Hum Genet (1991) 88:179-184

Springer-Verlug 1991

Molecular deletion patterns in families from southern France with Duchenne/Becker muscular dystrophies Mireille Ciaustres I, Sylvie Tufferyt, Marie- Pierre Chevron i, Marie-Pierre J o z e l o n l, Patricia Martinez I, Bernard E c h e n n e 2, and Jacques Demaille 1 ~INSERM U. 249. CRBM/CNRS UPR 8402. Institut de Biologie, and 2Neuropediatric Department. H6pital Gui de Chauliac, F-34000 Montpellier, France Received April 3, 1991

Summary. We studied 38 unrelated patients from southern France with Duchenne ( D M D ) or Becker (BMD) muscular dystrophy for intragenic deletions of the D M D / BMD gene. We used both multiplex amplification of selected exons and c D N A probes. Of the 26 (68%) unrelated individuals found to have deletions, 24 (92%) were detected by multiplex polymerase chain reaction. All these deletions have been delineated with regard to the exon-containing HindIII fragments revealed by cDNA probes, and in two cases, junction fragments of altered size were seen. The correlation between phenotype and type of deletion agreed with the reading frame theory, except for two BMD and two D M D cases.

Introduction X-linked muscular dystrophy is a degenerative disorder of the skeletal muscle comprising both a severe form leading to death in the early twenties (Duchenne muscular dystrophy, D M D ) and a milder form with a much slower course (Becker muscular dystrophy, BMD), which has been shown by linkage analysis and deletion studies to arise from mutations in the same gene (Kingston et al. 1983: Kunkel et al. 1986). The gene, located at Xp21, is the largest gene so far identified in humans and consists of at least 75 exons distributed over a region spanning 2.3 Mb (Koenig et al. 1987; van O m m e n et al. 1987). The full-length D M D coding sequence (14 kb cDNA, average size of each exon: 200bp) was completely sequenced by Koenig et al. (1987). The gene product, called dystrophin, was identified and localized inside the muscle fibers (Hoffman et al. 1987). Carrier detection and antenatal diagnosis were made possible by the use of restriction fragment length polymorphisms (RFLPs) very close to the D M D locus (BakOffprint requests to: M.Claustres, Laboratoire de Biochimie G6n6tique, Institut de Biologie, Boulevard Henri IV, F-34060 Montpellier. France

ker et al. 1985), and within the gene (Hejtmancik et al. 1986). However, the large gene size and the 5% intragenic recombination rate (Kunkel et al. 1986) made these analyses complex and very laborious. The discovery that intragenic deletions are the most common gene defect leading to BMD and D M D , now detected in over 60% of patients by the use of c D N A probes (Koenig et al. 1987), has led to direct and accurate diagnosis. Recently, an alternative to the complex hybridization procedure has been introduced with the use of the polymerase chain reaction (PCR; Saiki et al. 1988), which has been applied to the detection of deletions within the D M D gene by Chamberlain et al. (1988). This nonisotopic method, based on the simultaneous amplification of several deletion-prone exons within the D M D gene, now detects almost all the D M D gene deletions (Beggs et al. 1990). In the present study, we analyzed 47 D M D and BMD patients from southern France for intragenic deletions by using multiplex amplification of 18 exons and by using the c D N A probes described by Koenig et al. (1987). We mapped the breakpoints of the deletions relative to the exon-containing HindIII restriction fragments and examined the effect of the deletions on the translational reading frame of dystrophin m R N A . Dystrophin c D N A studies, in combination with flanking and intragenic RFLPs, allowed us to pinpoint the origin of D M D / B M D mutations in some families.

Materials and methods Patients

Forty-seven boys from 38 unrelated families were followed in the Hospital of Languedoc-Roussillon (southern France). The diagnosis of DMD or BMD was based on clinical findings including grossly raised serum creatine kinase (CK) activities, pseudohypertrophy of the calf muscles, electromyographic and muscular histological abnormalities consistent with a progressive muscular dystrophy. In some cases, dystrophin studies could be performed by Western blot and immunofluorescence. Some phenotypic charac-

180 T a b l e 1. P h e n o t y p i c i n f o r m a t i o n on affected p a t i e n t s from each of the 40 families studied. DMD, D u c h e n n e m u s c u l a r d y s t r o p h y : BMD, B e c k e r m u s c u l a r d y s t r o p h y : hi, inconclusive or m i s s i n g data Patients

