Arch Dis Child: first published as 10.1136/adc.59.3.195 on 1 March 1984. Downloaded from

Archives of Disease in Childhood, 1984, 59, 195-196

Annotations DNA markers and Duchenne muscular dystrophy

The localisation of the for Duchenne muscular DNA sequences selected from collections of DNA dystrophy using DNA polymorphisms has been one (DNA libraries); these may be derived from X of the most exciting episodes in muscular dystrophy chromosome specific DNA (for example probe research and marks the first clinical application of RC8) or from total human DNA (probe L1.28). these polymorphisms to a genetic disorder where the Returning to the use of these techniques in the primary gene product is unknown. Previously the muscular dystrophies; Duchenne muscular dystro- main applications of the new DNA technology had phy was first found to be linked to probe RC8, with been to identify , such as those for the around 15% recombination between the two loci;' haemoglobinopathies, whose function was already later probe L1.28 was found to be a similar distance well understood; the possibility of using allied on the opposite side (Figure).2 Similar studies on techniques for those numerous disorders about families with Becker muscular dystrophy have found which little or nothing except their Mendelian that, contrary to previous thinking, it shows similar inheritance is known, is particularly important linkage relations to Duchenne muscular dystrophy, clinically, since tests of predicition and prevention and may well be allelic with it.3 are usually lacking or inadequate. Thus the Duchenne (and Becker) muscular dys- The paper by Dr Pembrey and colleagues in this trophy genes have now been pinned down to a issue of Archives is likely to be the first of many restricted area of the and current applying DNA polymorphisms to genetic disorders, research is focusing on means of identifying the and some of the concepts and terminology will be actual mutational defects. This may prove a difficult unfamiliar to paediatricians, just as were the sym- task, though if a proportion of cases results from an bols used in the early days of cytogenetics. It is actual deletion of the gene, as has been shown for worth explaining some of the main terms here. some thalassaemias and some cases of The term polymorphism, meaning an inherited B,4 these should be readily detectable by a DNA variation where two or more forms are seen with probe that includes the disease locus. http://adc.bmj.com/ reasonable frequency, will be familiar from blood The present markers, each with a built in error groups and characteristics. Restriction frag- rate in prediction of at least 15% because of ment length polymorphism (RFLP) refers to the recombination between marker and disease, are not variably sized DNA fragments produced when DNA ideal for clinical application, but with care and using is cut with a specific restriction enzyme. The poly- them in combination with currentlv available tests, morphism is shown up as different positions of they can be of real help to families. Pembrey et al bands on electrophoresis (see Fig. 1 in the Pembrey estimated that 69 of 70 families in their series paper), the smaller fragments travelling faster than could be helped to some degree, and selected on October 4, 2021 by guest. Protected copyright. the larger. For convenience and stability the DNA is examples of clinical use have already been usually transferred to a nitrocellulose filter by the reported.5 process known as Southern blotting. To show up such a polymorphism among the myriad fragments of DNA in a sample from white blood cells (or chorionic villus) requires a gene Short arm Long arm probe. This is a specific DNA sequence which will XRC8 L1.28 seek out and bind to any corresponding sequence Xg DMD I * * v present in the patient sample on the filter; since it is radiolabelled it will produce a band or bands when a radiograph is exposed against the filter. Gene Figure Schematic map ofthe short arm ofthe X chromo- probes can be produced from various sources and if some showing the approximate location ofthe Duchenne the gene product is known, it may be possible to muscular dystrophy locus (DMD) in relation to two DNA construct a specific cDNA probe. If not (as in markers. The locus for Becker muscular dystrophy is Duchenne muscular dystrophy), one has to use similar or identical. 19. Arch Dis Child: first published as 10.1136/adc.59.3.195 on 1 March 1984. Downloaded from

196 Harper Where both marker sytems are informative an (3) Isolated cases are rarely likely to give helpful accurate prediction can be given, since if no recom- information at present but in the future closer bination is found between the two markers, it would markers will allow a valuable normal prediction by require the unlikely event of a double crossover to showing that a second child has not inherited the give an error in prediction for Duchenne muscular same maternal gene as was transmitted to a previous dystrophy. More commonly only one marker will affected child. give information, and here the value of DNA tests (4) Prenatal use should await closer markers will usually be in combination with creatine kinase except in the few families where both probes give testing which may produce a definitive prediction information. that would not have resulted from the use of either approach on its own. Meanwhile, research developments that will iden- The detailed example given by Pembrey et al tify the Duchenne muscular dystrophy gene itself illustrates both the advantages and the current are eagerly awaited and, given the rapidity of limitations of DNA techniques in Duchenne muscu- developments so far and the intense interest in the lar dystrophy. The prediction for heterozygotes is subject by molecular biologists as well as clinicians, unaffected by pregnancy or age (unlike creatine this now seems a very real prospect for the near kinase) and there is no problem of variation from X future. chromosome inactivation to contend with. Prenatal use of DNA markers in fetal tissue from chorion biopsy has already been validated for the probes in References question,6 and is already coming into clinical use for lMurray JM, Davies KE, Harper PS, Meredith L, first trimester fetal sexing, though the safety of Mueller CR,Williamson R. Linkage relationship of a cloned chorion biopsy remains to be established. The DNA sequence on the short arm of the X chromosome to some Duchenne muscular dystrophy. Nature 1982;300:69-71. example also illustrates, however, of the 2 Davies KE, Pearson PL, Harper PS, et al. Linkage analysis of disadvantages of the use of linked polymorphisms: two cloned DNA sequences flanking the Duchenne muscular the risk calculations may seem formidable and the dystrophy locus on the short arm of the human X chromosome. modification in the final risk is not always great. Nucleic Acids Res 1983;11:2303-12. with Duchenne 3Kingston HM, Thomas NST, Pearson PL, Sarfarazi M, How should the clinician working Harper PS. Genetic linkage between Becker muscular dystro- muscular dystrophy patients react to these new phy and a polymorphic DNA sequence on the short arm of the developments-by taking blood for DNA analysis X chromosome. J Med Genet 1983;20:255-8. from all the families under his or her care, or by 4Giannelli F, Choo KH, Rees DJG, Boyd Y, Rizza CR, Brownlee GG. Gene deletions in patients with haemophilia B + waiting until a really close marker is available that anti-factor IX antibodies. Nature 1983;303:181-2. will give an accurate prediction in all circumstances? 5Harper PS, O'Brien T, Murray JM, Davies KE, Pearson P, http://adc.bmj.com/ Personal experience suggests a few guidelines: Williamson R. The use of linked DNA polymorphisms for (1) Blood should be taken for DNA isolation genotype prediction in families with Duchenne muscular dys- trophy. J Med Genet 1983;20:252-4. from any boy with Duchenne muscular dystrophy 6 Elles RG, Wiliamson R, Niazi M, Coleman DV, Horwell D. likely to die in the near future, so that the Absence of maternal contamination of chorionic villi used for opportunity to utilise the approach in future for fetal gene analysis. N Engl J Med 1983;368:1433-5. relatives is not irretrievably lost. (2) Where there is a number of affected members P S HARPER in a family, particularly in more than one sibship, Section of Medical Genetics, on October 4, 2021 by guest. Protected copyright. serious consideration should be given to typing the Department of Medicine, kindred as a whole, including relevant spouses and Welsh National School of Medicine, healthy males. Such a family is most likely to Heath Park, provide clear cut predictions for carriers. Cardiff CF4 4XN