2008 Barcelona - European Society of Human Genetics

Molecular and biochemical basis of disease
of Friedreich’s ataxia . In cases with GAA expansions, ataxia, scoliosis
and pes cavus, cardiac abnormalities and some neurological findings
occurred more frequently than in our patients without GAA expansion .
mtDNA deletions were present in 76% of our patients representing
mtDNA damage, which may be due to iron accumulation in mitochondria.
Our findings showed that complex I activities and intracellular
ATP were significantly reduced (P=0.001) in patients compared with
control . 8 .6 kb deletion in mtDNA was detected in all of patients by
multiplex PCR but Southern blot analysis confirmed the presence of
deletion in 9 of 12 patients .
P06.182
the developmental changes in mitochondrial DNA content per
cell in human liver and muscle tissue during gestation
M. Pejznochova, M. Magner, M. Tesarova, H. Hansikova, J. Zeman;
Faculty of Medicine, Prague, Czech Republic.
The process of oxidative phosphorylation system (OXPHOS) maturation
is still not fully understood during the development of the fetus .
Since all mammalian cells except erythrocytes depend on mitochondrial
ATP production, knowledge of the OXPHOS maturation is an essential
query. Our previous study demonstrated significant changes
of mitochondrial DNA (mtDNA) content in cord blood leukocytes during
the gestation . To futher understand the role of mtDNA content in
human prenatal development, we analyzed mtDNA amount in human
fetal liver and muscle tissue during the gestation .
DNA was isolated from 26 liver and 26 muscle tissue samples obtained
at autopsy from miscarriages between 13 th and 28 th week of gestation .
The mtDNA amount was analyzed by the real-time PCR method using
SybrGreen I (Chromo4, Bio-Rad) .
The significant positive correlations were found between the gestational
age and the relative mtDNA amount in fetal liver tissue (r = 0,56;
p < 0,01) and mtDNA amount in fetal muscle tissue (r = 0,61; p < 0,01),
respectively .
In both fetal liver and muscle tissue, mtDNA content per cell was increasing
with onward fetal development . These results are in accordance
with the few of studies but there was never analysed as numerous
set of tissue samples as in our study . Presently we analyze a
relation among changes of the mtDNA content during prenatal development
and the transcription activation of some mtDNA maintenance
factors (NRF1, TFAM) in the same set of tissue samples .
This work was supported by grant GAUK 25755707, IGA MZ-NR
9410,GACR 305 08 H037 .
P06.183
Duplication analysis of the DmD gene by mLPA technique
J. Juan, M. Rodríguez, L. González-Quereda, L. Sedano, E. del Río, E. Tizzano,
M. Baiget, P. Gallano;
Hospital Sant Pau, Barcelona, Spain.
Duchenne and Becker muscular dystrophies (DMD/BMD) are the
most common form of dystrophinopathies, with a reported incidence of
1:3500 and 1:18000 birth males, repectively . About 65% of DMD/BMD
cases are attributable to large deletions of the DMD gene, whereas the
remaining cases are caused by duplications or point mutations of the
gene . The exact frequency of these two last types of mutations is not
known . The detection of duplications in Duchenne (DMD)/ Becker muscular
dystrophy (BMD) has long been overlooked . Recent techniques
such as multiplex ligation-dependent probe amplification (MLPA) have
simplified the detection of duplications.
We report here 20 duplications recently detected in BMD/DMD unrelated
patients using MLPA technique . Although the reading frame rule
could be applied in most patients, it was not applied in 2 DMD patients .
Special care was taken with one patient presenting non contiguous
rearrangements: c.[2623_2950dup; 11047_11055del], a duplication
of exons 21 and 22, and a deletion of exon 79 . The majority of the
duplications clustered towards 5’ end of the gene .
The availability of new quantitative methods including MLPA has
aroused interest in duplication detection analysis in DMD gene . The
MLPA technique enables us to test all the 79 exons, allowing the detection
of a significant number of new duplications. We report the duplications
in patients in whom a previous molecular screening did not
show any deletion .
