Hum Genet (1987) 75 : 32-40

© Springer-Verlag 1987

Genetic linkage study between the loci for Duchenne and Becker muscular dystrophy and nine X-chromosomal DNA markers

E. Wflichowski ~, M. Krawczak 1, E. Seemanova 2, F. Hanefeld 3, and J. Schmidtke 1

11nstitut for Humangenetik der Universit~it, Gosslerstrasse 12d, D-3400 G6ttingen, Federal Republic of Germany 2Research Institute of Child Development, Faculty of Pediatrics, Charles University, 15006 Prague, Czechoslovakia 3Universitfits-Kinderklinik, Humboldtallee 38, D-3400 G6ttingen, Federal Republic of Germany

Summary. A set of nine polymorphic loci defined by D N A probes was studied for linkage with the disease locus in ten families with a history of Duchenne muscular dystrophy (DMD), and three families with a history of Becker muscular dystrophy (BMD). The results confirm DMD and BMD link- age to all marker loci and suggest closer linkage of several probes than hitherto detected. This will be of practical interest for risk calculations in affected families.

Introduction

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders of the mus- cular system. DMD has an incidence of one in 3,000-5,000 live-born males. The clinical course is characterized by in- creased insufficiency of the affected muscle groups; most of the patients die at the end of their second decade of life. BMD, first described by Becker and Kiener in 1955, has a lower incidence of around one in 30,000-50,000 in live-born males and exhibits a more benign and prolonged course than DMD.

The DMD locus was mapped to within the band Xp21 of the human X chromosome by cytogenetic analysis of girls af- fected with this disease (Lindenbaum et al. 1979; Greenstein et al. 1980; Jacobs et al. 1981; Zatz et al. 1981; Moser 1984) and by detection of deletions of D N A loci localised to this re- gion by physical mapping (Franke et al. 1985; Monaco et al. 1985). Linkage studies using restriction fragment length poly- morphisms (RFLP) of marker loci localised to the short arm of the X chromosome support these findings (Murray et al. 1982; Davies et al. 1983; Sarfarazi et al. 1983; Brown et al. 1985a,b; Davies et al. 1985a,b; Dorkins et al. 1985; Wilcox et al. 1985).

Linkage data obtained from BMD-families suggest that the DMD- and BMD-loci might be allelic, a suggestion already made by Becker and Kiener in 1955 (Kingston et al. 1983; Brown et al. 1985a,b; Fadda et al. 1985; Dorkins et al. 1985; Sarfarazi et al. 1983; Wilcox et al. 1985). Since both the DMD/BMD locus and the primary defect leading to the dis- ease are unknown, there is no way to identify carriers of these X-linked muscular dystrophies directly. Risk estimates are however possible employing RFLP segregation analysis at marker loci closely linked to the DMD/BMD locus. We pre- sent here family studies of DMD and BMD using nine poly- morphic marker loci including eight "arbitrary" DNA seg-

Offprint requests to: J, Schmidtke

ments and the ornithine transcarbamylase (OTC) structural gene.

Materials and methods

Families, ten with a history of DMD and three with a history of BMD, were examined. Only families with at least two af- fected males were included. Cases occurring through new mu- tation were thus almost certainly excluded. The diagnosis of DMD or BMD in affected individuals was confirmed by fam- ily history, clinical course, levels of muscle specific enzymes, electromyography, and muscle biopsy. In mothers of affected boys, serum creatine phosphokinase (SCK) levels were deter- mined three times at intervals of at least one week, and elec- tromyography and sonography of the muscles of the thighs were performed.

Obligatory carrier status was ascertained in three different ways: (1) all mothers of at least two affected males, (2) all mothers of one affected male and at least one affected relative in the maternal line, (3) all female offspring with a mean SCK value of more than 100 U/1 (carried out with the test-kit "CK NAC-activated" from Boehringer, Mannheim, FRG, normal values for women at 25°C 10-70 U/l) as is the case in

Pedigree Member SCK mean (U/l)

DMD-2 III.2 489 DMD-3 III.2 237 DMD-9 II.3 198 BMD-3 III.6 166

Facultative carriers and their offsprings were included in the study. Their SCK-levels were ascertained three times at intervals of at least one week. From these values the relative probabilities h of not being a carrier were determined in ac- cordance with published data (Grimm 1982) and are included in the lod score calculations.

