Advances in exon skipping trial development towards proof-of-concept and systemic application in Duchenne Muscular Dystrophy
Topic:  10. Therapy for genetic disease
J. T. C. van Deutekom¹, H. Heemskerk¹, C. de Winter¹, P. van Kuik², S. de Kimpe², G. Platenburg², G. J. B. van Ommen¹;
¹Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands, ²Prosensa B.V., Leiden, The Netherlands.
Presentation Number: C56
Keyword: DMD, therapy, antisense
Antisense oligonucleotides (AONs) are emerging as small molecule drugs with a high, sequence-specific, corrective potential on RNA level. Especially for Duchenne muscular dystrophy (DMD), AONs have demonstrated to be an efficient and relatively simple and safe alternative to gene therapy approaches based on gene replacement. Over the last five years we have successfully developed this antisense approach towards clinical application. In the first half of 2006, a “proof-of-concept” clinical study, based on intramuscular injections of an exon 51 skipping AON, will be undertaken in a selected group of DMD patients, of which the set-up will be discussed. To facilitate full-body treatment of DMD patients, we are now focusing on the development of a safe and efficient systemic AON-delivery method. Using the mdx mouse model, we have compared efficacy, persistence and biodistribution of an AON (containing 2'-O-methyl RNA with a full-length phosphorothioate backbone) targeting the mutated exon 23, after intravenous administration of increasing doses and intervals. We obtained relatively high exon 23 skipping levels (up to 45%) in all muscle types, including diaphragm and heart, resulting in significant dystrophin expression as detected by Western blot analysis. There was an increased muscle-specific uptake and efficacy of AON in the mdx mice when compared to control mice. A highly sensitive ligation ELISA for AONs, allowed us to assess the stability of the AON in the different organs and tissues, showing a half life of 10 days in skeletal muscle. The overall therapeutic effect was indicated by significantly decreased creatin kinase levels in plasma of treated mice, and by improved performance in RotaRod studies. These results offer a favourable perspective for the first clinical Phase I/IIa studies on AONs in DMD patients, suggesting that these are within reach in the next few years.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy (DMD) by fluorescence in situ hybridization (FISH): a case report
Topic:  2. Cytogenetics
H. Filkova¹, I. Slamova¹, P. Kuglik², R. Gaillyova¹, B. Ravcukova¹;
¹Department of Medical Genetics, Faculty Hospital, Brno, Czech Republic, ²Department of Genetics and Molecular Biology, Faculty of Science, Brno, Czech Republic.
Presentation Number: P0352
Keyword: DMD, PGD, FISH
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with an incidence of approximately 1 in 3500 males, caused by mutation in the DMD gene, located on Xp21.2. About 2/3 of DMD cases are caused by gross DMD gene deletion mutations.Mutations in the DMD gene result in a progressive muscle degeneration and early death.
We reported a case of a family with a occurence of DMD. By means of PCR deletion of exon 45-50 was founded at one year son with clinical proved DMD. His healthy mother was detected as carrier for this mutation. The preimplantation genetic diagnosis (PGD) was used for detection of deletion in dystrophine gene at next pregnancy.
PGD is a principally new approach for the prevention of genetic disorders, which allows the selection of unaffected IVF embryos for establishing pregnancies in couples. PGD can be applied for monogenic disorders or chromozomal abnormalities using diagnostic protocols based on the PCR or fluorescence in situ hybridization.
Seven embryos were biopsied and four of them were analyzed by FISH method by using exon 46-47 DNA probe. Of these, two affected male embryos , one affected female embryo and one unaffected male embryo were detected, it was transferred.
Supported by grant MSM 0021622415.

Study of Dystrophin gene's hotspots in 23 Iranian families suspected to DMD or BMD
Topic:  5. Molecular and biochemical basis of disease
M. Dehghanmanshadi, T. Majidizadeh, M. Rostami, B. Hooshiar kashani, S. Saber, M. Houshmand;
National Institute Center Genetics Engineering & Biotechnology, Tehran, Iran (Islamic Republic of).
Presentation Number: P0683
Keyword: DMD, BMD, dystrophin gene
The dystrophinopathies_Duchenne muscular dystrophy (DMD)and Becker muscular dystrophy (BMD)_are the most common inherited disorders of muscle. Although reliable prevalence data are lacking, the prevalence of DMD is generally estimated at 1:3,500 live male births . Both DMD and BMD are due to mutations in the dystrophin gene , located at Xp21,which comprises 79 exons and 8 tissue-specificpromoters
distributed across [[Unsupported Character - Symbol Font ∼]]2.2 Mb of genomic sequence_making dystrophin the largest gene yet described.
Dystrophin gene deletions are found in [[Unsupported Character - Symbol Font ∼]]55%of patients with BMD and 65% of patients DMD; point mutations account for [[Unsupported Character - Symbol Font ∼]]30% of mutations, and duplications account for the remainder.
Genetic testing for deletions relies on a multiplex PCR technique, with amplification of fragments containing 20 of the gene’s 79 exons and with deletions detected as absent or size-shifted bands on poly Acryl amide gel analysis. Because deletions tend to occur in “hotspots” within the dystrophin gene, analysis of this limited number of exons can detect 98% of dystrophin deletions.
Hot spots are exons3-19 and 42-60.We studied all of these exons for 23 Iranian families.
In our study most common of deletion were in exon6, exon44, exon50, exon4 respectively.

