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

Molecular and biochemical basis of disease
P0706. MLPA assay as a screening tool for mutations in DMD/
BMD patients and their female relatives
E. M. Neagu 1 , G. Girbea 1 , A. Constantinescu 1 , C. Constantinescu 1 , D. Iancu 1 ,
E. Manole 2 , C. Matanie 3 , E. Ionica 3 , N. Butoianu 4 , S. Magureanu 4 , L. Barbarii 1 ;
1 National Institute of Legal Medicine, Bucharest, Romania, 2 Colentina Clinical
Hospital, Bucharest, Romania, 3 University of Bucharest, Bucharest, Romania,
4 “Al. Obregia” Clinical Hospital, Bucharest, Romania.
Our study, part of a current national research program, aims to evaluate
the dystrophin gene mutational pattern for the Duchenne/Becker
muscular dystrophies (DMD/BMD). Knowing the mutational pattern of
these diseases is proved to be of prognostic value and major importance
for genetic counseling. For this purpose we recently introduced
the multiplex ligation-dependent probe amplification (MLPA) method
to identify mutations on the dystrophin gene in a series of DMD/BMD
patients and their female relatives. We report our one year results and
experience with the MLPA DMD commercial kits, which allow scanning
of all 79 dystrophin exons in two PCRs. The amplicons were analysed
on a 3100 Avant ABI Genetic Analyzer. We investigated 102 DNA samples
from: 52 male patients with clinical and muscular biopsy picture of
DMD, 43 female relatives, one amniotic fluid sample from a possible
affected fetus. We assessed the extent and location of deletions and
duplication and ascertained frequency of de novo or familial mutations
with major importance for genetic counselling. The MLPA screen revealed
mutations in 75% of cases: 85.72% deletions, 14.28% duplications
and one non-contiguous deletion. The largest deletion detected
involved almost half of the gene (exons 3-42), while the others were
restricted to 2-5 exons, especially the exons 45 - 50. Because most the
detected mutations encompassed more exons, no additional analysis
was performed. The method proves to be easy to handle, accurate,
highly reproducible. Our results recommend the MLPA assay as a routine
screening tool for dystrophin mutations.
P0707. Identification of deletions and duplications of the DMD
gene in Macedonian patients using Multiplex Ligation-dependent
Probe Amplification (MLPA)
S. A. Kocheva 1,2 , S. Trivodalieva 1 , S. Vlaski-Jekic 3 , M. Kuturec 2 , G. D. Efremov
1 ;
1 Research Center for Genetic Engineering and Biotechnology, MASA, Skopje,
The former Yugoslav Republic of Macedonia, 2 Pediatric Clinic, Medical Faculty,
Skopje, The former Yugoslav Republic of Macedonia, 3 Department of Neurology,
Medical Faculty, Skopje, The former Yugoslav Republic of Macedonia.
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy
(BMD) are caused in the majority of cases by deletions of the DMD
gene. Mutational analysis is complicated by the large size of the gene,
which consists of 79 exons and 8 promoters spread over 2.2 million
base pairs of genomic DNA. The majority of recognized mutations are
however, copy number changes of individual exons, which traditionally
have been identified by three common multiplex polymerase chain
reaction. Here we report the use of the newly developed quantitative
assay multiplex ligation-dependent probe amplification (MLPA) to determine
the copy number of each of the 79 DMD exons. The sensitivity
and accuracy of MLPA were assessed and compared with multiplex
PCR in total of 92 subjects with DMD or BMD. MLPA was able to detect
all previously detected deletions. In addition, we detected five new
deletion and four duplications. The extend of the deletions and duplications
could be more accurately defined which in turn facilitated a
genotype - phenotype correlation.
P0708. Reduction of sperm DNA-fragmentation via Magnetic
Assisted Cell Sorter (MACS)-system
T. Winkle 1 , F. Gagsteiger 2 , T. Paiss 1 , N. Ditzel 1 ;
1 ReproGen- Ulm, Ulm, Germany, 2 IVF-Zentrum Ulm, Ulm, Germany.
In the course of an ART and especially an ICSI the used spermatozoa
are meticulously examined according to WHO-criteria, like concentration,
motility and morphology. So a fragmentation or other deterioration
of the DNA cannot be detected. The aim of our study is to determine
and, if required, reduce the amount of spermatozoa with DNA-fragmentation
(DNA-Fragmentation Index, DFI) within the ejaculate.