Pedigree Member SCK mean h (U/l)

DMD-1 III.2 40 4.7 IV.7 33.7 6.75

DMD-4 III.11 19.2 8.0 IV.4 42.0 3.9 IV.9 15.0 8.0 V.1 53.4 1.6 V.5 44.4 3.5

DMD-7 III. 1 40.0 4.7 DMD-10 III .6 31.8 7.2 BMD-2 III.2 (37 years) 35.0 15.6

33

Fig.1. Banding pattern of nine DNA-probes showing RFLPs

Table 1. Characterisation of the probes used in this study

Probe Locus Regional References name designation assignment

782 DXS 85 Xp22.2-Xp22.3 Hofker et al. 1985

RC 8 DXS 9 Xp22 Wieacker et al. 1984

xuT 23 DXS 16 Xp22 Willard et al. 1984

99-6 DXS 41 Xp22.1-Xp22.2 Kunkel et al. 1985

D 2 DXS 43 Xp22.1-Xp22.2 Kunkel et al. 1985

754 DXS 84 Xp21.1-Xp21.2 HoNer et al. 1985

HO 731 OTC Xp21 Horwich et al. 1984

L1.28 DXS 7 Xpl l .3 Wieacker et al. 1984

58-1 DXS 14 Xpll-centr Aldridge et al. 1985

22.3 1RC8 22.122"2 ]xuT 23

21.3 21.2 ]75/. 21.1 2 JOTC 11./. ] 11.3 11.23 58-1 11.22 11.21- 11.1~

782

Fig. 2. Regional assignment of used DNA probes

Table 2. Frequencies of alleles

Probe Enzyme RFLP (kb) Abbrevia- tions in pedigrees

Frequencies of alleles

(P, q)

782 Eeo RI 14 A 0.60 7 a 0.40

RC 8 TaqI 5.3 B 0.09 3.2 b 0.87 3.0 In pedigrees 0.04

not observed

xuT 23 BglII 17.5 C 0.80 12.5 c 0.20

99-6 PstI 22 D 0.56 13 d 0.44

D 2 PvuII 6.6 E 0.45 6.0 e 0.55

754 PstI 12 G 0.55 9 g 0.45

HO 731 MspI 6.6/5.1 H1 0.43 (OTC) 6.6/4.4 H2 0.25

6.2/5.1 H3 0.27 6.2/4.4 H4 0.05

L1.28 TaqI 12 I 0.67 9 i 0.33

58-1 MspI 4 J 0.79 2.5 j 0.21

DNA-extraction and analysis

Genomic D N A was extracted from venous blood taken up in E D T A according to conventional procedures including sodium dodecyl sulfate (SDS)- t reatment and repeated phenol and chloroform extractions (Schmidtke et al. 1984). Af t e r di-

34

Table 3. Lod scores for Duchenne muscular dystrophy and marker loci a. N, Number of informative families; 0, recombination fraction; z(0), maximum lod score

0 782 RC 8 xuT 23 99-6 D 2 754 OTC L1.28 58-1 N = 7 N = I N = 4 N = 5 N = 8 N = 7 N = 8 N = 4 N = I

0.01 2.89 0.51 0.54 3.70 --0.39 --1.03 --2.07 0.04 0.47

0.05 3.19 1.75 0.47 2.47 1.04--2.55 3.36 8.07 0.68--0.48 0.12 15.92 0.30 5.53 0.62--1.23 0.42

0.10 2.98 4.91 0.43 3.23 1.09--0.48 2.92 9.42 0.88 4.18 0.41 18.08 0.96 6.72 0.76 3.83 0.36

0.15 2.62 5.92 0.39 3.57 1.00 1.57 2.49 9.23 0.83 5.28 0.46 17.70 1.12 6.70 0.77 5.60 0.29

0.20 2.20 5.88 0.34 3.36 0.87 0.85 2.04 8.16 0.68 5.25 0.42 16.18 1.08 6.17 0.72 6.21 0.23

0.25 1.76 5.37 0.29 2.94 0.71 0.99 1.60 6.81 0.50 4.67 0.34 14.03 0.93 5.35 0.63 5.98 0.17

