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Report on the 16th ENMC workshop—Carrier diagnosis of Duchenne and Becker muscular dystrophy
Neuromusc. Dist~rd., Vol. 3, No. 3, pp. 2,11 242, 1993 Printed in Great Britain
0960-8966/93 $6.00 t 0.00 i 1 9 9 3 Pergamon Press Ltd
WORKSHOP REPORT R E P O R T ON T H E 16th E N M C W O R K S H O P - - C A R R I E R D I A G N O S I S OF DUCHENNE AND BECKER MUSCULAR DYSTROPHY Baarn, The Netherlands, 1 8 ~ 0 September 1992
The principal aims for molecular diagnostic laboratories working on Duchenne and Becker muscular dystrophy are to determine which females in a family are at risk of having an affected child, and to be able to offer them prenatal diagnostic testing of male fetuses. The first stage of this process is to identify the causative mutation in the family, after which female relatives can be tested for the presence or absence of the mutation, allowing accurate carrier diagnosis. In approximately 65% of cases, the mutation involves a partial deletion of the dystrophin gene. This can readily be detected in males by amplification of several loci in multiplex polymerase chain reaction (PCR) assays, since deleted loci will simply fail to amplify. However, determining whether a female carries a deletion on one of her X chromosomes is not so straightforward, as the normal X chromosome "masks" the presence of any deletion on the mutant X. The practical difficulties in determining female carrier status for deletional mutations are evident from the number of techniques which have been described for this purpose. At least seven methods are available, but none are currently applicable to all families. Each has its advantages and disadvantages, and the accuracy of the methods varies. The workshop brought together several workers, each with expertise in one or more of the specific methodologies used for carrier diagnosis. The aim was to discuss the advantages and disadvantages of the various techniques, particularly concentrating on difficulties and potential misdiagnoses that each method could show. After an introduction outlining the methods that were used prior to the application of molecular biology (H. Moser), the various molecular techniques were discussed, starting with hybridization methods then moving on to the more recently developed procedures using the 241
polymerase chain reaction. Finally, the importance of using immunological methods for the confirmation of diagnosis and the consequences on carrier diagnosis were discussed. The molecular methods discussed for detecting deletion heterozygotes were: dosage analysis and detecting junction fragments by Southern hybridization analysis with cDNA probes (E. Bakker); detecting junction fragments using cosmid probes (L. Blonden) or pulse field gel electrophoresis (C. van Broeckhoven); in situ hybridization (M. Cremer); the use of polymorphic markers that lie within deleted loci (A. Read); dosage analysis by PCR amplification, detecting the products with ethidium bromide staining (S. Abbs), radioactively (K. Panayides), or by automated fluorescent analysis (P. Ray); PCR amplication of reverse-transcribed mRNA (R. Roberts); and immunological analysis of the gene product, dystrophin (L. Nicholson). The advantages and disadvantages of these varied techniques, and the use of the M L I N K computer program for calculating carrier risks (A. Read), were discussed. It was agreed that some of the methods were very specialized, and required experienced workers to perform them competently. Even in the hands of such people, some of the techniques are not 100% accurate. The simplest, most robust method is unquestionably the use of polymorphic markers within deletions. The highly polymorphic short tandem repeat markers, such as described by Clemens et al. [1], are the most useful type available, but at present insufficient have been discovered to cover all deletion prone loci. In addition to this method, it was thought useful to employ one of the quantitative techniques, since a single assay covers a large number of deletions, particularly if a multiplex PCR assay is used, thus providing the most efficient method of searching for unidentified mutations in female relatives. A small number of presentations were given on recent research work that were relevant to carrier
242
Workshop Repor!
diagnosis. These included: methods for identifying point mutations (R. Roberts, V. Nigro): studies on dystrophin in the retina (P. Ray): recent data on intragenic recombination frequencies (C. Oudet); and data on male and female mutation rates (S. Abbs). A consortium was established in order to keep participants informed of developments relating to carrier diagnosis. A list will be collated detailing resources available for diagnosis, such as polymorphisms, cosmid and YAC clones from within the gene. Further workshops will be organized, if deemed necessary, in the future. Workshop participants S. Abbs (London, U.K.), E. Bakker (Leiden, The Netherlands), L. Blonden (Leiden, The Netherlands), M. Bobrow (London, U.K.), C. van Broekhoven (Antwerpen, Belgium), A. Covone (Genova, Italy), P. Gallano (Barcelona, Spain), F. Letureq (Paris, France), H. Moser (Bern, Switzerland), C. R. Mtiller (Wurzburg, Germany), L. Nicholson (Newcastle upon Tyne, U.K.), V. Nigro (Napoli, italy), C. Oudet (Strasbourg, France), K. Panayides (Nicosia, Cyprus), L. Politano (Napoli, Italy), P. Ray
(Toronto, Canada), A. Read (Manchester, U.K.), D. Recan (Paris, France), R. Roberts (London, U.K.). On heha(/ qf E N M C . A. E. H. Emery (Edinburgh, U.K.), Y. S. Poortman (Baarn, The Netherlands), M. R. Rutgers (Baarn, The Netherlands). Acknowledgements The authors wish to thank the members of ENMC for their excellentorganization of the workshop, and ENMC, Association Francaise contre les Myopathies IAFM) and the Italian "Comitato Promotore Telethon" for financial support of the workshop. STEPHEN ABBS and MARTIN BOBROW Paediatric Research Unit Division of Medical and Molecular Genetics UMDS, Guy's Hospital London, SE1 9RT U.K. REFERENCE
Clemens P R, Fenwick R G, Chamberlain J S, eta/. Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Gem't 1991: 49:951 960.
