Hum Genet (1988) 80 : 379-381

© Springer-Verlag 1988

Segregation of linked probes to myotonic dystrophy in a family demonstrating that 152 and APOC2 are on the same side of DM on 19q

K. Joh nson I , E . N i m m o I , P . Jones 1, M. Weiss 1, M.-L. Savontaus 2 , M. A n v r e t 3 , R. Bartlett 4, A . Roses 4 , D . Shaw s , P. S. Harper s,

E. Koivunen-Tapio% and R. Wi l l iamson 1

1Department of Biochemistry, St. Mary's Hospital Medical School, Paddington, London W2 1PG, UK 2Department of Biology, University of Turku, Turku, Finland 3Department of Clinical Genetics, Karolinska Institute, Stockholm, Sweden 4Department of Neurosciences, Duke University, Durham, North Carolina, USA 5Department of Medical Genetics, University Hospital of Wales, Cardiff, UK 6Department of Neurology, Central Hospital of Central Finland, Jyvaskyla, Finland

Summary. The two markers most closely linked to the myo- tonic dystrophy (DM) locus on chromosome 19 are the gene that codes for apolipoprotein CII (APOC2) and the anonym- ous probe D19S19 (LDR152). Both of these markers show tight linkage to DM, with maximum lod scores of > 20 at re- combination fractions of less than 0.05. We have identified, in a family in which DM segregates, an affected individual where a meiotic recombination event has occurred in which both of these linked markers have crossed over with the gene defect. This demonstrates that APOC2 and D19S19 are probably on the same side of DM.

Introduct ion

Myotonic dystrophy (DM) is an autosomal dominant disease; the basic defect is not known. There is a variable age of onset and many different tissues are affected (Harper 1979). Link- age analysis first identified linkage between the DM gene and the loci for Lutheran and the A B H secretor blood groups; DM was the first inherited disease of unknown basic defect for which linkage to a polymorphic marker was demonstrated (Mohr 1954; Renwick et al. 1971). The polymorphic comple- ment component 3 locus (protein C3) was subsequently linked to these markers (Eiberg et al. 1981) and the linkage group was assigned to chromosome 19 (Whitehead et al. 1982). Linkage was also shown between DM and C3 using D N A polymorphisms (Davies et al. 1983).

More recently polymorphic DNA probes have been iso- lated that show tighter linkage to the DM locus than the mark- ers described above, the gene that encodes apolipoprotein CII (APOC2) (Myklebost et al. 1984; Humphries et al. 1984) and an anonymous D N A fragment D19S19 (LDR152) (Roses et al. 1986; Bartlett et al. 1987). These are the two markers that are most tightly linked to the DM gene, showing maximum lod scores of more than 20 for recombination fractions of 0-0.05. The order of these probes with respect to the DM locus has not been determined as no verifiable cross-overs between these loci have been observed in individuals with the disease.

Offprint requests to: K. Johnson

It is important to determine which markers flank the DM locus in order to formulate strategies to clone the DM gene. This analysis is important not only to localise the DM gene so that it can be cloned but also to provide accurate family data for prenatal diagnosis and presymptomatic testing. Further data from cross-over events is also required to order probes proximal or distal to the DM gene. Knowledge of probe order and distance permits strategies for locus isolation to be evalu- ated, using directional chromosome walking utilising cosmid overlapping techniques (Estivill et al. 1987), chromosome mediated gene transfer (Pritchard and Goodfellow 1987), and linking and jumping libraries (Collins et al. 1987).

In this paper we describe a meiotic recombination in which both of the tightly linked markers APOC2 and D19S19 have clearly crossed over with the DM locus.

Materials and methods

DNA probes

Probe D19S19 (LDR152) recognises restriction fragment length polymorphisms (RFLPs) for the enzymes MspI and PstI (Roses et al. 1986). Both cDNA and genomic polymorph- ic probes exist for apolipoprotein CII (APOC2 and p S C l l ) (Humphries et al. 1984; Wallis et al. 1984). These probes detect RFLPs for the enzymes TaqI, NcoI and BanI (APOC 2) and BglI (pSC11). D19S7 (4.1) and D19S8 (17.1) have been described by Shaw et al. (1986)). D19S13 (pHW60) has been described by Hulsebos et al. (1986). CYP2A, D19S9 (pIJ2), D19S l l (13-1-82), LDLR, C3, INSR and D19S6 (pBam34) were all described in Human Gene Mapping 8 (1985).

