Hum Genet (1988) 80:49-52

© Springer-Verlag 1988

Tight linkage between myotonic dystrophy and apolipoprotein E genes revealed with allele-specific oligonucleotides

Bert Smeets, Jozef Poddighe, Han Brunner, Hans-Hilger Ropers, and B~ Wieringa

Department of Human Genetics, Radboud Hospital, University of Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands

Summary. In 16 families with myotonic dystrophy (DM) a novel approach based on use of allele-specific oligonucleotides has been employed to study the linkage relationship between the apolipoprotein E (APOE) gene and DM. Synthetic oligo- nucleotides, designed to discriminate between APOE alleles ~3 and ~4, enabled us to distinguish heterozygous carriers in a hybridization assay. In a subset of families, the relevant seg- ment of the APOE gene was enzymatically amplified to in- crease the sensitivity of the method. For DM and APOE, a maximum lod score (Zmax of 7.47 was obtained at a recombina- tion frequency (0) of 0.047 (male 0 = female 0). No recombi- nation (maximum lod score of 5.61 at 0 = 0.0) was found be- tween APOE and the apolipoprotein CII (APOC2) gene. These results suggest that, in addition to APOC2, APOE is a useful marker for presymptomatic DM diagnosis.

Introduction

In the absence of reliable clues to the functional defect under- lying myotonic dystrophy (DM), direct approaches to unravel the molecular basis of this disorder are not available, and bio- chemical diagnosis is not possible, Therefore, recent efforts have concentrated on the search for closely linked genetic markers, which can be employed as diagnostic tags and as pos- sible starting points for the molecular isolation of the DM gene.

The apolipoprotein CII (APOC2) gene is one of the few markers that is both closely linked to DM and highly informa- tive: restriction fragment length polymorphisms (RFLPs) have been described with seven different endonucleases (Humphries et al. 1983; Myklebost et al. 1984; Wallis et al. 1984; Meredith et al. 1986; Frossard et al. 1986; Komeluk et al. 1987). APOC2 is located on chromosome 19q (Hulsebos et al. 1986) as are two other genes encoding apolipoproteins, apolipoprotein CI (APOC1) and apolipoprotein E (APOE) (Lusis et al. 1986). For both of these, RFLPs have been described recently (Fros- sard et al. 1987a, 1987b; Klasen et al. 1987a). Studies involv- ing molecular cloning and physical mapping have indicated that APOC1 and APOE are only 4 kb apart (Myklebost and Rogne 1986; Davison et al. 1986). Tight genetic linkage has been reported for APOE and APOC2 (Humphries et al. 1984; Myklebost et al. 1984; Naylor et al. 1985) and all three genes are found to be part of a gene cluster (Smit et al. 1988).

However, studies to determine the genetic distance between APOE and APOC2 or DM have not fully exploited the infor-

Offprint requests to: B. Wieringa

mational content of the numerous DNA polymorphisms re- ported for the APOE gene region. As yet only the use of allelic isoproteins, resulting from amino acid substitutions that lead to variation in the isoelectric charge, has been described. The three most common alleles are a3 (cysna, arglss), ~4 (argn2, arglss), and a2 (cysu2, cyslss), where ~3 is considered the most common allele and a4 and ¢2 have resulted from mutations in- volving the first base of the codons 112 and 158, respectively (Breslow et al. 1982).

Here we report on the use of allele-specific oligonucleotides (ASOs) for detection of DNA sequence variation at codon 112. This technique, which is analogous to methods originally developed for the identification of point mutations in the J3 globin gene (Connor et al. 1983), has been used in combina- tion with primer-directed enzymatic DNA amplification by the polymerase chain reaction (PCR) (Saiki et al. 1985) and employed to obtain accurate estimates of the linkage relation- ships between APOE, APOC2 and the DM gene.

Materials and methods

Materials

Peripheral blood samples of individuals with known APOE type (analysed by isoelectric focusing) were a kind gift of Drs. Stuyt and Stalenhoef (Department of Internal Medicine, Nijmegen). Myotonic dystrophy families were ascertained through various neurological centres in the Netherlands, as re- ported elsewhere (Friedrich et al. 1987).