0.30 1.30 4.50 0.24 2.43 0.54 1.29 1.17 5.31 0.32 3.70 0.26 11.38 0.73 4.34 0.52 5.31 0.12

0.35 0.87 3.45 0.18 1.83 0.37 0.79 0.77 4.02 0.16 2.75 0.18 7.58 0.50 3.22 0.40 4.27 0.07

0.40 0.49 2.29 0.13 1.20 0.23 0.58 0.42 2.56 0.05 1.76 0.11 4.93 0.29 2.07 0.27 3.01 0.04

0.45 0.19 0.07 0.10 0.16 --0.01 0.05 0.12 0.13 0.02

t) 0.04 0.15 0.01 0.15 0.09 b 0.01 0.10 0.11 0.15 0.15 0.10 0.16 0.10 0.13 0.20 0.01

Z(t~) 3.19 5.92 0.51 3.57 1.09 3.70 9.42 0.88 5.28 0.46 18,08 1.13 6.72 0.78 6.21 0.47

a Left column, present study; right column, combined data b No single ~ observed

Table 4. Lod scores for Becker muscular dystrophy and marker loci a. maximum lod score

N, Number of informative families; 0, recombination fraction; z(0),

0 782 RC 8 xuT 23 99-6 D 2 754 OTC L1.28 58-1 N = 2 N = 2 N = 3 N = 2 N = 2 N = I N = I N = 2 N-=2

0.01 0.55 -1.93 -4.00 1.13 -3.90 0.58 1.20 1.30 2.07

0.05 0.49-1.56 -0.66 0.61 -2.00 -2.44 1.03 4.17 -1 .91-2 .88 0.55-1.33 1.10 0.40 1.20-0.11 1.91

0.10 0.40 0.17 -0.22 1.86 -1.19 -1.38 0.90 4.94 -1.12 1.27 0.50 1.70 0.99 1.95 1.06 3.18 1.70

0.15 0.32 0.89 -0.03 2.22 -0.77 -0.83 0.77 4.86 -0.70 2.92 0.45 3.12 0.86 2.45 0.92 4.39 1.49

0.20 0,25 1.19 0.06 2.20 -0.50 -0.49 0.65 4.54 -0.45 3.44 0.40 3.07 0.73 2.51 0.77 4.71 1.26

0.25 0,18 1.24 0.09 1.99 -0.31 -0.26 0.53 3.90 -0.28 3.36 0.35 2.91 0.59 2.37 0.61 4.50 1.03

0.30 0.12 1.11 0.08 1.62 -0.19 -0.12 0.41 3.24 -0.16 3.01 0.29 2.47 0.44 2.18 0.46 3.97 0.79

0.35 0.07 0.89 0.06 1.21 -0.10 -0.03 0.29 2.42 -0.09 2.34 0.23 1.87 0,29 1.87 0.30 3.17 0.55

0.40 0.03 0.55 0.03 0.76 -0.04 0.02 0.19 1.58 -0.04 1.55 0.16 1.19 0.15 1.35 0.15 2.20 0.32

0.45 0.01 0.01 0.01 0,09 -0.01 0.08 0,04 0.87 0.04 0.13

6 0.01 0.25 0.25 0.15 0.45 0.40 0.01 0.10 0.45 0.20 0.01 0.15 0,01 0.20 0.01 0.20 0.01

z(6) 0.55 1.24 0.09 2.22 0.01 0.02 1.13 4.94 -0.01 3.44 0.58 3.12 1.20 2.51 1.30 4.71 2.07

a Left column, present study; right column, combined data

gestion with restriction enzymes using the manufacturer 's re- commended conditions, resulting D N A fragments were sepa- rated by agarose electrophoresis and transferred to nitrocel- lulose filters (Southern 1975). D N A was hybridised with ap- propriate DNA-probes radiolabelled by nick-translation (Rigby et al. 1977) and the resulting banding patterns were de- tected by autoradiography for be tween 6 and 72 h (Fig. 1).