Birth date

Diagnosis"

S e r u m creatine kinase (IU/I) ~

Family history c

No. of years D y s t r o p h i n wheelchair bound d

Exons deleted r

D2 D3 D5 D6 D7 D9 D 10 C B D 12 D13 D 14 T JM D15 D 16 D19 D20 D22 D23 D25 D26 D27 D28 D29 D30 D35 D37 D 3 8 JN T P D50 D51 D52 D43 D44 D47 B31 R JP E S B32 B33 B34 B39 D B B42 A N B45 B46 B49

1975 1981 1970 1985 1967 1973 1986 1983 1969 1978 1971 1975 1983 1971 1974 1976 1972 1970 1979 1974 1972 1976 1978 1984 1974 1980 1986 1987 1989 ni ni 1973 1976 1974 1973 1944 1949 1974 1980 1979 1948 1974 1966 1969 1967 1974 1948 1967 1964

DMD, DMD. DMD, DMD, DMD, DMD, DMD. DMD. DMD, DMD, DMD, DMD, DMD, DMD. DMD. DMD, DMD, DMD, DMD. DMD, DMD, DMD, DMD, DMD. DMD, DMD. DMD. DMD, DMD, DMD, DMD. DMD, DMD, DMD. DMD, BMD, BMD, BMD. BMD, BMD. BMD. BMD, BMD, BMD, BMD. BMD. BMD, BMD. BMD.

ni 4960 (8) ni 11300 (3) 600 (18) 3400 (15) 19400 (2) 3860 (5) 1500 (17) 8000 (7) ni ni 5400 (4) 280 (18) 6000 (6) 9000 (8) 3000 (4) 14200 (3) 3292 (8) 1490 (5) 975 (9) 1252 (10) ni ni 5000 (8) ni 10450 (4) 10220 (3) 9010 (2) ni ni ni 7000 (6) 3600 (7) 1900 (7) 3600 (16) ni 800 (15) ni 8770 (10) ni 1300 (10) 1500 (18) 1000 (15) ni ni 970 (15) 4000 (13) ni

+ + + + + + + + + ni ni + + + + + + ni ni + + + + + + + + +

12 11 11

No 12-44 No No 45 49-50 No No No 48-50 48-50 48-50 No 8-37 49-50 50-52 45 8-27 47-52 19-44 No 10-11 20-25 45-50 45 21-34 No No No No DNA No DNA 45-50 48-52 No 45-52 45-53 45-53 45-53 45-53 3- 7 No No 45-48 45-48 3- 4 3- 4 45-48 45-48 3- 7

5 4 5 4 6 4 2 5 6 6 4 3 4 7 6 7 6 3 8 5 9 4 4 5 5 5 6 3 2 ni 4 5 6 7 7 16 15 4 2 10 ni 4 18 12 15 12 15 13 ni

~'A g e at d i a g n o s i s (years) b N o r m a l v a l u e s for s e r u m creatine kinase activity are less t h a n 75 or 150 IU/I d e p e n d i n g on t h e laboratory: age in y e a r s at time of det e r m i n a t i o n given in p a r e n t h e s e s ~ C o n s i d e r e d positive if t h e r e were o t h e r affected m a l e s a n d obligate carriers a n d n e g a t i v e if t h e a f f e c t e d m a l e was an isolated case d D a s h indicates p a t i e n t is still a m b u l a t o r y

-

13 12 -

Absent

-

11 10 11 10 11.5 11 10 11 12 10 8 10 9,5 9,5 ni ni 11 9 -

ni ni 11,5 12 11 10 38 ni 16 39 -

Absent

Absent Absent Absent Absent Absent

Absent

Present Smaller Smaller Smaller

Reading frame f

Shift. + 1

Shift, + 2 Shift. + 1

Shift, + 1 Shift, + 1 Shift. + 1 Shift, + 2 Shift. + 1 Shift, + 1 Shift, + 2 '~ Shift. + 1 In f r a m e Shift, + 2 Shift, + 1 Shift, + 2 In f r a m e

Shift, + 1 shift, + 1 Shift. + l In f r a m e In f r a m e In f r a m e In f r a m e Shift. + 1