Duplications in the DMD gene in DMD/BMD patients in Serbia and Montenegro
Topic:  5. Molecular and biochemical basis of disease
T. Lalic¹, R. H. A. M. Vossen², J. Coffa³, J. P. Schouten³, M. Guc-Scekic¹, D. Radivojevic¹, M. Djurisic¹, M. H. Breuning², S. J. White², J. T. den Dunnen²;
¹Mother and Child Health Institute, Belgrade, Serbia and Montenegro, ²Center for Human and Clinical Genetics,Leiden University Medical Center, Leiden, The Netherlands, ³MRC-Holland, Amsterdam, The Netherlands.
Presentation Number: P0685
Keyword: duplications, DMD/BMD, MLPA
Although duplication mutations in the DMD gene were reported to be relatively frequent early on, the considerable effort to detect them made them largely neglected. This situation has changed only recently with the development of MAPH and MLPA, two easy and versatile methods for the detection of both deletions and duplications.We present here a retrospective study of 123 unrelated DMD/BMD patients (already screened for deletions in the hot spot regions using modified multiplex PCR kits).Rescreening these with MLPA revealed nine duplications, dispersed over the whole gene. This corresponds to a duplication rate of 17% among deletion-negative and 7% among all patients, an ocerall duplication frequency comparable with what previous studies had suggested.The majority of cases contain a simple contiguous duplication, but we also detected one non-contiguous duplication/triplication. In most non-contiguous duplications reported thus far the 3' end of the gene is affected. Whilst potentially disturbing the reading frame of the mRNA, these mutations would go undetected using standard multiplex PCR screening. These findings emphasize the importance of screening the entire gene for rearrangements and that duplications, compared to deletions, need to be treated with special care.

Mutant DMD allele carried by different maternal X chromosomes in two brothers
Topic:  6. Genetic analysis, linkage, and association
M. Czakó¹, E. Endreffy², E. Morava³, G. Kosztolányi¹;
¹Clinical Genetics Research Group, Department of Medical Genetics and Child Development, University of Pécs, Pécs, Hungary, ²Department of Pediatrics, Albert Szent-Györgyi University Medical School, Szeged, Hungary, ³Institutes of Neurology and Pediatrics, University Radboud Nijmegen Medical Center, Nijmegen, The Netherlands.
Presentation Number: P0915
Keyword: Duchenne muscular dystrophy, DMD
Segregation analysis of chromosomes carried by affected individual is a helpful approach for genetic counseling when the mutation itself is not identifiable. The interpretation of the finding, however, may be erroneous, since the mutant allele could be transmitted from one chromosome to the other by recombination event during homologous pairing, as the case presented indicates.
In two brothers Duchenne muscular dystrophy was proved by mutation analysis of the dystrophin (DMD) gene showing deletion of exons 45-50. We performed microsatellite marker analysis of the X chromosomes in the family, in order to identify the X chromosome carrying the mutation in the obligate carrier mother. The markers in (5’ DYS MSA, 3’ DYS MS, IVS44SK21, DMD-45, DMD-49, DMD-50, DysI, DysII, STR-44, STR-45, STR-49, STR-50) and around (DXS451) the DMD gene showed that the two brothers have, as expected, identical X chromosomal region. Surprisingly, however, markers on the distal short and long arms (DXS207, DXS691, DXS1283E) could be assigned to different maternal X chromosomes in the two sons, indicating that the DMD gene carrying the deletion might have been transferred between the two maternal X chromosomes.
Moreover, further extended marker analyses of samples taken from the maternal grandparents and other members of the mother’s family suggested germinal mosaicism of the mother. Our case shows that well-constructed microsatellite marker analyses, in addition to help genetic counseling, may shed lights on the nature and mechanism of disease causing mutations as well.

Deletion/Duplication screening of the DMD gene in 98 individuals using MLPA technique
Topic:  8. Genomics, technology, bioinformatics
P. Jorge, S. Brás, L. Rodrigues, E. Vieira, J. Oliveira, R. Santos;
Instituto de Genética Médica, Porto, Portugal.
Presentation Number: P1191
Keyword: DMD, MLPA, Duplications/deletions
MLPA, multiplex ligation-dependent probe amplification, is now widely recognized as a reliable method to detect both deletions and duplications in several genes, including DMD [MIM 300377]. Mutations in this gene are responsible for one of the most common neuromuscular disorders - the allelic Duchenne and Becker Muscular Dystrophies (D/BMD) [MIM 310200 and 300376].
In our laboratory, the commonly used multiplex PCR reactions, Southern blot and hybridization techniques have been used in the routine molecular diagnosis of D/BMD. In total, there were 373 unrelated referrals, including cases later seen to have been misdiagnosed, presenting defects in other genes (ex: sarcoglycans, CAPN3). These methods enabled the characterization of DMD deletions in 137 patients of which 45% involved exons 45-52, 13% exons 3-19 and 16% a single exon (either 44 or 45).
We used MLPA assay to screen for mutations in 68 unrelated patients (partially retrospective study). All 21 deletions were confirmed, and a further 15 duplications were detected (8 undocumented changes), two of which were already suspected based on routine screening results. Additionally, carrier status was ascertained and/or clarified in some families, also enabling determination of de novo versus familial mutations in sporadic cases, as well as the detection of gonadal mosaicism.
The authors highlight the added value of complementing routine methods with the MLPA technique in the molecular diagnosis of D/BMD, considering that gross deletions and duplications comprise the majority of mutations in the DMD gene.