DNA-probes

Nine DNA-probes detecting eight arbitrary D N A loci and the OTC-gene , all localised to the short arm of the human X chro- mosome, were used for linkage analysis. Their regional as- signments and sources are given in Table 1 and Fig. 2. F r o m a populat ion study comprising 51 healthy unrelated persons (17 females, 34 males) f rom West Germany and Czechoslovakia, the allelic frequencies were est imated (Table 2) and used for lod score calculation. No differences of the allelic frequencies derived f rom German and Czech subjects were observed.

Linkage analysis

Two point l inkage analysis was carried out using the computer program L I P E D (Ott 1974) with lod scores being calculated in steps of 0.05 and, in addition, 0 .0i at recombinat ion fractions be tween 0.0 and 0.5. By this procedure the maximum likeli- hood estimate ~) of the recombinat ion fraction 0 was deter- mined, i .e. , that value of 0, at which the lod score is highest.

Results

Ten DMD-famil ies and three BMD-famil ies were variously informative for RFLPs at nine marker loci around the D M D / B M D locus as indicated in Appendixes 1 and 2. Only the ge- notypes of informative family members used for calculation are shown here. Lod scores calculated in steps of 0.05 and 0.01, respectively, of the recombinat ion fraction 0 are shown in Table 3 for D M D and in Table 4 for B M D . Our results were