DNA isolation and Southern blot analysis

Chromosomal DNA from peripheral blood was isolated ac- cording to Aldridge et al. (1984), with minor modifications. Chromosomal DNA (15 ~tg) was digested with restriction en- zyme EcoRI and resolved by electrophoresis on a 0.7% (w/v) agarose gel. Gels were soaked in 0.25M HC1 for 10min to partly depurinate the DNA and DNAs were denatured in 0.4M NaOH and transferred onto BioTrace (Gelman Science) mem- brane in the same solution (6-12 h). DNA blots were washed in 2 × SSC (0.3M NaC1, 30mM sodium citrate) and dried at room temperature.

In vitro D N A amplification

For amplification of the relevant APOE gene segment two oligonucleotide primers complementary to codons 89-96 and 159-165 were used. Starting from l~tg of genomic DNA, the

50

90 95 100 g±u glu thr arq a±a arg leu ser lys glu leu gln ala ala GAG GAG ACG CGG GCA eGG CTG TCC AAG GAG CTG CAG GCG GCG~/

AE-I 5'~G CGG GCA CGG CTG T C C A ~ G ~ / / ~ /

arg / 110 CGC 115

,~asp met glu asp val cys gly arg leu val gln GAC ATG GAG GAC GTG TGC GGC CGC CTG GTG CAG

AE-t112 AE-clI2

f 5' ~G arG rGc aGc cGc crl GAC 5'a-CgZG a^c GTG cac aGC CGC crJN~

/ ~ 160 165 ~ g l n IVs arg leu ala val tyr gln ala gly ala arg

CAG AAG CGC CTG GCA GTG TAC CAG GCC GGG GCC CGC

]GAC CGT CAC ATG-GTC CGG cC~'5 AE-2

Fig. 1. Structure of DNA and protein across the polymorphic region in the APOE gene and sequences of oligonucleotides used for allele-specific hybridization and in vitro amplification. Positions of homology for amplification oligonucleotides (AE-1 and AE-2) and for ~3- and ~4-specific oligo- nucleotides (AE-t112 and AE-c112) are indicated by boxes

pertinent segment of the APOE exon IV gene sequence was amplified in 16 consecutive PCR cycles (Saiki et al. 1985). De- tailed reaction conditions will be described elsewhere (Smeets et al., 1988). Products from the reaction mixture were precipi- tated with ethanol, dissolved in water, separated on a 4% (w/v) agarose gel and transferred to Gene Screen Plus membranes by conventional Southern blotting techniques.

Generation of oligonucleotide probes and hybridization procedure

Oligonucleotides were designed and synthesized as described (Smeets et al., 1988). Oligonucleotides (2pmoles of either AE-t112 or AE-c112; Fig. 1) were 5' end-labelled using 7-[32p] ATP (4pmoles, > 5000 Ci/mmol, Amersham) and T4 poly- nucleotide kinase (11.5 units, Pharmacia-P&L) in a 10gl reaction mixture containing 50mM Tris-HC1 pH 9.5, 10ram MgC12, 2 mM dithiothreitol. Unincorporated 7-[32P] ATP was removed by passage through a Sephadex G50 column. Specif- ic activity was between 10 s and 109cpm/~tg.

Blots were pre-hybridized overnight at 65°C in 5 x SSPE (1 x SSPE is 0.15 M NaC1, 0.01 M sodium phosphate, 0.001 M EDTA), 0.3% SDS and 0.5mg/ml single-stranded herring sperm DNA. Hybridization was performed under similar con- ditions at probe concentrations of 0.3-1 x 106cpm/ml. The blots were washed twice for 5min each in 5 x SSPE, 0.3% SDS at 65°C and once for 10min in 1 x SSPE, 0.3% SDS at 65°C. Gene Screen Plus membranes carrying amplified DNA were treated similarly, but pre-hybridization was restricted to 2-4 h and lower concentrations of herring sperm DNA (10 gg/ ml) were used. Blots containing total genomic DNA or amplified DNAs were exposed to Kodak X-OMat S film with two intensifying screens for periods of up to 7 days or for 4 - 16 h, respectively.

