2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
mately 56 mutations have been described in the CYP17A1 gene . We
present the molecular characterization of two novel mutations in this
gene in a mexican-mestizo 46,XY female with CAH and severe hypertension
who does not respond to treatment . Gene sequence revealed
a compound heterozygous (K110X/ R362H) leading to a complete lack
of enzyme activity . The patient was heterozygote for four SNPs . We
suggest that these polymorphisms could be related to the failure of
blood pressure treatment . We propose that all DSD patients require a
multidisciplinary team to determine the etiology and orient the therapeutic
approach to minimized medical, psychological and social complications
.
P05.031
molecular genetic analysis of cYP2D6 gene and used methods
E. Flodrova1,2 , A. Žourková3,2 , T. Kašpárek3 , J. Juřica2 , R. Gaillyová1,2 ;
1Department of Medical Genetics, University Hospital, Brno, Czech Republic,
2 3 Faculty of Medicine, Masaryk University, Brno, Czech Republic, Department
of Psychiatry, University Hospital, Brno, Czech Republic.
The cytochrome CYP2D6 is enzyme responsible for metabolisation
of many commonly used drugs such as tricyclic antidepressants, neuroleptics,
beta blockers and antiarhythmics . CYP2D6 is localized together
with pseudogenes CYP2D7 and CYP2D8 at chromosome 22
and it is highly polymorphic . The polymorphisms leads to different individual
responses following drug administration and increased risk of
adverse reactions or the lack of the therapeutic response . According
to the enzymatic activity the population has been grouped as - poor
metabolizers, intermediate metabolizers, effective metabolizers, ultrarapid
metabolizers. There are studied influences of polymorphisms to
the antidepressants treatment for a long time in Department of Medical
Genetics and Department of Psychiatry .
Methods such as direct sequencing and agarose electroforesis are
subsequently combinated with more modern, faster and more effective
method - Real-Time PCR and High Resolution Melting using the Light
Cycler 480 System . Recently we can detect the most frequent null alleles
3* 4* 6* 7* and 8* using the specific fluorescent labeled probes.
Up to 99% of poor metabolizers in the Caucasian population can be
detected with genetic testing for only 5 alleles (plus allele 5*, which can
be detected by using the Sybr green) . The High Resolution Melting has
been performed for the most frequent SNP´s in CYP2D6 gene . Heterozygous
samples are readily distinguished from homozygous and
wild-type samples with used dyes . This paper provides an overview of
current technologies available for assessing polymorphisms in Department
of Medical Genetics .
Supported by research project MSM 0021622404 (2005 - 2011)
P05.032
the clinical Use of Fluorescent Repeat-Primed PcR Assay in the
Diagnosis of myotonic Dystrophy type 1
E. L. Spriggs 1,2 , P. Frosk 1 , R. Ray 1 ;
1 Molecular Diagnostic Laboratory, Diagnostic Services of Manitoba, Winnipeg,
MB, Canada, 2 Departments of Biochemistry & Medical Genetics and Pediatrics
& Child Health, University of Manitoba, Winnipeg, MB, Canada.
Some trinucleotide repeat expansion disorders require Southern blot
analysis to confirm the presence or absence of a large repeat expansion
adding days to turnaround time . Cagnoli et al . (2004) successfully
applied the use of fluorescent repeat-primed PCR (RPPCR) method to
differentiate between patients who are homozygous for a normal-sized
allele and those who are heterozygous for a very large expansion for
Friedreich ataxia and spinocerebellar ataxia types 10 and 12 . Using
a slight modification of this RPPCR method, we were able to consistently
detect the presence of large repeat expansions in the 3’UTR of
the DMPK gene, the mutation underlying myotonic dystrophy type 1 .
To assess the validity of this assay in a clinical setting, all 63 patient
samples and 9 quality assurance samples received over 18 months for
myotonic dystrophy type 1 were analyzed by the usual “gold standard”
method as well as by RPPCR . In the standard method, the number of
CTG repeats is determined using fluorescent PCR followed by capillary
electrophoresis . For a situation where only one normal-sized repeat is
observed or two normal-sized repeats that are only one trinucleotide
repeat apart in size, the sample is further tested by Southern blot analysis
. A review of all data showed 100% correlation between the results
obtained from the “gold standard” and RPPCR . As RPPCR can only
show the presence of the expansion and cannot provide information
regarding size of the expanded allele, Southern blot must be pursued
for RPPCR-positive patients . For RPPCR-negative patients, Southern
blot analysis is unnecessary .