35

Table 5. Lod scores for marker loci

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.45 0.45

782 versus

RC 8 1.17 1.06 0.95 0.84 0.73 0.61 0.50 0.37 0.25 0.13

xuT 23 - 0.50 2.31 2.26 2.07 1.82 1.53 1.23 0.91 0.60 0.29

D 2 -0 .65 2.13 2.03 1.81 1.53 1.23 0.92 0.63 0.37 0.16

99-6 4.20 3.77 3.33 2.88 2.42 1.94 1.47 1.01 0.59 0.24

754 - ~ 0.30 0.60 0.65 0.61 0.53 0.43 0.32 0.21 0.10

OTC - ~ 1.19 1.47 1.43 1.27 1.04 0.78 0.52 0.29 0.11

L1.28 1.05 0.94 0.84 0.74 0.64 0.54 0.44 0.34 0.23 0.12

RC 8 versus

xuT 23 - co 1.37 1.43 1.36 1.23 1.06 0.86 0.65 0.44 0.22

D 2 - ~ 0.35 0.52 0.55 0.53 0.47 0.38 0.29 0.19 0.09

99-6 - m -0 .52 -0 .28 -0 .16 -0 .09 -0 .05 -0 .03 -0 .01 -0 .01 0.00

754 -4 .59 -0 .75 -0 .47 -0 .31 -0 .21 -0 .13 -0 .08 -0 .04 -0 .01 0.00

OTC - c~ 0.65 0.79 0.79 0.73 0.64 0.53 0.40 0.27 0.13

L1.28 - co 0.20 0.37 0.40 0.38 0.33 0.27 0.19 0.12 0.05

xuT 23 versus

D 2 3.87 3.49 3.10 2.70 2.29 1.87 1.45 1.03 0.63 0.28

99-6 -c~ 0.99 1.08 1.03 0.93 0.78 0.63 0.46 0.29 0.14

754 - co - 1.33 -0 .62 -0 .28 -0 .09 0.00 0.05 0.07 0.06 0.04

OTC - ~ -1 .67 -0 .62 -0 .13 0.12 0.24 0.28 0.27 0.21 0.12

L1.28 - co - 2.10 - 1.07 - 0.55 - 0.24 -0 .05 0.06 0.10 0.10 0.06

D 2 versus

99-6 - ~ 0.94 0.98 0.89 0.75 0.59 0.43 0.28 0.16 0.06

754 -c~ -4 .04 -2 .28 -1 .39 -0 .87 -0 .54 -0 .33 -0 .19 -0 .10 -0 .04

OTC -co -2 .96 -1 .40 -0 .79 -0 .46 -0 .28 -0 .19 -0 .13 -0 .10 -0 .05

L1.28 - ~ -3 .29 -1 .92 -1 .17 -0 .70 -0 .37 -0 .16 -0 .03 0.04 0.05

99-6 versus

754 - ~ 1.11 1.17 1.11 0.99 0.86 0.71 0.55 0.37 0.19

OTC - ~ -0 .28 0.21 0.38 0.41 0.37 0.29 0.18 0.08 0.01

LI.28 -co -0 .24 -0 .21 -0 .18 -0 .14 -0 .11 -0 .08 -0 .06 -0 .03 -0 .02

754 versus

OTC 1.67 1.70 1.56 1.35 1.12 0.89 0.66 0.45 0.27 0.12

L1.28 - o~ 0.35 0.46 0.45 0.40 0.33 0.24 0.17 0.10 0.04

OTC versus

L1.28 - co 0.90 1.03 0.97 0.83 0.64 0.45 0.26 0.11 0.01

58-1 versus

782 - 8.74 - 1.87 - 1.20 -0 .83 - 0.58 - 0.40 - 0.27 - 0.17 - 0.10 - 0.04

RC 8 - co -0 .90 -0 .42 -0 .19 -0 .07 -0 .00 0.02 0.02 0.01 0.00

xuT23 -co -2 .28 -1 .42 -0 .94 -0 .62 -0 .40 -0 .25 -0 .13 -0 .06 -0 .02

D 2 - oo -3 .74 -2 .16 - 1.34 -0 .83 -0 .50 -0 .28 -0 .14 -0 .05 -0 .01

99-6 0.77 0.71 0.64 0.57 0.49 0.41 0.33 0.25 0.17 0.08

754 -co -1 .17 -0 .43 -0 .08 0.11 0.21 0.25 0.24 0.19 0.11

OTC 1.04 0.94 0.84 0.73 0.61 0.49 0.36 0.23 0.11 0.02

L1.28 - ~ 1.21 1.27 1.20 1.05 0.86 0.66 0.44 0.23 0.07

c o m b i n e d wi th the da ta p r e s e n t e d at the H u m a n G e n e Map-

p ing C o n f e r e n c e 8, He ls ink i 1985 ( G o o d f e l l o w et al. 1985)

wi th the excep t ion o f the locus D X S 14 (58-1) w h e r e n o o t h e r

l inkage da ta have b e e n p r e s e n t e d . T h e 13 D M D / B M D - p e d i -

g rees and t h r ee f u r t h e r ped ig rees ( p r e s e n t e d in A p p e n d i x 3)

w e r e used for two po in t l inkage analysis o f the n ine m a r k e r

loci a m o n g themse lves . T h e resu l t s o b t a i n e d by c o m p u t e r

analysis are p r e s e n t e d in Tab le 5.

36

Discussion

The results confirm linkage between DMD and the following loci: DXS 85 (782), DXS 9 (RC 8), DXS 16 (xuT 23), DXS 41 (99-6), DXS 43 (D 2), DXS 84 (754), OTC (HO 731), DXS 7 (L1.28), and DXS 14 (58-1). Examinat ion of three BMD- families demonstrate linkage with DXS 85 (782), DXS 41 (99- 6), DXS 84 (754), OTC (HO 731), DXS 7 (L1.28), and DXS 14 (58-1), as shown in Table 4, while lod scores of the loci DXS 9 (RC 8), DXS 16 (xuT 23), and DXS 43 (D 2) are too weak. When the present data and published data (Goodfellow et al. 1985) were pooled, the recombinat ion fractions 0 were altered for the following loci:

D MD versus DXS 85 DXS 43 DXS 41 (782) (D 2) (99-6)

Goodfellow et al. (1985) 0 0.20 0.20 0.15 Combined data 0 0.15 0.15 0.10 Total lod scores z(0) 5.92 5.28 9.42

BMD versus DXS 41 DXS 84 (99-6) (754)

Goodfellow et al. (1985) 0 0.15 0.15/0.20 Combined data 0 0.10 0.15 Total lod scores z(0) 4.94 3.12

These alterations are of particular interest for risk calcula- tions, for carrier assessment, and for prenatal diagnoses.