Linkage analysis

Two-point linkage analyses were performed employing ver- sion 4.6 of the L INKAGE program package (Lathrop and Lalouel 1984).

Results

The sequences of the oligonucleotide probes AE-t112 and AE-c112 are shown in Fig. 1. Discriminative hybridization and washing conditions were determined empirically by dot blot

hybridization of plasmid pEB4 (Wallis et al. 1983), which con- tains an APOE3 cDNA insert spanning the entire gene area between codon 45 and the termination codon. Next, these hy- bridization conditions were employed for oligonucleotide screening on blots of EcoRI-cleaved genomic DNAs from var- ious individuals with known APOE type. Figure 2a shows that the signals were of the expected size (2 kb) and concordant with the known APOE phenotype.

A major problem encountered in the direct oligonucleotide screening assay is that generally bands are weak and show up only after prolonged periods of autoradiography. To increase the sensitivity and speed of the ASO hybridization procedure and to avoid any ambiguity in the interpretation of the results, DNA from several individuals was re-typed after amplifica- tion of the relevant APOE gene region by the PCR technique. We have recently adapted this technique to obtain 10,000-fold amplification of the APOE exon IV gene region spanning the polymorphic codons at positions 112 and 158 (H. Smeets et

Fig. 2. a Direct haplotyping of individuals with known APOE pheno- type by hybridization with APOE allele-specific oligonucleotides. DNA blots of genomic DNAs digested with restriction enzyme EcoRI were sequentially hybridized with oligonucleotide AE-t112 (top) and AE-cll2 (bottom) and exposed for autoradiography for 7 days. Only the relevant portions of the autoradiographs containing the 2000-bp signals are shown, b APOE haplotyping of DM (cross-over) families using hybridization of APOE allele-specific oligonucleotides com- bined with in vitro amplification of DNAs. Small samples of genomic DNAs of individuals from DM families 10 and 13 were amplified and reaction products were blotted as given in Materials and methods. Oligonucleotide hybridization was as given for (a) and autoradiography was for 16 h. Only the portions of the autoradiographs containing the 228-bp signals of the amplified DNAs are shown. Relevant parts of DM pedigrees containing the APOE-DM cross-over are given on top of both panels. Known (a) or determined (b) APOE phenotypes (3/3, 3/4 or 4/4) are given below each lane

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Table 1. Two point linkage data for DM, APOE and APOC2: A shows the combined lod scores (z) calculated under the assumption that male and female recombination rates are equal; B gives the maximum lod scores at independent estimation of optimum male and female recom- bination frequencies. Tested were 16 families with myotonic dystrophy comprising 156 individuals

A

Locus vs locus

Lod score at recombination frequency (0) (male 0 = female 0)

0.00 0.02 0.04 0.06 0.08 0.10

Zmax (Omax)

DM- -~c 7.22 7.46 7.44 7.32 7.12 7.47 (0.047) APOE

APOC2- 5 . 6 1 5 .39 5 .16 4 .93 4 .70 4.46 5.61 (0.00) APOE

DM- 8.30 7 .98 7 .65 7 .31 6 .95 6 .59 8.30 (0.00) APOC2

B

Locus vs locus Zmax 0max males Omax females

DM-APOE 8.35 0.001 0.117 APOC2-APOE 5.60 0.001 0.001 DM-APOC2 8.29 0.001 0.001

al., 1988). Because the method is as yet too laborious to be applied routinely in a large number of pedigrees we have only re-analysed a selected subfraction of the DNAs. Haplotypes of eight individuals that were re-examined in this way were in agreement with those of the direct assay (see Fig. 2b).

In a sample of 45 unrelated Caucasian individuals, allele frequencies of the cys112 (a3) and the a rg l l2 (a4) variant were 88.9% and 11.1%, respectively. To estimate the genetic dis- tance between the myotonic dystrophy locus and the APOE gene, 16 myotonic dystrophy families were screened with the AE-c112 and AE-t112 probes. Seven families were informa- tive for the polymorphism. Only two cross-overs - doubly checked both by direct hybridization and after combination with primer-directed enzymatic DNA amplification (see Fig. 2b) - were observed in a total of 42 fully or partly informative meioses, yielding a maximum lod score of 7.47 for a recombi- nation fraction of 0.047 (male 0 = female 0).