P05.033
Human miRNAs on chromosome 21 are differentially expressed
in Down syndrome fetal hearts
F. Fabbrini, A. Izzo, R. Negri, R. Cicatiello, A. Ferraro, R. Genesio, A. Conti, L.
Nitsch;
University Federico II, Napoli, Italy.
We previously demonstrated dosage-dependent upregulation of chromosome
21 (Hsa21) genes and dysregulation of mitochondrial and
ECM genes in heart tissues of Down syndrome (DS) fetuses . Some
of these dysregulated genes might be responsible for the DS cardiac
phenotype, but it is evident that also other functional non-coding sequences,
such as miRNAs, might play an important role . MiRNAs are
highly expressed in the heart and regulate cardiac development and
function . Five miRNAs, according to Sanger miRBase, are on Hsa21:
miR-99a, miR-125b, let-7c, miR-155 and miR-802 . Nothing is known
about their expression in trisomic tissues .
We evaluated by qRT-PCR the expression of Hsa21 miRNAs in heart
tissues from DS fetuses and controls . We found that miR-99a, miR-
125b, let-7c and miR-155 were expressed in 20-22 weeks fetal heart .
MiR-802 was not expressed . MiR-99a, miR-155 and let-7c were overexpressed
in trisomic hearts, whereas miR-125b was normoregulated .
Target genes of upregulated miRNAs were obtained by merging Pic-
Tar, TargetScan and MiRanda prediction lists . As miRNAs could affect
protein expression by either interfering with RNA translation or promoting
mRNA degradation, we evaluated the mRNA expression of target
genes of overexpressed miRNAs by using the data set of our previous
study . Seventeen targets of miR-99a, 12 of miR-155 and 15 of let-7c
were expressed in fetal heart and downregulated in trisomic samples .
Target genes possibly involved in DS phenotype were found such as
SLC25A4, let-7c target, downregulated in DS hearts and involved in
mitochondrial function, and CYP26B1, miR-99a target, showing a dosage-dependent
effect on ventricular septal defects .
P05.034
study of folate genes alteration CBS 844ins68 and MTR A2756G
as maternal risk factor of Down syndrome among iranian cases
M. T. Mirgani, A. Aleyasine;
National Institue for Genetic Engineering and Biotechnology, Tehran, Islamic
Republic of Iran.
Down syndrome is the most common chromosomal abnormality occured
1 in 700 live birth . Abnormal segregation of chromosome 21 in
meiosis is the cause of trisomy 21 . Common polymorphisms in enzymes
coding genes in folic acid pathway have been suggested to
play role in etiology of chromosome 21 nondisjunction . In this study,
the role for 844ins68 polymorphism in cystathionine beta synthase
(CBS) gene and A2756G polymorphism in methionine synthase gene
(MTR) in folic acid pathway has been investigated as maternal genetic
risk factor of Down syndrome among Iranian patients . CBS 844Ins68
polymorphism is a 68 bp insertion in exon 8 of this gene and MTR
A2756G polymorphism causes an A>G transition at 2756 bp . We have
studied 93 mothers having DS children and 116 aged matched control
mothers for the frequency of above polymorphisms using RFLP
PCR analysis . Genomic DNA was extracted from blood Leukocytes by
salting out procedure . Frequencies of 844Ins68 polymorphism were 8
(8 .6%) in mother cases and 18 (15 .5%) in control mothers with no homozygousity
in both groups . Frequency of A2756G polymorphism was
observed as AA in 56 (60 .2%) AG in 33 (35 .5%) and GG in 4 (4 .3%) in
cases mothers and were 56%, 35 .3% and 10% among control mothers
respectively . Statistical analysis showed no association between
844ins68 and A2756G polymorphisms and risk of Down syndrome in
Iranian mothers (P-value for 844Ins68 and A2756G were 0 .146 and
0 .451 respectively) . Combination of these polymorphisms also have
no effect on risk of Down syndrome’s mother (P-Value was 0 .216 ) .