several genes
to action but the biological mechanisms underlying sperm production
are still largely unknown. In this view, it is crucial to obtain a full picture
of the global expression profile of normal testis.
In order to identify the list of testis up and down regulated genes directly
linked to testis metabolism and function towards the human general
metabolism, we start to analyse, using an 21,329 spotted oligonucle-
20
otides microarray, global gene expression pattern of healthy testis biopsies
versus an home made RNA universal reference.
Our preliminary research revealed a group of 2.718 up-regulated and
2.654 down-regulated annotated UniGene in testis as compared to the
reference RNA pool. These annotated genes were grouped together
using the EASE software, which allowed us to identify a list of biological
process. Among these the most abundant categories were those
related to physiological processes and metabolism, which are very unspecific
classes, while less represented but more specific were those
containing genes involved in fuction related to human testis, such as
reproduction, meiosis, imprinting. Among these, of interest is the presence
of genes mapped within the AZF loci of the Y chromosome such
as DAZ, BPY, DBY, SMCY as well as DAZL autosomal homolog gene.
Our results clearly demostrates that expression profiling using microarray
technology is able to evidence genes specifically involved in testicular
fuction, and to provide a general pattern of the human normal
testis trascriptome, which could be certainly useful for understanding
also the pathological pathway.
P0786. Gene expression studies in testicular tissue.
Suitability of PBGD and HPRT as internal references genes for
normalization
E. Terribas 1 , S. Bonache 1 , E. Guinó 2 , J. Sánchez 3 , E. Franco 4 , L. Bassas 3 , S.
Larriba 1 ;
1 Centre de Genètica Mèdica i Molecular-IDIBELL, L’Hospitalet de Llobregat,
Barcelona, Spain, 2 Unitat de Bioinformàtica i Bioestadística-ICO-IDIBELL,
L’Hospitalet de Llobregat, Barcelona, Spain, 3 Servei d’Andrologia-Fundació
Puigvert, Barcelona, Spain, 4 Servei d’Urologia-Hospital Universitari de Bellvitge,
L’Hospitalet de Llobregat, Barcelona, Spain.
Semiquantitative gene expression experiments require normalization
to compensate for differences in the amount and quality of biological
material and reverse transcription reaction efficiency in the tested
samples. An extended strategy is to normalize to internal reference
genes, which should show similar expression in the samples investigated.
We have assessed the suitability of PBGD and HPRT genes as candidates
for gene normalization in both pathological and normal testicular
tissue. We have analysed their expression levels in testicular
biopsies of 13 non-obstructive infertile men, who showed either severe
hypospermatogenesis or maturation arrest (patient group 1), 7 men
diagnosed with germ-cell tumour (patient group 2) and 10 infertile men
with obstructive azoospermia (control group). The quantitative realtime
PCR reactions were performed in a LightCycler ® 1.5 Instrument
(Roche), using SYBR Green I fluorescence dye. The Mann-Whitney
U test was used to analyse gene expression differences in case and
control patients.
Non-significant differences were observed in PBGD expression levels
in both patient groups 1 and 2 compared to controls (P= 0.077 and P=
0.262, respectively) and in HPRT expression profile between patient
group 1 and control group (P= 0.515). Interestingly, HPRT was found
to be differently expressed in patient group 2 compared to controls (P=
0.001) suggesting that HPRT would not be a suitable reference gene
to be used for normalization of target genes expression data in testicular
malignant phenotype. Hence, while choosing reference genes,
the testicular phenotype under study should be considered to avoid
erroneous normalizations.
Supported by FIS (PI05/0759, CP03/00088, C03/07)
P0787. Microdeletions in AZF region of Y chromosome in
infertile males, a study from Latvian population
A. Puzuka 1 , A. Krumina 1 , V. Baumanis 2 , N. Pronina 3 , V. Lejins 4 , J. Erenpreiss 5 ,
J. Bars 6 , I. Grinfelde 3 ;
1 Riga Stradins University, Department of Medical Biology and Genetics, Riga,
Latvia, 2 Latvian Biomedical Research and Study Centre, Riga, Latvia, 3 Children
University Hospital, Medical Genetics Clinic, Riga, Latvia, 4 EGV Clinic, Riga,
Latvia, 5 Riga Stradins University, Laboratory of Andrology, Riga, Latvia, 6 Clinic
of Medical Genetics, Department of Prenatal Diagnostics, Riga, Latvia.
Male factor infertility accounts for about half the cases of couple infertility
in Latvia. The most common genetic cause of human Y-linked
male infertility is partial or complete deletions of the AZF region on the
Y chromosome. AZF region consists of three intervals, AZFa, AZFb,
and AZFc, which are associated with different spermatogenic failure.
The aims were to introduce the molecular screening method of Y-