In f r a m e In f r a m e In f r a m e In f r a m e In f r a m e In f r a m e Shift, + 1

r No indicates that n o deletion was d e t e c t e d by p o l y m e r a s e chain reaction or c D N A probing: exon n u m b e r s a c c o r d i n g to K o e n i g et al. (1989) f Shift occurs w h e n the deletion disrupts t h e o p e n translational f r a m e ( O R F ) , c a u s i n g a c h a n g e in t r a n s l a t i o n ( n u m b e r indicates predicted f r a m e s h i f t of t h e r e s u l t a n t m R N A ) : in f r a m e indicates that the c o n s e q u e n c e of t h e deletion o n t h e r e a d i n g f r a m e could not be d e t e r m i n e d

181

teristics are shown in Table 1. Five DMD patients originated from North Africa (D7, D25, D26, D44, D47).

a

b

9

3'

D NA analysis D N A used for PCR and Southern blot studies was extracted from blood leucocytes using standard phenol/chloroform procedures. D N A concentrations were quantified by using a spectrophotome-

I1~ 388

8

50~

547 268 357

5b-7

Dystrophin analysis. The presence and distribution of dystrophin in muscle could be analyzed for some patients by immunofluorescence and immunoblotting methods (Hoffman et al. 1988; Arahata et al. 1989). Antidystrophin antibodies were generous gifts from JJ L6ger (INSERM U300, Montpellier. France).

Results

Gene deletions Using the nine pairs of primers described by Chamberlain et al. (1988, 1990), we detected exonic deletions in 22 unrelated patients (57.8%). With the second multiplex PCR using set primers of Beggs (1990), 2 additional D M D patients were found to have deletions. All these deletions were systematically checked and their extent determined by c D N A probing using 6 subclones ( 1 - 9 ) on HindlII restriction digests. In most of the cases, another restriction enzyme was used (TaqI or PstI) to avoid ambiguous results. In the 14 remaining patients in w h o m deletions were not found with PCR analysis, we were able to demonstrate, for 2 D M D patients, a deletion when c D N A probe 4 - 5 a was used. Twenty-six molecular deletions removing portions of the c D N A were detected in this sample of 38 unrelated B M D and D M D patients, corresponding to an overall deletion detection rate of 68%. Thus, the overall efficiency of multiplex amplification in detecting deletional mutations of the D M D gene was 92% in our series. The deletion endpoints were mapped on the established HindlII fragments map (Koenig et al. 1987) as il-

1.5 t t 0.0 0.5 4.I

45 44 43 I 1.0 4.2 I 42 6.2 1 411+-I1 0,1 38+39 i ~ r'36

4-5a

Southern blot analysis. D N A from affected males and their families was digested with the appropriate restriction enzyme, sepa-

The c D N A subclones 12b-14 were not used, as they detect an untranslated region9 The cDNA probes were obtained from Dr. L. M. Kunkel's laboratory or from the American Type Culture Collection.

47

0"4 I 3 5 1.8 34 18.0 3O-33 12.01 29 4.7 ]- 28

ducts, Rockland, M e . ) + 1 % agarose gels.

rated on 0.8% agarose gels by electrophoresis, and transferred to Hybond-N or Hybond-N+ membranes (Amersham). The membranes were hybridized with 32P-dCTP random-primed c D N A or anonymous Xp21-p22 genomic probes, then autoradiographs were processed at -70~ for 2-7 days after adequate washings.

It,!

381 48

I

-181

ter, as well as by monitoring the intensity of ethidium bromide staining lanes to determine the dosage difference of the hybridization intensity. Multiplex amplification of exons in vitro by PCR. Synthetic oligonucleotide primers were prepared by the phosphoramidite method on an automated DNA synthesizer (Applied Biosystems), from the sequences described by Chamberlain et al. (1988, 1990) and Beggs et al. (1990). Multiplex DNA amplifications were carried out exactly according to these authors, except that 100pmol of each primer of exon 48 was used instead of 50pmol. Amplification products (151al) were analyzed on 2% NuSieve (FMC Biopro-

271

d C 8.3 d 54 1,0 .~ 53 7.8 .l 7~/ I 52 51 3. , 5O 49

459 416

2b-3 238 331

5.2 I 21~'0

26+27 22"2521

7.3 3.0]12.0r i.71 6.[)l ~ ' 6.6 I 4..: i 10.5 I

20

7S

1-2a

202 [ 196 410 5'