Ejaculate samples of 55 patients were purified via the MACS-system.
The native and the purified part of the sample were then measured in
a cytometer. The purification was carried out with the help of magnetic
marked Annexin V and an appropriate column (the MACS-System).
The DNA was stained by 4´-6-diamidino-2-phenylindole (DAPI). In
1 1
each sample 20,000 cells were measured and the percentage of cells
with DNA-fragmentation was determined. In comparison, samples of
37 additional patients were analyzed as described above, both natively
and according to density gradient centrifugation.
The average DFI was 17.6% in native ejaculate, while, after purification
by MACS, this percentage was reduced to 11.29%. By purification
via density gradient centrifugation a percentage of 11.83% was
obtained, while the percentage in native ejaculate was 15.96%. So
we obtained a reduction via MACS of 35.85% and via density gradient
centrifugation of 25.88%.
On the basis of our results it has been found that by means of MACS
the DFI can be reduced distinctly (35.85% vs. 25.88%). Furthermore,
the high DFI in the samples purified by density gradient shows that
DNA-fragmentation cannot be reduced by this common method.
P0709. Folate gene alteration: Dose it influence the chromosome
21 nondisjunction in Down syndrome
S. Aleyasin, S. Rezaee, F. Jahanshad;
National Institute for Genetic Engineering and Biotechnology, Tehran, Islamic
Republic of Iran.
Common polymorphisms in the MTHFR (C677T and A1298C) and
MTRR (A66G) genes have been reported to be a maternal genetic risk
factor for Down syndrome in some populations. However, considering
parental origin of trisomy 21 in mother of Down syndromes has drawn
less attention in previous studies. This may cause reduction in degree
of associations of those maternal genetic risk factors and occurrences
of chromosome 21 trisomy. In this study parental origin of chromosome
21 were tested in 260 families of Down syndromes using five STR
markers related to chromosome 21 (D21S11, D21S1414, D21S1440,
D21S1411, D21S1412). Parental origins were determined successfully
for 226 of cases. Mothers have been categorized in maternal (198)
and paternal (28) groups. All mothers of Down patients totalled 226
individuals, and a normal control group contaned 176 mothers with
halthy children. These were tested for common polymorphisms C677T,
A1298C in the MTHFR and C66G in the MTRR gene. A significant
association was detected between maternal derived chromosome 21
mothers and A1298C of the MTHFR gene (P12.06). This
study has showed for the first time the importance of parental origin
determination in the study of maternal genetic risk factor of Down syndrome.
The significance of A1298C polymorphism of MTHFR gene as
maternal genetic risk factor of Down syndrome in Iranian population
increases the knowledge about etiology of Down syndrome and helps
in better prevention of Down syndrome.
P0710. Homozygous Dubin-Johnson Syndrome. A Novel
Mutation in MRP2 Gene in a Large Family from Slovakia
L. Barnincova1 , J. Behunova2 , P. Martasek1 ;
1Department of Pediatrics and Center of Applied Genomics, 1st School of Medicine,
Prague 2, Czech Republic, 2Children´s Hospital, Kosice, Slovakia.
Hepatobiliary excretion of conjugated bilirubin is mediated by an ATP
dependent canalicular transporter, the multidrug resistence associated
protein 2 (MRP2/cMOAT). MRP2 is responsible for biliary excrection
of glucuronide and glutathione conjugates of endogenous and exogenous
compounds.
Dubin-Johnson syndrome (DJS) is an inherited autosomal recessive
disorder characterised by the absence of functional protein MRP2 at
the canalicular membrane of hepatocytes. Known mutations in this
gene cause impaired maturation and trafficking of the mutated protein
from the endoplasmic reticulum to the Golgi complex.
As a result, conjugated hyperbilirubinemia, increased urinary excrection
of coproporphyrinogen isomer I, and deposition of melanin-like
pigment are found. Otherwise liver function is normal.
Here we present a study of a large Slovak family with unconjugated
hyperbilirubinemia Dubin-Johnson type. These subjects were found to
be homozygous for the novel mutation 1012delGT, which causes a
frameshift.
Dubin-Johnson syndrome is very rare disorder, and the homozygous
form of mutations has been reported only in unique cases.
Supported by MSMT 0021620806