0960-8966/93 $6.00 t 0.00 i 1 9 9 3 Pergamon Press Ltd
WORKSHOP REPORT R E P O R T ON T H E 16th E N M C W O R K S H O P - - C A R R I E R D I A G N O S I S OF DUCHENNE AND BECKER MUSCULAR DYSTROPHY Baarn, The Netherlands, 1 8 ~ 0 September 1992
The principal aims for molecular diagnostic laboratories working on Duchenne and Becker muscular dystrophy are to determine which females in a family are at risk of having an affected child, and to be able to offer them prenatal diagnostic testing of male fetuses. The first stage of this process is to identify the causative mutation in the family, after which female relatives can be tested for the presence or absence of the mutation, allowing accurate carrier diagnosis. In approximately 65% of cases, the mutation involves a partial deletion of the dystrophin gene. This can readily be detected in males by amplification of several loci in multiplex polymerase chain reaction (PCR) assays, since deleted loci will simply fail to amplify. However, determining whether a female carries a deletion on one of her X chromosomes is not so straightforward, as the normal X chromosome "masks" the presence of any deletion on the mutant X. The practical difficulties in determining female carrier status for deletional mutations are evident from the number of techniques which have been described for this purpose. At least seven methods are available, but none are currently applicable to all families. Each has its advantages and disadvantages, and the accuracy of the methods varies. The workshop brought together several workers, each with expertise in one or more of the specific methodologies used for carrier diagnosis. The aim was to discuss the advantages and disadvantages of the various techniques, particularly concentrating on difficulties and potential misdiagnoses that each method could show. After an introduction outlining the methods that were used prior to the application of molecular biology (H. Moser), the various molecular techniques were discussed, starting with hybridization methods then moving on to the more recently developed procedures using the 241
polymerase chain reaction. Finally, the importance of using immunological methods for the confirmation of diagnosis and the consequences on carrier diagnosis were discussed. The molecular methods discussed for detecting deletion heterozygotes were: dosage analysis and detecting junction fragments by Southern hybridization analysis with cDNA probes (E. Bakker); detecting junction fragments using cosmid probes (L. Blonden) or pulse field gel electrophoresis (C. van Broeckhoven); in situ hybridization (M. Cremer); the use of polymorphic markers that lie within deleted loci (A. Read); dosage analysis by PCR amplification, detecting the products with ethidium bromide staining (S. Abbs), radioactively (K. Panayides), or by automated fluorescent analysis (P. Ray); PCR amplication of reverse-transcribed mRNA (R. Roberts); and immunological analysis of the gene product, dystrophin (L. Nicholson). The advantages and disadvantages of these varied techniques, and the use of the M L I N K computer program for calculating carrier risks (A. Read), were discussed. It was agreed that some of the methods were very specialized, and required experienced workers to perform them competently. Even in the hands of such people, some of the techniques are not 100% accurate. The simplest, most robust method is unquestionably the use of polymorphic markers within deletions. The highly polymorphic short tandem repeat markers, such as described by Clemens et al. [1], are the most useful type available, but at present insufficient have been discovered to cover all deletion prone loci. In addition to this method, it was thought useful to employ one of the quantitative techniques, since a single assay covers a large number of deletions, particularly if a multiplex PCR assay is used, thus providing the most efficient method of searching for unidentified mutations in female relatives. A small number of presentations were given on recent research work that were relevant to carrier
242
Workshop Repor!
diagnosis. These included: methods for identifying point mutations (R. Roberts, V. Nigro): studies on dystrophin in the retina (P. Ray): recent data on intragenic recombination frequencies (C. Oudet); and data on male and female mutation rates (S. Abbs). A consortium was established in order to keep participants informed of developments relating to carrier diagnosis. A list will be collated detailing resources available for diagnosis, such as polymorphisms, cosmid and YAC clones from within the gene. Further workshops will be organized, if deemed necessary, in the future. Workshop participants S. Abbs (London, U.K.), E. Bakker (Leiden, The Netherlands), L. Blonden (Leiden, The Netherlands), M. Bobrow (London, U.K.), C. van Broekhoven (Antwerpen, Belgium), A. Covone (Genova, Italy), P. Gallano (Barcelona, Spain), F. Letureq (Paris, France), H. Moser (Bern, Switzerland), C. R. Mtiller (Wurzburg, Germany), L. Nicholson (Newcastle upon Tyne, U.K.), V. Nigro (Napoli, italy), C. Oudet (Strasbourg, France), K. Panayides (Nicosia, Cyprus), L. Politano (Napoli, Italy), P. Ray
(Toronto, Canada), A. Read (Manchester, U.K.), D. Recan (Paris, France), R. Roberts (London, U.K.). On heha(/ qf E N M C . A. E. H. Emery (Edinburgh, U.K.), Y. S. Poortman (Baarn, The Netherlands), M. R. Rutgers (Baarn, The Netherlands). Acknowledgements The authors wish to thank the members of ENMC for their excellentorganization of the workshop, and ENMC, Association Francaise contre les Myopathies IAFM) and the Italian "Comitato Promotore Telethon" for financial support of the workshop. STEPHEN ABBS and MARTIN BOBROW Paediatric Research Unit Division of Medical and Molecular Genetics UMDS, Guy's Hospital London, SE1 9RT U.K. REFERENCE
Clemens P R, Fenwick R G, Chamberlain J S, eta/. Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Gem't 1991: 49:951 960.