Acknowledgements. This work was supported by a grant from the Bundesminister for Forschung und Technologie (SVG 07065025/ 9 366 482) to J.S. We thank Drs. K. Davies, L. Kunkel, P. L. Pearson, and L. E. Rosenberg for providing probes. Thanks are also due to Dr. D. N. Cooper for reading the manuscript and to Mrs. J. Sist for secre- tarial help,

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 : 74-77

Becker PE, Kieuer F (1955) Eine neue X-chromosomale Muskeldys- trophie. Arch Psychiatr Nervenkr 193 : 427-448

Brown CS, Pearson PL, Thomas NST, Sarfarazi M, Harper PS, Shaw DJ (1985a) Linkage analysis of a DNA polymorphism proximal to the Duchenne and Becker muscular dystrophy loci on the short arm of the X chromosome. J Med Genet 22:179-181

Brown CS, Thomas NST, Sarfarazi M, Davies KE, Kunkel LM, Pear- son PL, Kingston MH, Harper PS (1985b) Genetic relationships of seven DNA probes with Duchenne and Becker muscular dys- trophies. Hum Genet 71 : 62-74

Davies KE, Pearson PL, Harper PS, Murray JM, O'Brien T, Sar- farazi M, Williamson R (1983) Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the X chromosome. Nucleic Acids Res 11 : 2303-2312

Davies K, Briand P, Ionasescu V, Williamson R, Brown C, Cavard C, Cathelineau L (1985a) Gene for OTC: characterisation and link- age to Duchenne muscular dystrophy. Nucleic Acids Res 13 : 155- 165

Davies K, Speer A, Herrman F, Spiegler AWJ, McGlade S, HoNer MH, Briand P, Hauke R, Schwartz M, Steinbicker V, Szibor R, Korner H, Sommer D, Pearson PL, Coutelle C (1985b) Human

X-chromosome markers and muscular dystrophy. Nucleic Acids Res 13 : 4319-3426

Dorkins H, Junien C, Mandel JL, Wrogemann K, Moison JP, Mar- tinez M, Old JM, Bundey S, Schwartz M, Carpenter N, Hill D, Lindlof M, De la Chapelle A, Pearson PL, Davies K (1985) Segre- gation analysis of a marker localized Xp21.2-Xp21.3 in Duchenne and Becker muscular dystrophy families. Hum Genet 71 : 103-107

Fadda S, Mochi M, Roucuzzi L, Sangiorgi S, Sbarra D, Feriini A, Nobile C, Zatz M, Romeo G (1985) Definitive localization of Becker muscular dystrophy in Xp by linkage to a cluster of DNA polymorphism (DXS43 and DXS9). Hum Genet 71 : 33-36

Francke U, Ochs HD, de Martinville B, Giacolone J, Lindgren V, Cisteche C, Pagon RA, HoNer MH, van Ommen G-JB, Pearson PL, Wedgwood RJ (1985) Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa and McLeod syndrome. Am J Hum Genet 37 : 250-267

Goodfellow PN, Davies KE, Ropers HH (1985) Report of the com- mittee on the genetic constitution of the X and Y chromosome. Cytogenet Cell Genet 40 : 296--352

Greenstein RM, Reardon MP, Chan TS, Middleton AB, Mulivor RA, Green AE, Coriell LL (1980) A (X;ll) translocation in a girl with Ducheune muscular dystrophy. Cytogenet Cell Genet 27 : 268

Grimm T (1982) Probleme in der genetischen Beratung yon X- chromosomalen Mnskeldystrophien. Medizinische Habilitations- schrift, Wiirzburg

HoNer MH, Wappenaar MC, Goor N, Bakker B, van Ommen GJB, Pearson PL (1985) Isolation of probes detecting restriction frag- ment length polymorphisms from X-chromosome specific libra- ries: potential use for diagnosis of Duchenne muscular dystrophy. Hum Genet 70 : 148-156

Horwich AK, Fenton WA, Williams KR, Kalousek F, Kraus JP, Doolittle RF, Konigsber W, Rosenberg LE (1984) Structure and expression of a complementary DNA for the nuclear coded pre- cursor of human mitochondrial ornithine transcarbamylase. Science 224 : 1068-1074

Jacobs PA, Hunt PA, Mayer M, Bart RD (1981) Duehenne muscular dystrophy (DMD) in a female with an X/autosome translocation: further evidence that the DMD locus is at Xp21. Am J Hum Genet 33 : 513-518

Kingston HM, Harper PS, Pearson PL, Davies KE, Williamson R, Page D (1983) Localisation of gene for Becker muscular dys- trophy. Lancet II : 120