APOC2-TaqI haplotypes were determined in the same families. No recombination was found between APOC2 and DM (Zmax = 8.30) nor between APOC2 and APOE (Zmax = 5.61). The two families showing cross-overs between APOE and DM, were not informative for the APOC2-TaqI poly- morphism. Results of two-point linkage analysis with lod scores calculated for males and females combined (male 0 = female 0) or calculated separately are given in Table 1, parts A and B respectively. A multi-point linkage map spanning the DM gene and a variety of other chromosome 19 markers will be presented elsewhere.

Discussion

Oligonucleotide hybridization procedures have been used pre- viously for detection of mutant alleles in various disorders like sickle cell anaemia and phenylketonuria. As demonstrated here, this procedure can also be employed for routine linkage analysis. In principle, oligonucleotide hybridization allows de-

tection of virtually any kind of allelic DNA sequence variation, whether in expressed genes or in anonymous loci, and unlike the RFLP approach is not confined to sequence variation in restriction enzyme cleavage sites. Methodological and techni- cal progress has rendered oligonucleotide synthesis both rapid and affordable. The only major drawback in the use of oligo- nucleotides for routine linkage analysis lies in their limited sensitivity. This limitation can be overcome by use of the tech- nique for in vitro D N A amplification, which tremendously in- creases both speed and sensitivity of the method and enables reliable haplotyping, even when only very limited amounts of D N A are available (Saiki et al. 1985; Embury et al. 1987). The polymerase chain reaction, as described by Saiki et al. (1985), is a quick, but rather laborious, process requiring se- rial pipetting. Therefore, we have only applied it to D N A samples from a selected subset of individuals and to samples that could not be scored unambiguously in the direct hybrid- ization assay. Recent improvements of this technique involv- ing the application of heat-stable DNA polymerase of Ther- mus aquaticus (Saiki et al. 1988) and the automation of vari- ous steps involved in consecutive reaction cycles soon should pave the way for facile parallel processing of greater numbers of samples and, consequently, should widen the scope for oligonucleotides as tools for diagnosis and linkage studies.

In the population sample tested, allele frequencies were 88.9% for APOE-t112 and 11.1% for APOE-c l l2 . This agrees well with the published (joint) frequencies of the alleles ~2 and ~3 and the frequency of ~4, respectively, as determined by protein typing (Breslow 1985; Klasen et al. 1987b).

Compared to other chromosome 19 markers, surprisingly little has been published about the linkage between the APOE gene and DM. In the only other study that we are aware of, Laberge et al. (1985) examined protein polymorphisms in a large French-Canadian kindred and obtained a lod score of 7.2 at a recombination fraction of 0.10 (males and females combined). APOE and APOC2 have been found to be very tightly linked by others (Myklebost et al. 1984; Naylor et al. 1985), which is confirmed by our data (Zmax = 5.61 at 0 = 0.0). Only one recombination event between these two markers has ever been reported (Berg et al. 1985). This provides further circumstantial evidence for APOE and DM being closely linked, as suggested by our present study, because of the tight linkage between APOC2 and DM (0max = 0.04, Zmax>30; Naylor et al. 1985; Pericak-Vance et al. 1986; Wieringa et al. 1988; Shaw and Eiberg i987). Physical evidence in support of APOE, APOC2 and APOC1 being part of a gene cluster has been published recently (Smit et al. 1988). Already earlier it was known that all three genes map into a small segment of chromosome 19 in the 19q131-q132 region (Schonk et al., un- published results; Hulsebos et al. 1986; Wieringa et al. 1988).

Because of its close linkage, APOC2 is currently being used as one of the most useful diagnostic markers of DM. Un- fortunately, however, as a result of linkage disequilibrium be- tween the various RFLPs that have been detected, this marker is not informative in a substantial proportion (approximately 50%) of DM families. Preliminary evidence from our labora- tory indicates that indeed, useful diagnostic linkage informa- tion could be obtained by APOE typing in those families that were not informative for any of the APOC2 markers (B. v. Oost and H.Brunner, unpublished results). Therefore, it appears that APOE can be added to the list of polymorphic D N A markers that can be employed profitably for presymptomatic diagnosis of myotonic dystrophy.