!i

19

I~ 17 16 14+15 13 12 I0"-I 1

I 8+9

4,6l 7 8.01 -4 ~,ll 5 8.51 4 4.2 I 3 3.2~ 2 3.2 I 1

e,l_

Fig.1. Deletion pattern observed in the 25 unrelated Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients by using the dystrophin cDNA. a Dystrophin cDNA probes used for the detection and delineation of the deletions (map according to Koenig et al. 1989); only part of the gene is shown, b Position and size (in bp) of fragments amplified by the multiplex PCR reactions used in this study. The corresponding exons are indicated in d. c HindIII genomic fragments (sizes in kb) detected by cDNA probes 1-9 (Koenig et al. 1989). d exon numbers. Each deletion is marked with a vertical line (solid bars, DMD; open bars. BMD). J indicates that a junction fragment of abnormal size was observed. The dot above the bars indicates the deletions that do not follow the reading-frame rule

lustrated in Fig. 1. Of the 25 deletions, 17 were localized to the region detected by probes 7 and 8, which define the hot-spot region of the middle part of the c D N A . Of the deletions, 46% had one of their intronic breakpoints b e t w e e n the genomic HindIII fragments 4.1 and 0.5 kb, the p20-containing large intron (Wapenaar et al. 1988). In two cases, an altered-size restriction fragment was detected, one with probe 8 (patients D 1 4 ) , another with probe 4 (patient D37). The presence of a junction fragment in some females of the corresponding pedigree allowed an easy identification of the carriers. The deletions involved from 1 to 32 exons; this size variation s e e m e d to be greater among D M D patients than among those with B M D . In the four familial deletion cases where D N A from the other affected males could be studied, we found the

182

deletions to be identical in all the affected males from the same family. We did not observe deletions of the D M D gene in 21 healthy males in our series, No duplication of any exon-containing HindlII fragment was found in our patients.

Frameshift and in-frame deletions We could predict the consequences of deletions on the translational open reading frame (ORF) of the dystrophin m R N A from the published sequences of the entire c D N A

and of the intron/exon boundaries of main parts of the D M D gene (Koenig et al. 1989; Malhotra et al. 1988: Baumbach et al. 1989). As exons of the D M D gene do not necessarily contain an integral number of triplet codons ( M o n a c o et al. 1988: Malhotra et al. 1988), deletion of a D N A segment consisting of one or several exons with a nonintegral number of codons is expected to cause a shift in the O R F , producing a prematurely truncated protein. The O R F is maintained if the deletion juxtaposes exons with exon/intron borders that would restore the normal triplet codons in the D N A sequence. The predicted effects that deletions would exert on the O R F are presented with clinical data in Table 1. Of 19 D M D cases deleted, 17 could be explained by frameshifts followed by premature translation termination, 1 had an apparently in-frame deletion (D26), and 2 could not be determined. T w o brothers (D 14) displayed a deletion of exons 48-50, with an apparent discrepancy between the c D N A / S o u t h e r n blot (absence of the 3.1-kb HindlII restriction fragment) and the multiplex PCR, which revealed the exon 51 and its flanking introns to be

99.6 C7 p20 JBir p87.30 p 8 7 15 p87. I x J2.3 754 754. 754

Pstl EcoR\ Ec~)R ~, ~ a m H) 13gll I Xmnl Taql Ec(~R\ Xmn[

D7

9

Taql Pstl EcoRI [~gl] I

Fig. 2. D M D deletions of patients D 14. Left, representative autoradiography displaying the pattern of hybridization obtained with HindlII-digested D N A after probing with cDNA probe 8. The number in parentheses to the left of the HindllI fragments indicates the exon contained in each fragment. Right, analysis of the deletion D14 by PCR. From top to bottom, the amplified fragments correspond to the exons indicated on the left. Arrows point to missing exons (top exon 50, bottom exon 48). Lane 1 control male. lanes 2, 3 the two DMD brothers (D14)

2 2 2 2 2

2 2 2 2 2

I I

2 I

I

!

2

2

I

!