Restriction digests, gel electrophoresis and Southern blotting

Genomic D N A samples (2gg) prepared from lymphocytes isolated from peripheral blood were digested with restriction enzymes according to the conditions specified by the suppliers (except for MspI) utilizing 10 units of enzyme per digest and

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Fig.1. a The pedigree of family 100 showing the genotypings for APOC2 (upper line) and D19S19 (lower line). The typings for APOC2 are haplotypes generated from the four RFLPs associated with the two probes for this locus, pSCll and APOC2. Haplotypes were assigned as a, Taq 1, Nco 2, Ban 1, Bgl 2; b, Taq 2, Nco 1, Ban 1, Bgl 1; c, Taq 2, Nco 1, Ban 2, Bgl 1; d, Taq 1, Nco 1, Ban 1, Bgl 2; e, Taq 2, Nco 2, Ban 1, Bgl 2, f, Taq 1, Nco 1, Ban 1, Bgl 1. In these haplotypes 1 refers to the higher molecular weight allele, b MspI poly- morphism for members of family 100 detected by the anonymous probe D19S19. e TaqI polymorphism for family 100 detected using the cDNA probe APOC2

digesting for 6 h. Digests with MspI (BRL) were performed at room temperature in "core" buffer for 6-8 h using 5 units/gg DNA. Samples were fractionated by gel electrophoresis in Tris/acetate buffer using 0.8% agarose gels. D N A was vis- ualised by ethidium bromide straining and UV transillumina- tion. DNA was transferred from denatured and neutralised gels onto Hybond-N filters (Amersham) by standard methods (Southern 1975). The D N A was covalently cross-linked to the membrane either by UV irridiation of the filter for 5 min or by baking at 80°C for 2h. Hybridization using oligo-labelled probes (Feinberg and Vogelstein 1984) was carried out at 65°C in the presence of 10% dextran sulphate (Sigma) under standard salt conditions. Final filter washes were at 0.1 × SSC (15mM NaC1, 1.5mM sodium citrate) at 65°C for 10min. Autoradiography was performed at -70°C for 2-3 days using pre-flashed Fuji RX film.

Results

Clinical details of the family

The pedigree of family 100 in which myotonic dystrophy is segregating is shown in Fig. 1. The grandfather, individual I-1, war born illegitimately in 1909 and his mother never married; his father is unknown. He has one sister, by a different father. His mother came from southern Finland and there are no clin- ical details available concerning her status of health. His wife 0-2) comes from central Finland and there is little chance of them being related. She is clinically healthy and extraordinar- ily brisk considering that she is in her eighties. There is no one

on her side for the family with muscle disease. The daughter of II-7 (!II-6) shows the typical EMG pattern for DM.

In 1954 I-1 was diagnosed as having "atrophia musculorum" with gradual weakening of the hands and legs and observed at- rophy. His vision deteriorated. By 1964 his movements were slow and stiff. Considerable thenaratrophy was present and atrophy of the interosseous muscles was observed, particularly in the right hand. The ability to press was extremely weak in both hands. The patient was considerably demented. At this time he was not diagnosed as DM, but the medical records of other family members presenting later are more detailed, and in these cases electromyography (EMG) confirmed a diag- nosis of DM.

Genetic analysis of family 100

The genotypes of the living members of this pedigree for the probes D19S19 and APOC2 are shown. Figure lb and c shows the autoradiographs of the genomic Southern blots probed with D19S19 (Fig. lb) or APOC2 (Fig. lc). It is clear from the data that the affected daughter II-7 is exhibiting a different ge- notype for the paternal (DM) chromosome 19 to her affected sisters II-2 and II-3 with the probe D19S19. Assuming phase from the other members of this sibship, gives a consistent pat- tern of segregation of the disease locus with the 2 allele for D19S19, whereas individual II-7 is homozygous at this locus for the i allele. This is the first reported crossover in an affect- ed individual between this probe and DM.

By haplotyping the APOC2 locus with both APOC2 and P S C l l using all four of the published RFLPs it is clear that in the affected sibs II-2, II-3, II-9 and II-11 the DM gene is seg- regating with the paternal haplotype b, whereas in individual II-7 the paternal haplotype which has been inherited with the disease is haplotype a. Both APOC2 and D19S19 have there- fore undergone meiotic recombination with the disease locus on the paternal chromosome in individual II-7. They are therefore likely to be on the same side of the disease locus on this chromosome.

Discussion

The DM gene has been localized to chromosome 19, and poly- morphic D N A markers APOC2 and D19S19 have been shown to be tightly linked to the disease locus. The recombi- nation data presented in this paper exclude APOC2 and D19S19 from flanking the DM gene. This case is unequivocal, since the person studied is affected, and the chromosome showing the crossover has been inherited by her affected daughter. To our knowledge this is the first example of a crossover between DM and D19S19 and therefore makes this family a key resource for ordering loci around the DM gene. Although other recombinants with APOC2 have been report- ed, none of those individuals has proved to be informative with D19S19 (HGM9 1987). It is not possible to identify flanking probes at this time, as a probe on the proximal side of DM that is close enough to the gene to be useful has not been reported.

These results make the determination of the gene order on 19q even more pressing. Accurate gene order and linkage will permit the combined use of family data with chromosome walking and jumping strategies to delineate and isolate the gene (Collins and Weissman 1984; Estivill et al. 1987).