Kunkel LM, Lalande M, Monaco AP, Flint A, Middlesworth W, Latt SA (1985) Construction of a human X-chromosome-enriched phage library which facilitates analysis of specific loci. Gene 33 : 251-258

Lindenbaum RH, Clarke G, Patel C, Moncrieff M, Hughes JT (1979) Muscular dystrophy in an X/1 translocation suggests that Duchenne locus is on X chromosome short arm. J Med Genet 16 : 389-392

Monaco AP, Bertelson CJ, Middlesworth W, Coletti C-A, Aldridge J, Fischbeck KH, Bartlett R, Pericak-Vance MA, Roses AD, Kunkel LM (1985) Detection of deletions spanning the Duchenne muscular dystrophy locus using a tightly linked DNA segment. Nature 316: 842-845

Moser H (1984) Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Hum Genet 66 : 17.40

Murray JM, Davies KE, Harper PS, Meredith L, Mueller CR, Wil- liamson R (1982) Linkage relationship of a cloned DNA sequence and the short arm of the X chromosome to Duchenne muscular dystrophy. Nature 300 : 69-71

Ott J (1974) Estimation of the recombination fraction in human pedi- grees: efficient computation of the likelihood for human linkage studies. Am J Hum Genet 26 : 588-597

Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237-251

Sarfarazi M, Harper PS, Kingston HM, Murray JM, O'Brien T, Davies KE, Williamson R, Tippett P, Sanger R (1983) Genetic linkage relationships between the Xg blood group system and two X chromosome polymorphisms in families with Duchenne and Becker muscular dystrophy. Hum Genet 65 : 169-171

Schmidtke J, Pape B, Krengel U, Langenbeck U, Cooper DN, Breyel E, Mayer H (1984) Restriction fragment length polymorphisms at the human parathyroid gene locus. Hum Genet 67 : 428-431

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

Wieacker P, Davies KE, Cooke HJ, Pearson PL, Williamson R, Bhattacharya S, Zimmer J, Ropers H-H (1984) Towards a linkage map of the human X chromosome: regional assignment of 16 cloned single copy DNA sequences employing a panel of somatic cell hybrids. Am J Hum Genet 36 : 265-276

Wilcox DE, Affara NA, Yates JRW, Ferguson-Smith M (1985) Multi- point linkage analysis of the short arm of the human X chromo-

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some in families with X-linked muscular dystrophy. Hum Genet 70: 365-375

Willard HF, Schmeekpeper B J, Smith KD, Goss SJ (1984) High reso- lution regional mapping of the human X chromosome by radiation induced segregation analysis of X-linked DNA-probes. Cytogenet Cell Genet 37 : 609-610

Zatz M, Vianna-Morgante AM, Campos P, Diament AJ (1981) Translocation (X;6) in a female with Duchenne muscular dys- trophy: implications for the location of the DMD locus. J Med Genet 18 : 442-447

Received May 12, 1986 / Revised August 7, 1986

Appendix

I DMD-1

II

I l l

1

a I A~ A D /d id / d / e .LEJe ,.Le A ~i .~(JGIg 31 I G Z.(,,J 6 ( 1 3 " T H3/H3 H3 5

1 Ill I J J/j J

e E A ,.I.. Ele ~L, -~ L_] GzL~ g 6 -;,(._)GIG ~L.J " HE" H3 - H3/H3"

I I Ill J J J/j

e

)7 I J

o;

2S:

d

F AIA

8 ~ Did E/e GIG

f 7 ~ HIlH3jIjI/I

9_11 [~ 12 t ~3 (

I J

DMD-2

I ,£

- ,~D/d ~] II 1 1 '~ ]H I /H2 3 L

III I~

I DMD-3 I~F 2;p

!t l! :i 12 1:

7-9

38

Ill

IV

DMD-/.