2

2 2 2 I

2 I 2

2 Ile 2

I

i

"

2

2

2

! 1

I I

2 1

2

2

1

I -

I I

1 1

"~

2

2

ni I

1

I

1

I

2

2 2 2

2 De 2

2 2 2

2 I 2

2 2 2

2 De 2

I I

2 2

I

I

2

2

2

2

2

2

I 1

2 1

1 I

I I

2 I

2

I

2

2

1 1

I 1

1 I

1 I

2

2

2

2

I I 2 1 I 2

2 I)e

2

Fig.3A, B. Muscle extracts from patients with DMD or BMD. Blots (A) and sections (B) were probed with polyclonal antibody to dystrophin (gift from J. J. Leger): N, normal control

I 1 I

2

2 2 I)e

2 2 2 2 2 I ! 1 I 2

C7 F~'oR ~, p20 I:,coR%, p87A I.;coR'~

Fig. 4. Genotyping of family D3 to illustrate the determination of parental origin of neomutations using genomic probes. Distinct haplotypes are represented by vertical bars. 1 Absence of restriction site. 2 presence of restriction size, De absence of band on the autoradiogram. The RFLPs are listed according to the gene order

183 intact (Fig. 2). This finding suggests that the distal breakpoint of this deletion is within the intronic 3.1-kb HindIII fragment, the proximal breakpoint between the 10kb HindIII fragment and the 1.25/3.8kb fragments that contain exon 48. All the B M D deletions were in-frame, except for patients B32 and B49, who were found deleted for exons 3 - 7 , and fell in the category of B M D exceptions described by Malhotra et al. (1988), Koenig et al. (1989), and others. Muscle specimens from eight patients could be analyzed using polyclonal antibodies directed against the D M D protein, dystrophin. The pattern of dystrophin labeling on blots of muscle extracts and on muscle sections was in complete agreement with the phenotype. Dystrophin was undetectable in samples from the D M D patients D20, D38, D44, all with "out-of-frame" deletions, and in samples from the D M D patients D50 and D51 (deceased patients, no D N A available). Four B M D patients could be analyzed for muscular expression of the gene: one (B39) had reduced abundance of a 429kDa dystrophin on immunoblotting and showed m a r k e d intrafiber variations with immunochemistry: the three other (B42A, B45, B46) had a smaller dystrophin in their muscle samples (Fig. 3). In some of our 26 families with deletions, the grandparents of the proband were available for study. By using Xp21 RFLPs and the dystrophin c D N A , we could trace the origin of the chromosome in which the deletion arose, as illustrated in Fig. 4. One deletion was a new mutation in the affected individual (D3, maternal gamete), two deletions were new mutations in the carrier m o t h e r (D7, D14, grandmaternal gametes), and four deletions were transmitted to carrier grandmothers. For four families without grandparental D N A available, all the mothers of affected children were found to have gene deletions by Southern/cDNA analysis.

Discussion As predicted by Monaco et al. (1988), collaborative studies (Koenig et al. 1989: Gillard et al. 1989) have demonstrated that the effect of deletions on the translational reading frame of dystrophin m R N A m a y account for the phenotypic differences between B M D and D M D , rather than the size and location of deletions. O u r data, with four exceptions, agree with most of the published series (Gilgenkrantz et al. 1989; B a u m b a c h et ai. 1989). The severe phenotype in the D M D patients who had apparent in-frame deletions could be explained in several ways: effects on transcription, splicing, or translation of the D M D gene, or very simply because the deletion may invisibly affect the edge of an exon. The mild phenotype of the two B M D patients having an out-of-frame deletion of exons 3 - 7 could be due to differential splicing creating an in-frame mutation (Chelly et al. 1990), or to the use of one of the translational initiation A T G codons in exon 8 (Malhotra et al. 1988). Detecting deletions of the D M D gene is important both for the light they throw on the pathogenesis and because they permit much more accurate risk estimation

and prenatal diagnoses in families (Darras et al. 1988: den Dunnen et al. 1989). Screening of each D M D or B M D patient for deletion using multiplex P C R was very accurate and reproducible (we had no technical problems with P C R in this study, and no discrepancy with c D N A probing was found). For a routine diagnosis service laboratory, the detection of almost all the D M D deletions by two series of 1-day multiplex P C R is a fantastic alternative to the very difficult and time-consuming Southern technique with multiple hybridization with c D N A subclones. However, we still continue to use c D N A probing for males and females from each family to delineate each deletion and to try to obse~rve the presence of absence of the 50% reduction in hybridization signal intensity that should occur in the carrier females. Due to the technical difficulties of reproducing such an analysis in a modest hospital laboratory, we are still using the analysis of segregation of several gene-specific or flanking R F L P markers. Also, we think that defining the parental origin of mutations is fundamentally and clinically important.

Acknowledgements. This study was supported in part by grants from the Association Franc aise contre les myopathies (AFM). The authors are grateful to Drs.L.M.Kunkel, J.L.Mandel, and R. Worton for providing DNA probes: Dr.E.Bakker for excellent technical advice, and Dr. M. Koenig for unpublished information on primer sequences. We would like to thank the numerous physicians who provided clinical information and contributed to the gathering of blood and muscle samples. The manuscript was typed by M. Nicolas.