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Acknowledgements. The authors thank Drs. Sue Chamberlain, Martin Farrall and Be Wieringa for helpful discussions. K.J. and P.J. thank the Denver Fund for Medical Research and the Muscular Dystrophy Association of America. E.N. thanks the Leukaemia Research Fund for support. M. A. was supported by a grant from the Swedish Medical Research Council (No. 3681). M.-L. S. and E. K.-T. acknowledge the support of the Finnish Medical Research Council.

References

Bartlett R, Pericak-Vance M, Yamaoka L, Gilbert J, Herbstreith M, Hung W-Y, Lee J, Mohandas T, Bruns G, Laberge C, Thibault M-C, Ross D, Roses A (1987) A new probe for the diagnosis of myotonic muscular dystrophy. Science 235 : 1648-1650

Collins FS, Weissman SM (1984) Directional cloning of DNA frag- ments at a large distance from an initial probe: a circularisation method. Proc Natl Acad Sci USA 81 : 6812-6816

Davies KE, Jackson J, Williamson R, Harper PS, Ball S, Sarfarazi M, Meredith L, Fey G (1983) Linkage analysis of myotonic dystrophy and sequences on chromosome 19 using a cloned complement 3 gene probe. J Med Genet 20 : 259-263

Eiberg H, Mohr J, Nielsen LS, Simonsen N (1981) Linkage relation- ships between the locus for C3 and 50 polymorphic systems: as- signment of C3 to the DM-SE-LU linkage group: confirmation of C3-LES linkage: support of LES-DM synteny. 6th World Con- gress of Genetics, Jerusalem 1981 (abstr)

Estivill X, Farrall M, Scambler PJ, Bell GM, Hawley KMF, Lench NJ, Bates GP, Kruyer HC, Frederick PA, Stanier P, Watson EK, Williamson R, Wainwright B (1987) A candidate for the cystic fribrosis locus isolated by selection for methylation-free islands. Nature 326 : 840-845

Feinberg A, Vogelstein B (1984) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137 : 266-267

Harper PS (1979) Myotonic dystrophy. Saunders, Philadelphia Hulsebos T, Westphal H, Peek R, Coerwinkel M, Wieringa B (1986)

pHW60, and anonymous single copy clone from the proximal por- tion of the long arm of chromosome 19 (HGM assignment no D19S13). Nucleic Acids Res 14:7137

Human Gene Mapping 8 (1985) 8th International Workshop on Human Gene Mapping. Cytogenet Cell Genet 40 : 244-246

Human Gene Mapping 9 (1987) 9th International Workshop on Human Gene Mapping. Cytogenet Cell Genet 46 : 243-244

Humphries SE, Berg K, Gill L, Cumming AM, Robertson FW, Stalenhoef AFH, Williamson R, Borresen A-L (1984) The gene for apolipoprotein C-II is closely linked to the gene for apolipop- rotein E on chromosome 19. Clin Genet 26 : 389-396

Mohr J (1954) A study of linkage in man. Munksgaard, Copenhagen Myklebost O, Williamson R, Markham AF, Myklebost SR, Rogers J,

Woods DE, Humphfies SE (1984) The isolation and characterisa- tion of cDNA clones for human apolipoprotein CII. J Biol Chem 259:4401-4404

Pritehard CA, Goodfellow PN (1987) Investigation of chromosome- mediated gene transfer using the HPRT region of the human X chromosome as a modell. Genes Dev 1 : 172-178

Renwick JH, Bundey SE, Ferguson-Smith MA, Izatt MM (1971) Confirmation of linkage of the loci for myotonic dystrophy and ABH secretion. J Med Genet 8 : 407-416

Roses A, Pericak-Vance M, Ross D, Yamaoka L, Bartlett R (1986) RFLPs at the D19S19 locus of human chromosome 19 linked to myotonic dystrophy (DM). Nucleic Acids Res 14 : 5569

Shaw D J, Meredith AL, Sarfarazi M, Harley HG, Hnson SM, Brook JD, Bufton L, Litt M, Mohandas T, Harper PS (1986) Regional localization of and linkage relationships of seven RFLPs and myo- tonic dystrophy on chromosome 19. Hum Genet 74 : 262-266

Southern E (1975) Detection of specific sequences among DNA frag- ments separated by gel electrophoresis. J Mol Biol 98 : 503-517

Wallis SC, Donald JA, Forrest LA, Williamson R, Humphfies SE (1984) The isolation of a genomic clone containing the apolipo- protein CII gene and the detection of linkage disequilibrium be- tween two common DNA polymorphisms around the gene. Hum Genet 68 : 286-289

Whitehead AS, Solomon E, Chambers S, Bodmer WF, Povey S, Fey G (1982) Assignment of the structural gene for the third compo- nent of human complement to chromosome 19. Proc Natl Acad Sci USA 79: 5021-5026

Received August 5, 1987 / Revised June 8, 1988