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Acknowledgements. We thank Drs.P. Stuyt and A. Stalenhoef for making available blood samples of various APOE phenotypes and for helpful discussions and we thank Dr. S. Humphries for help, for useful comments on the manuscript and for providing us with APOEB 4 plas- mid DNA. This work was supported by Grant RO 389/15-1 of the Deutsche Forschungsgemeinschaft, by Grant 8%2694 of the Dutch Prinses Beatrixfonds, and by the Dutch Praeventiefonds Grant 28- 1165.

References

Aldridge J, Kunkel L, Bruns G, Tantravahi U, Lalande M, Brewster T, Moreau E, Wilson M, Bromley W, Roderick T, Latt SA (1984) A strategy to reveal high-frequency RFLPs along the human X chromosome. Am J Hum Genet 36 : 546-564

Berg K, Pedersen JC, BCrresen AL, Heiberg A, Solaas MH, Hum- phries S (1985) Concerning some chromosome 19 markers in families with hypercholesterolemia with xanthomatosis. Cyto- genet Cell Genet 40 : 582

Breslow JL (1985) Human apolipoprotein molecular biology and ge- netic variation. Ann Rev Biochem 54 : 699-727

Breslow JL, McPherson J, Nussbaum AL, Williams HW, Lofquist- Kahl F, Karathanasis SK, Zannis VI (1982) Identification and DNA sequence of a human apolipoprotein E cDNA clone. J Biol Chem 257:14639-14641

Conner B J, Reyes AA, Morin C, Itakura K, Teplitz RL, Wallace RB (1983) Detection of sickle cell 13S-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci USA 80 : 278- 282

Davison PJ, Norton P, Wallis SC, Gill L, Cook M, Williamson R, Humphries SE (1986) There are two gene sequences for human apolipoprotein CI (APO CI) on chromosome 19, one of which is 4kb from the gene for APO E. Biochem Biophys Res Commun 136 : 876-884

Embury SH, Scharf S J, Saiki RK, Gholson MA, Golbus M, Arnheim N, Ehrlich HA (1987) Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis. N Engl J Med 316: 656-661

Freidrich U, Brunner H, Smeets D, Lambermon E, Ropers H-H (1987) Three-point linkage analysis employing C3 and 19cen markers assigns the myotonic dystrophy gene to 19q. Hum Genet 75 : 291-293

Frossard PM, Coleman RT, Assman G (1986) NcoI RFLP at the human apolipoprotein CII gene locus. Nucleic Acids Res 14:5120

Frossard PM, Lim DW, Coleman RT, Funke H, Assman G, Malloy MJ, Kane JP (1987a) Human apolipoprotein CI (ApoCI) gene locus: BglI dimorphic site. Nucleic Acids Res 15:1344

Frossard PM, Coleman RT, Malloy MJ, Kane JP, Levy-Wilson B, Appleby VA (1987b) Human apolipoprotein CI (apoCI) gene locus: DraI dimorphic site. Nucleic Acids Res 15 : 1884

Hulsebos T, Wieringa B, Hochstenbach R, Smeets D, Schepens J, Oerlemans F, Zimmer J, Ropers H-H (1986) Toward early diag- nosis of myotonic dystrophy: construction and characterization of a somatic cell hybrid with a single human der(19) chromosome. Cytogenet Cell Genet 43 : 47-56

Humphries SE, Jowett NI, Williams L, Rees A, Vella M, Kessling A, Myklebost O, Lydon A, Seed M, Galton D J, Williamson R (1983) A DNA polymorphism adjacent to the human apolipoprotein CII gene. Mol Biol Med 1 : 463-471

Humphries SE, Berg K, Gill L, Cumming AM, Robertson FW, Stalenhoef AFH, Williamson R, B0rresen AL (1984) The gene for apolipoprotein C-II is closely linked to the gene for apolipo- protein E on chromosome 19. Clin Genet 26 : 389-396