P06.184
A novel mLPA technique for copy number analysis on small
amounts of DNA
K. M. Sorensen 1 , P. S. Andersen 1 , L. A. Larsen 2 , M. Schwartz 3 , J. Schouten 4 ,
A. Nygren 4 ;
1 Statens Serum Institut, Kbh. S, Denmark, 2 Wilhelm Johannsen Centre of Functional
Genome Research, Kbh. N, Denmark, 3 Department of Clinical Genetics,
Rigshospitalet, Kbh. N, Denmark, 4 MRC Holland, Amsterdam, The Netherlands.
Background: The MLPA technique was introduced in 2002 as a new
sensitive technique for relative quantification of up to 40 different nucleic
acid sequences in a single, extremely easy to perform, reaction .
Today the technique is routinely used for copy number analysis in various
syndromes and diseases .
Aim: The aim is to exploit the potential of MLPA in areas where DNA
material is limited . The DNA concentration required in standard MLPA
analysis is not attainable from dried blood spot samples (DBS) often
used in neonatal screening programs . By redesigning the MLPA probes
we attempt to perform MLPA analysis on small amounts of DNA .
Patients and methods: 7 patients with Congenital Adrenal Hyperplasia
(CAH) were used in this study . DNA was extracted from both whole
blood and DBS, and subjected to MLPA analysis using SALSA P050B
probemix (normal and modified probes). Results were analysed using
GeneMarker (Softgenetics) and Excel spreadsheet analysis .
Results: We found a 99 .6% concordance of the results obtained with
DNA extracted from whole blood compared with DNA from dried blood
spot samples .
Conclusion: We demonstrate that MLPA reactions with modified probes
are functional and reliable at very low DNA concentrations . This broadens
the diagnostic perspectives of biobanks consisting of DBS allowing
for CNV analysis in general and particularly testing for CAH .
This study is supported by the SAFE Network of Excellence
P06.185
is sNPs in MSX gene playing a role in the development of cleft
palate (CP) or cleft lip/palate (CLP)?
B. Lace 1 , I. Vasiljeva 2 , I. Dundure 1 , B. Barkane 3 , I. Akota 3 , A. Krumina 1 ;
1 Dept. of Med.Biology and Genetics, Riga, Latvia, 2 Biomedical study and research
centre, Riga, Latvia, 3 RSU, Institute of Stomatology, Riga, Latvia.
MSX1 gene role in the development of cleft palate and cleft lip/palate
and oligodontia has been confirmed in several studies. MSX1 (member
of muscle segment homeobox gene family) gene expression is related
to transcription repression during embriogenesis and craniofacial
development .
It was confirmed, that mutations in MSX1 gene approximately in 2%
can cause nonsyndromic CL/CLP . Results were replicated in studies
involving different populations .
Objective of our study was to evaluate role of mutations and also SNPs
in MSX1 gene in Latvian nonsyndromic CP and CLP patients .
Materials and methods . DNA from 101 persons with CP or CLP was
extracted from venous blood (CP- 21, CL/CLP-80) . Promoter region,
exons, introns boundaries and 3` UTR region were sequenced .
Results . One mutation 457G>C in exon 2 and 16 SNPs in MSX1 gene
was identified. Thirteen of them were described earlier and were commonly
met in Caucasian populations, three of them were not observed
in previous studies .
Interestingly two SNPs, who were localized in intron 1 451+(41-51)del
and 3`UTR 3969 A>G showed higher frequencies in nonsyndromic
CLP patients (0 .41;0 .38 respectively) than in CP patients (0 .17;0 .14) .
Conclusions . Considerable difference observed between SNPs in patients
with CP and CLP, who had localised in the non-coding regions,
raise suggestions about their possible role in the development of nonsyndromic
CLP .
P06.186
Association of c677t and A1298c polymorphisms in the
methylenetetrahydrofolate reductase (mtHFR) gene with cervical
dysplasia in Yucatan, mexico
L. J. Gonzalez-Herrera1 , P. Rodriguez-Morales1 , V. Fernández-Gonzalez1 , I.
Rosado-Lopez2 , T. Canto de Cetina1 ;
1 2 Universidad Autonoma de Yucatan, Merida, Mexico, Clinica de displasias del
Hospital O´Horán, Merida,Yucatan, Mexico.
The influence of MTHFR activity on DNA methylation, synthesis and
repair, presents MTHFR as a candidate cancer-predisposing gene .
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