I Med Genet 1992; 29: 785-788 785 Specific molecular prenatal diagnosis for the CTG mutation in myotonic dystrophy J Med Genet: first published as 10.1136/jmg.29.11.785 on 1 November 1992. Downloaded from Jenny Myring, A Linda Meredith, Helen G Harley, Gertrude Kohn, Gail Norbury, Peter S Harper, Duncan J Shaw Abstract Methods The results of DNA analysis for the speci- All prenatal diagnoses were undertaken on fic mutation of myotonic dystrophy are chorion villus samples taken between 9 and 12 reported in eight pregnancies (two studied weeks' gestation; blood samples were also retrospectively) in six families. Four re- taken from all relevant available family mem- sults were normal; in the other four, large bers. DNA was extracted from chorion villus DNA expansions were found, comparable samples and venous blood by standard to the range seen in severely affected chil- methods. The DNA was digested to comple- dren with congenital onset of the disorder. tion using the restriction enzyme EcoRI. The The results agreed with those obtained by fragments were size fractionated by electro- linked DNA markers in the six cases phoresis on 0-8% agarose gels run in TAE where they were available. We conclude buffer pH 77 (Tris-HCl 40 mmol/l, glacial that specific molecular prenatal diagnosis acetic acid 250 mmol/l, EDTA 1 mmol/l) at of myotonic dystrophy is feasible, and 1-4 V/cm for 30 hours. The buffer was changed that an abnormal result may also give a once during this time. Gels were blotted using guide to possible severity, though this Hybond N, and hybridised using the 32p should be interpreted with caution until labelled DNA probe pM1OM6, which is a greater experience is available. clone from the region D19S95 containing the (J Med Genet 1992;29:785-8) unstable CTG sequence described by Harley et al6 and Brook et al.'0 The filters were washed down to 0-1 x SSC and autoradiography car- Myotonic dystrophy is one of the most vari- ried out overnight. The probe pMlOM6 de- able of the muscular dystrophies' with severe tects an EcoRI polymorphism showing 9 and and frequently fatal congenital onset of the 10 kb fragments in the normal population; in myotonic dystrophy a band of variable size is Institute of Medical disorder occurring in a proportion of offspring Genetics, University of affected women,2 while in the older genera- seen resulting from the expansion of the 10 kb of Wales College of tions subjects may have cataract and no signi- fragment by up to 6 kb. Medicine, Heath Park, ficant neuromuscular abnormalities. Prenatal J Myring diagnosis of myotonic dystrophy has been http://jmg.bmj.com/ A L Meredith feasible using linked DNA markers since the Results H G Harley detection of close linkage with the APOC2 The overall results of the study are summar- D J Shaw locus on chromosome 19,34 but its application ised in the table. Of the eight pregnancies has been limited by a combination of factors, analysed (two retrospectively on stored DNA Edith Wolfson including inadequate family structure, unin- from earlier chorion biopsies), four were pre- Hospital, Holon, Israel. formativeness of markers, and the possibility dicted to be affected, each with a clear expanded DNA sequence, while the remaining G Kohn of inaccuracy from genetic recombination.5 on October 1, 2021 by guest. Protected copyright. The recent detection of an unstable CTG four samples showed no abnormality using Medical Genetics, tandem repeat sequence as the basis for the restriction enzyme EcoRI. Churchill Hospital, mutation in myotonic dystrophy6'0 has, for the Oxford. first time, provided the possibility of making a G Norbury specific molecular prenatal diagnosis in this Case reports Correspondence to condition. We report here our initial experi- FAMILY 1 Professor Harper. ence with this and discuss the advantages and This family from Israel has already been men- Received 8 June 1992. Revised version accepted potentialo ikdmresproblems by comparison with the use tioned briefly in a previous report.9 Two sis- 29 June 1992. of linked markers. ters, both moderately affected with myotonic dystrophy from late teenage years, requested prenatal diagnosis in a total of four pregnan- cies, the last occurring shortly after detection of the unstable CTG sequence. Summary ofpregnancies studied. The first pregnancy of the older sister had Affected Mutation Linkage ended in the neonatal death of a congenitally Family Pregnancy parent analysis prediction affected child, an event which led to the dia- 1 1 Mother 9kb+E High gnosis ofmyotonic dystrophy in the mother and 2 (R) Mother 9kb+E High 3 (R) Mother 9 kb, 9 kb Low other family members. In her subsequent two 2 1 Mother 9kb, 9kb Low pregnancies prenatal diagnosis was requested 3 1 Father 9kb, 