2008 Barcelona - European Society of Human Genetics

Clinical genetics
P01.037
Validating assays for relative quantification of CFTR cDNA from
nasal epithelial brushing
L. Masvidal1 , A. Alvarez2 , L. Ruano2 , X. de Gracia2 , T. Casals1 ;
1 2 Medical and Molecular Genetics Centre. IDIBELL, Barcelona, Spain, Cystic
Fibrosis Unit. Hospital Vall d’Hebron, Barcelona, Spain.
Real-time PCR has proven to be a useful method to quantify gene expression
in samples with small number of cells . We have applied this
technology to validate the relative quantification of CFTR RNA from
nasal brushing of CF patients in a Taqman assay (ABI 7300) . Four
endogenous genes have been evaluated (HPRT1, beta2-M, GUSB,
PMCA4) . Standard curves were performed for CFTR and endogenous
genes from the cell line HEK293 over expressing CFTR, nasal polyp
and nasal epithelial (NE) samples . The selection of suitable endogenous
genes for normalization is a prerequisite for accurate determination
of expression level. Hence, PCR efficiencies have been determined
and two different software programs (NormFinder, qBase) have
been used to evaluate expression stability in NE samples from CF
patients (n=9) and controls (n=9) . DNAs from all individuals were analyzed
for CFTR gene . RNA quality was determined and only samples
with a RIN above 5 .2 were included . The experiment has been carried
out twice from two independent RT reactions and each sample
was analyzed in triplicate. We have identified three endogenous genes
suitable for accurate normalization of CFTR data expression .
Supported by Spanish ISCiii project PI050804 .
P01.038
A diagnostic approach for characterization of the cFtR gene
defects by mRNA analysis
L. Porcaro, L. Costantino, V. Paracchini, D. Coviello, L. Claut, M. Di Cicco, A.
Monti, P. Capasso, D. Degiorgio, C. Colombo, M. Seia;
Fondazione Policlinico Mangiagalli e Regina Elena, Milan, Italy.
A significant percentage of CF alleles remain unidentified in most population,
even after extensive studies of the CFTR gene by PCR based
procedure . We believe that mRNA analysis may allow researchers to
define the pathogenic role of sequence variations not yet defined and
particularly splicing defects . After an extensive analysis at DNA level, in
a cohort of 745 CF patients, 81 alleles (6%) were still unknown . Aim of
this work was to evaluate the role of the CFTR analysis at mRNA level
as a diagnostic method for the characterization of molecular defects in
CF patients who still had one or two unidentified alleles. RNA was extracted
from nasal epithelial cells and collected using cyto-brush from
7 CF patients and 3 non-CF controls. The cDNA was amplified in six
overlapping fragments spanning the entire CFTR gene .Disease-related
mutations were identified in two patients; mRNA analysis performed
on two other related patients with a deletion of exon 2 at DNA level,
showed two novel transcription products carrying a deletion of exon
2-3 and an insertion of intron sequence of about 80bp near exon 6b,
respectively . Two patients presented low level of mRNA product and
should be analyzed by quantitative technique . One patient showed a
normal profile. In conclusion our data suggest that the defects at RNA
level could explain the pathogenic role of abnormal mRNA products in
Cystic Fibrosis onset . In our experience, CFTR mRNA analysis represents
an effective diagnostic tool for the identification of unknown
molecular defects of the CFTR gene .
P01.039
mALDi-tOF based multiplex assay for cystic Fibrosis newborn
screening
P. Raña 1 , C. Colon 2 , A. Carracedo 1 , F. Barros 1 ;
1 Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela,
Spain, 2 Hospital Clinico Universitario de Santiago, Santiago de Compostela,
Spain.
Cystic fibrosis (CF) is a heterogeneus disease and one of the most
common autosomal recessive deseases known in the European population.
Since the identification of the responsible gene, CFTR, more
than one thousand mutations have been identified, most of which are
very rare . The number and the selection of mutations tested for vary
among laboratories and countries, being common to tailor the mutation
panels to local patiente population served . Limited sets of mutations,
as the ACMG/ACOG 25 mutations and commercially available
panels, are insufficiently sensitive for certain groups within a diverse
population . We developed and evaluated a MALDI-TOF based mul-
tiplex genotyping assay to detect 185 CF mutations . The methodology
is extremely sensitive, allowing the genotyping of very small dried
blood spot samples usually employed in newborn screening programs .