H1/H3 i/i J/j

A b C D

3,(

A/A [A] bib [b] C/C [c]

[g] 7-9 [HI] [I]

HI/H1 I/i Jd

AIA bib Clc DID

) E/E gig HI/H3 I/I J/J

T A A/o b bib C C/c D Did

l e 5 ( ~E/e /" G G/g

H1 i I/i J

1 Ale A b/b / \

I c,c / \

/ /,",', I

g H2 i J

21

A/A bib ClC DID ele GIG HIlH2 Ili J/J

o B c D

l i 3 A/O Ala A B/b Bro b CIc CIc C DID DID D

I( elelff )e le l l [~E G/G-- G/G--[ G HIlH3 H2/H3 I H2 Ill Ill | I JIJ ~ J

o [ A 5-~A/o S I b Bib q I C CIc D .L D DID • Z,~ • )Ele G G GIG H3 H2 H2/H3 I I III J J JIJ

1

DMD-5 I

II

Ill

a I Aa c / C/c

I--I E (-~ E/e 1LI-J g 2"~' 61g | H 2 | HIlH3 L _ ~ Ill

I

3(

'A a

H3 H1 I i

D M D - ? [c] [C/c]

I 1 ~ [El 2 ,~ ' [E /e ]

c / = | c r'-ie ('lh EIE i"~ e

II 1y i 2Ti, i 3 " i

III 1 ~ JIj J

JLI

T

~() 5-7

D M D - 8 Ele

. , ,c?

II 1 H I ~ 3=L~1

D M D - 6

I 1 2

I I 1

I I I l ~ g 1 u 6 H3 H2

D HIlH2

I I I 1mine 2 ( O H2

AIo DM D -9 ....,[d] DID

. I/1 [e] - ( '~Ele '"F tgl "T~6 / I H ' ! /H2m4

A/o IA i Did / D ~ele L F"l • 5

Gig ~ G HIlH2 H2

I DMD - 10

~ A/a

?! 1 2 E/e G,/g

L _ ~ HI/H2

T

I ~,o [Ioi o,o l o,o I~ ,o I I C~e / [ c ] c~c / c~ /

P1 .~ Did FIpf'ld ~ DID f--I ,,~ did I'-"1 f'JE "[Dld] III 1LTJ 2~E/E3X-rJ 4yEleSy 6yEle?y 8,x~

/ / g ig / [g [ Gig I I Gig I I L~F_ J H2tH3 [ / ~ Hl/H3 L___~ [H2/H31

I I c I c': I c'c I I I I c j D FLhDIJ fJhDId F~ J. ~ j D

IV l',J 2~m E 3".JEI~L"v'EleS"-J 6" 7)I(~, y 8mm E 9 g Gig glg g H3 H2

Appendix 1. Pedigrees and genotypes of DMD families (abbreviations see Table 2)

9 ( ) l~ill

39

BMD-1

II I[ E le GIG J/j

I Clc / c

III i[ 1 2 l ' G/gEleD/dj/j 3DDGej ~[ 50

A;A B b c C d 2 d e E G 6 J J

IV ~I

I

I

617n;

BMD-2 I

to] I Alo I [b] / Bib I

I Clc .j., . j ~ Dld .C/)

II 1 Ill "~P"

I°,° I ~~ [ bib I B

2LPdj..dg~ L D m ~ E ] ,j, cI~ .1,

L_ I l l I

IV 1 d 2 3 I

l

BMD -3

b C

II 11i l

III

IV 1 7

I I+; b+ ++ E H1 3 H3

J I i

/ Ctc

(Jh E/e l 2 3 5,,~r HIlH3 6 7 8 I I I/i I I J~J

/ c / ~ / e l--I (~E/e l-Lie

5 T 6TH~H:~THI / / vi / r

3,._J 4".] 5LJ

Appendix 2. Pedigrees and genotypes of BMD families (abbreviations see Table 2)

40

M a r - 1 Mar-2 gE GIgele I Mar-3 1 ~ j

Ili r'~ e Ele I 1[~] 2( P J/J I--1 G ~ Gig l

' 1 y H 3 2 y H I I H 2 • / E / e / E / e / I I

1'-i-' i 2 ,? ~n 3 z~i t.

I I I 1 2 3 /.

/ l l i I I I ( ~ 61g I~' IV 1 2 3 1" i Iii 2 ~ JIj

e

I Ala A 3 clc c

~,a ,..L AIA s m c 6 ~ c / c

H2 H31H4

Appendix 3. Pedigrees and genotypes of families used for lod score calculation marker versus marker (abbreviations see Table 2)