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184 Darras BT, Blatmer P, Harper JF, Spiro A J, Alter S, Francke U (1988) Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: location of breakpoints on HindIII and BgIII exon-containing fragment maps. meiotic and mitotic origin of the mutations. Am J Genet 43 : 620-629 Dunnen JT den, Grootscholten PM, Bakker E, Blonden LAJ, Ginjaar HB, Wapenaar MC, Paassen HMB van, Pearson PL, Ommen GJB van (1989) Topography of the DMD gene: FIGEand cDNA-analysis of 152 cases, involving 85 deletions and 11 duplications. Am J Hum Genet 45 : 835-847 Gilgenkrantz H, Chelly J, Lambert M, R~can D, Barbot JC, Ommen GJB van, Kaplan JC (1989) Analysis of molecular deletions with cDNA probes in patients with Duchenne and Becker muscular dystrophy. Genomics 5 : 574-580 Gillard EF, Chamberlain JS, Murphy EG, Duff CL, Smith B, Burghes AHM, Thompson MW, Sutherland J, Oss I, Bodrug SE. Kalmut HJ, Ray PN. Worton RG (1989) Molecular and phenotypic analysis of patients with deletions with the deletionrich region of the Duchenne muscular dystrophy (DMD) gene. Am J Hum Genet 45 : 507-520 Hejtmancik JF, Harris SG, Tsao CC, Ward PA, Caskey CT (1986) Carrier diagnosis of Duchenne muscular dystrophy using restriction fragment length polymorphisms. Neurology 36 : 15531562 Hoffman EP, Brown RHJ, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919-928 Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterson R, Brooke M. Specht L, Kupsky W, Chamberlain J, Caskey CT, Shapiro F, Kunkel LM (1988) Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy patients. N Engl J Med 318: 1363-1368 Kingston HM, Harper PS, Pearson PL, Davies KE. Williamson R. Page D (1983) Localization of the gene for Becker muscular dystrophy. Lancet II : 1200 Koenig M, Hoffman E. Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organi-

zation of the DMD gene in normal and affected individuals. Cell 50:509-517 Koenig M, Beggs AH. Moyer M, Scherpf S, Heindrichs K. Bettecken T, Meng G. Muller CR. Lindlof M, Kaariainen H, Chapelle A de la. Kiuru A. Savontaus ML, Gilgenkrantz H, Recan D, Chelly J, Kaplan JC, Covone AE, Archidiacono M, Romeo G, Liechti-Gallati S, Schneider V. Braga S, Moser H, Darras BT, Murphy P. Francke U, Chen JD. Morgan G, Denton M, Greenberg CR, Wrogemann K. Blonden LA, Paassen HMB van, Ommen GJB van. Kunkel LM (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Genet 45 : 498-506 Kunkel LM, et al (1986) Analysis of deletions in DNA of patients with Becker and Duchenne muscular dystrophy. Nature 322: 73-77

Malhotra SB. Hart KA, Klamut HJ, Thomas NST, Bodrug SE, Burghes AHM. Bobrow M, Harper PS, Thompson MW, Ray PN. Worton G (1988) Frame-shift deletions in patients with Duchenne and Becker muscular dystrophy. Science 242:755759

Monaco AP, Bertelson CJ, Liechti-Gallati S. Moser H, Kunkel LM (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2 : 90-95 Ommen GJB van, Bertelson C. Ginjaar HB, Dunnen JT den, Bakker E. Chelly J. Matton M, Essen AJ van, Bartley J. Kunkel LM, Pearson PL (1987) Long-range genomic map of the Duchenne muscular dystrophy (DMD) gene: isolation and use of J66 (DXS268), a distal intragenic marker. Genomics 1 : 329336 Saiki RK, Gelfand DH, Stoffel S. Scharf SJ, Higuchi R, Horn GT. Mullis KB, Erlich HA (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science 239 : 487-491 Wapenaar MC. Kievits T, Hart KA, Abbs S, Blonden LAJ, Dunhen JT den, Grootscholten PM, Bakker E, Verellen-Dumoulin CH, Bobrow M, Ommen GJB van, Pearson PL (1988) A deletion hot spot in the Duchenne muscular dystrophy gene. Genomics 2 : 101-108