Klasen EC, Talmud PJ, Havekes L, Wit E de, Kooij-Meijs van der, Smit M, Hanson G, Humphries SE (1987a) A common restriction fragment length polymorphism of the human apolipoprotein E gene and its relationship to type III hyperlipidaemia. Hum Genet 75 : 244-247

Klasen EC, Smit M, Knijff P de, Gevers Leuven J, Kempen-Voogd R, Havekes L (1987b) Apolipoprotein E phenotype and gene dis- tribution in The Netherlands. Hum Hered 37 : 340-344

Korneluk RG, MacLeod HL, Leblond SC, Monteith NL, Boralle FE, Hunter AGW (1987) AvaII RFLP at the human apolipoprotein CII (APOCII) gene locus. Nucleic Acids Res 15 : 6769

Laberge C, Gaudet D, Morisette J, Moorjani S, Thibault MC (1985) Linkage of myotonic dystrophy and apoE in a French Canadian isolate. Cytogenet Cell Genet 40: 675

Lathrop GM, Lalouel JM (1984) Easy calculations of lod scores and genetic risks on small computers. Am J Hum Genet 36 : 460-465

Lusis AJ, Heinzmann C, Sparkes RS, Scott J, Knott TJ, Geller R, Sparkes MC, Mohandas T (1986) Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1,-C2, and E and LDLR) and the genes C3, PEPD, and GPI. Proc Natl Acad Sci USA 83 : 3929-3933

Meredith AL, Huson SM, Lunt PW, Sarfarazi M, Harley HG, Brook JD, Shaw DJ, Harper PS (1986) Application of a closely linked polymorphism of restriction fragment length to counselling and prenatal testing in families with myotonic dystrophy. Br Med J [Clin Res] 293 : 1353-1356

Myklebost O, Rogne S (1986) The gene for human apolipoprotein CI is located 4.3 kilobases away from the apolipoprotein E gene on chromosome 19. Hum Genet 73 : 286-289

Myklebost O, Rogne S, Olaisen B, Gedde-Dahl T Jr, Prydz H (1984) The locus for apolipoprotein CII is closely linked to the apolipo- protein E locus on chromosome 19 in man. Hum Genet 67:309- 312

Naylor S, Lalouel J-M, Shaw DJ (1985) Report of the committee on the genetic constitution of chromosomes 17, 18 and 19. Cytogenet Cell Genet 40: 242-267

Pericak-Vance MA, Yamaoka LH, Assinder RIF, Hung W-Y, Bartlett RJ, Stajich JM, Gaskell PC, Ross DA, Sherman S, Fey GH, Humphries S, Williamson R, Roses AD (1986) Tight linkage of apolipoprotein C2 to myotonic dystrophy on chromosome 19. Neurology 36:1418-1423

Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 : 1350-1354

Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplifi- cation of DNA with a thermostable DNA polymerase. Science 239 : 487-491

Shaw D, Eiberg H (1987) Report of the committee for chromosomes 17, 18, 19. (9th International Workshop on Human Gene Map- ping) Cytogenet Cell Genet 46: 242-256

Smeets HJM, Poddighe J, Stuyt PMJ, Stalenhoef AFH, Ropers HH, Wieringa B (1988) Identification of apolipoprotein E polymorphism by using synthetic oligonucleotides. J Lipid Res (in press)

Smit M, Kooij-Meijs E van der, Frants RR, Havekes L, Klasen EC (1988) Apolipoprotein gene cluster on chromosome 19. Definite localization of the APOC2 gene and the polymorphic HpaI site as- sociated with type III hyperlipoproteinemia. Hum Genet 78 : 90- 93

Wallis SC, Rogne S, Gill L, Markhyam A, Edge M, Woods D, Williamson R, Humphries S (1983) The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues. EMBO J 12: 2369-2373

Wallis SC, Donald JA, Forrest LA, Williamson R, Humphries 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

Wieringa B, Brunner H, Hulsebos T, Schonk D, Ropers HH (1988) Genetic and physical demarcation of the locus for Dystrophia Myotonica. Adv Neurol 48: 47-69

Received December 7, 1987 / Revised April 12, 1988