Furthermore, the Sequenom MALDI-TOF platform is a rapid and high
throughput system, allowing to process a high number of samples simultaneusly
in a cost-effective manner . We validated the system by
performing the assay on 348 dried blood samples in the Galician CF
newborn screening pilot program, and 75 samples in the American CF
proficience testing (CDC) and the European CF aqality assessment
scheme (CF European Network) . In the 2006-2007 period we detected
11 new CF cases, of wich 4 cases would be missed using the ACMG/
ACOG minimal panel of 25 mutations .
P01.040
A retrospective analysis of patients tested for cystic fibrosis
mutations in a reference Genetics center in izmir, turkey
B. Durmaz1 , H. Onay2 , G. Itirli2 , H. Akin2 , O. Cogulu1 , F. Ozkinay1 ;
1Department of Pediatrics, Ege University, Faculty of Medicine, Izmir, Turkey,
2Department of Medical Genetics, Ege University, Faculty of Medicine, Izmir,
Turkey.
Cystic fibrosis (CF) is an autosomal recessive disorder of epithelial ion
transport caused by mutations in the cystic fibrosis transmembrane
conductance regulator (CFTR) gene . The CFTR gene is located at
7q31 .2 and functions as a chloride channel and controls the regulation
of other transport pathways . More than 900 mutations and variants
have been described in the CFTR gene . We have restrospectively
evaluated the molecular test results of 550 CF patients referred to the
Molecular Genetics Laboratory of Medical Genetics Department, Faculty
of Medicine, Ege University, Izmir - Turkey in the last three years .
Patients had been tested for 36 mutations in the CFTR gene using the
strip assay method (Innogenetics, Belgium) . Out of 550 patients (1100
alleles) tested, 488 (88 .73%) did not carry any mutations, while 27
patients (4 .91%) were either homozygous or compound heterozygous,
35 (6 .36%) carried only one detected mutation . Allelic frequencies for
the six most common mutations in the positive groups were 41 .57%
(F508del), 14 .61% (I148T), 11 .24% (2183AA-G), 7 .87% (N1303K),
4 .49% (W1282X) and 4 .49% (R347P) . The rest of the alleles (15 .73%)
showed rare mutations which were 3210+1G-A, 3199del6, 621+1GT,
G85E, 2789+5GA, G542X, R117H, 3849+10kbCT . No patient showed
the mutations 2184delA, I507del, 1717-1GA, R334W, 3659delC,
G551D, 1078delT, R1162X, R560T, A455E, 711+5GA, R553X, Q552X,
394delTT, E60X, 2143delT, 3905insT, CFTRdele2,3(2 .1kb), 711+1G-T,
3272-26A-T, 1898+1G-A . In conclusion, the referrals for CF mutation
analysis increased annually . The low mutation detection rate may be
associated with the physicans’ attitudes as they started to use molecular
testing in the differential diagnosis of the diseases .
P01.041
Allelic heterogeneity of glycogen storage disease type iii: a
study of 34 patients
M. Hebert 1 , F. M. Petit 1 , A. Nadaj 2 , L. Capel 1 , F. Parisot 1 , A. Mollet-Boudjemline
3 , P. Laforêt 2 , P. Labrune 3 ;
1 Department of biochemistry, hormonology and genetics, Antoine Béclère Hospital
(AP-HP), Clamart, France, 2 Institute of Myology, Pitié-Salpêtrière Hospital
(AP-HP), Paris, France, 3 Department of paediatrics and genetics, referral Center
for Inherited Metabolic Liver Diseases, Antoine Béclère Hospital (AP-HP),
Clamart, France.
Introduction
Glycogen storage disease type III (GSDIII) is due to the deficiency of
the glycogen debranching enzyme (AGL). Deficiency of AGL activities
causes an incomplete glycogenolysis resulting in the accumulation, in
liver and/or muscle of an abnormal glycogen . Clinical symptoms include
variable tolerance to fasting, hypoglycaemia and hepatomegaly,
frequently accompanied by muscular hypotonia and hypertrophic cardiomyopathy
. To date, 71 genetic alterations of AGL gene have been
described in GSDIII patients .
Material
Here we report the molecular characterisation of 34 GSDIII patients .
The 33 AGL gene coding exons were screened by single strand conformation
polymorphism and sequenced when an abnormal electrophoretic
profile was observed. The allelic distribution of the c.3199C>T
and c .3343G>A polymorphisms were determined on 64 control healthy
patients by PCR-amplification and enzymatic digestion.