10kb Not done 4 1 Mother 10kb+E High and a low risk was predicted in both by the use 5 1 Mother 9kb, 10kb Low of linked DNA markers on DNA from chorion 6 1 Mother 9kb+E Not done biopsies. Healthy children without signs of E = DNA expansion present. R = retrospective. myotonic dystrophy were subsequently born. 786 Myring, Meredith, Harley, Kohn, Norbury, Harper, Shaw The younger sister later requested prenatal DNA expansion, while large expanded bands diagnosis in her first pregnancy; a high risk was are present in the two congenitally affected predicted by linked markers and the pregnancy children. The CVS sample shows a single 9 kb was terminated. She again requested prenatal band, suggesting the fetus has received this J Med Genet: first published as 10.1136/jmg.29.11.785 on 1 November 1992. Downloaded from diagnosis in her second pregnancy, the CVS allele from both parents (fig 2). There is no sample being received soon after the recogni- evidence of an expanded fragment. Linked tion of the CTG unstable sequence in myotonic markers also predicted a low risk and the dystrophy and before we had experience of its pregnancy continues. use in a diagnostic situation. After considerable discussion it was decided to rely primarily on genetic linkage rather than FAMILY 3 on the hitherto untried specific mutation test; This family was referred by Dr M Zatz, Sao the closely linked marker D19S63 again pre- Paulo, Brazil. The healthy wife of a moder- dicted a high risk and the pregnancy was termi- ately affected man with myotonic dystrophy nated on the basis ofthis report. DNA from this requested prenatal diagnosis and underwent CVS sample was simultaneously analysed for chorion biopsy at 91 weeks ofpregnancy. Sam- the unstable sequence (fig 1) along with DNA ples were sent by air to Cardiff and the results from family members and residual CVS DNA are shown in fig 3. Both the proband and his from the previous high risk pregnancy. DNA affected brother show an abnormal expanded available from one of the two low risk pregnan- band, while the unaffected wife of the proband cies of the sister was tested separately. is homozygous for a normal 10 kb band. The It can be seen that both high risk pregnan- fetus is heterozygous with normal 9 and 10 kb cies show an abnormal band expanded by bands; the fact that it is the 9 kb band that has around 3 kb, in the range found to be associ- been received from the affected father makes it ated with severe disease9; the two mothers even less likely that the fetus would be affec- show abnormal bands of moderate size, while ted, since in all cases studied so far, the expan- the affected grandfather also shows a small sion has been of a 10 kb band.6 DNA expansion. No abnormal band was seen in the CVS sample from the pregnancy pre- dicted to be at low risk (not shown in fig 1). FAMILY 4 In this British family the affected mother had already given birth to a severely affected child, FAMILY 2 and also had an affected sister and father. All In this British family, referred from Birm- affected members showed an expanded frag- ingham by Dr Ian Glass, the affected mother ment, largest in the severely affected child. already had two congenitally affected children The CVS sample taken at 11 weeks' gestation and requested prenatal diagnosis in her third showed an expanded band slightly larger than pregnancy. The mother shows a moderate that of the severely affected sib. Linked markers also predicted an affected fetus and termination was carried out. http://jmg.bmj.com/ FAMILY 5 In this family, the affected mother had pre- viously undergone prenatal diagnosis using linked probes (D19S63), the outcome of which on October 1, 2021 by guest. Protected copyright. N 3114b - 9 kb I-'--1 I- k F " .I 4b ko ..kti L; a Figure I Family 1. Abnormal DNA expansions are present in the affected adult Figure 2 Family 2. A DNA expansion is present in members of the family including the mother (lane 5), her sister (lane 6), and maternal the affected mother (lane 1), while the two congenitally grandfather (lane 2). A large expansion is present in both the previous affected affected children (lanes 3 and 4) show large expansions. pregnancy (lane 10) and the present pregnancy (lanes 8 and 9). The faintness of The pregnancy shows a single 9 kb band, with no alleles in lanes 8 to 10 is from uneven lane loading owing to the minimal quantity of evidence of DNA expansion. A normal control (lane 6) available DNA. shows normal heterozygous pattern. Specific molecular prenatal diagnosis for the CTG mutation in myotonic dystrophy 787 status of the affected mother, being greater for mothers with more severe disease, whereas congenital myotonic dystrophy was not seen where the mother was neurologically normal.