Hum Genet (1992) 89 : 635-639

9 Springer-Verlag1992

DNA analysis in Turkish Duchenne/Becker muscular dystrophy families Esra Battalo~lu a, Milhan Telatar 1, Feza Deymeer 2, Piraye Serdaro~iu 2, Faik Kuseyri 3, Co~kun 0zdemir 2, Memnune Apak 3, and Ashhan Tolun 1 1Biology Department, Bo~azi~i University, Bebek Istanbul, Turkey 2Neurology Department, Istanbul University Medical School, (~apa Istanbul, Turkey 3Institute for Child Health, Istanbul University, ffapa Istanbul, Turkey Received February 3, 1992 / Revised March 16, 1992

Summary. The molecular genetics of Duchenne/Becker muscular dystrophy was investigated in 81 affected Turkish families. Deletions were detected by multiplex polymerase chain reaction assays and c D N A Southern analyses. The distribution of the deletions along the gene and their correlation to clinical phenotype were different from the studies reported on other populations. Moreover, D N A polymorphisms in mothers were determined using 8 D N A probes and three C A repeat sequences, and a high degree of informativeness was observed.

Introduction Duchenne muscular dystrophy ( D M D ) is an X-linked recessive disorder, affecting about 1 in every 3400 males. This progressive disease causes death around the age of 20. Becker muscular dystrophy (BMD) is the more rare and clinically less severe form. The allelic disorders arise as a result of mutations affecting the dystrophin protein, which is coded by the largest h u m a n gene identified so far (for a review, see Darras 1990). We have undertaken a large study in Turkish D M D families, the goal being to analyze and determine whether a genetic difference exists in the population. Such an analysis should illuminate a n u m b e r of questions regarding the molecular genetics of this disorder. Moreover, the results should help to determine the most efficient strategy for carrier detection and prenatal diagnosis in Turkish D M D families. D N A analyses were p e r f o r m e d on 81 unrelated families with affected boys. The purpose of the study was to determine (1) the frequencies of the deletions involving different gene regions, (2) the deletion breakpoints and their correlation to clinical phenotype, and (3) the frequencies of restriction enzyme fragment length polymorphisms (RFLP) and C A repeat sequence {(CA)n} length polymorphisms in mothers. Correspondence to: A. Tolun

Patients and methods Patients were diagnosed in the Medical School of Istanbul University, having been referred by either the Neurology Department or the Institute of Child Health. Diagnosis for DMD was based on clinical findings, high creatine kinase levels and electromyographic findings. DNA was isolated from peripheral blood leukocytes either by extraction with phenol-chloroform or by salting-out the proteins as described by Miller et al. (1988). Southern analysis was performed by standard methods, except that nonhybridized cDNA probes were removed by washing in a buffer containing 2 • SSC (1 • SSC = 150mM NaCl/15mM sodium citrate, pH7.0) and 0.1% SDS. Occasionally, hybridization buffer was re-used after heat-denaturing the contained probe. Dystrophin cDNA probes 1-2a, 2b-3, 4-5a, 5b-7, 8 and 9-14 have been described by Koenig et al. (1987). Instead of the whole cDNA9-14, a 1.9-kb HindII fragment containing the cDNA region 9-10 was used (Liechti-Gallati et al. 1990). DNA samples from patients were digested separately by the restriction enzymes HindlII and BgllI for Southern analysis. The orders of the fragments are according to Darras et al. (1988). The DNA probes used for RFLP analyses, the allele sizes, and the restriction enzymes detecting the polymorphisms are shown in Table 1. Multiplex polymerase chain reaction (PCR) assays (designated as multiplex I and multiplex II for simplicity) were according to Chamberlain et al. (1988) and Beggs et al. (1990), respectively. A modified reaction buffer described by Abbs et al. (1991) was also used in these assays. The two 5' (CA), length polymorphisms 5'DYS MSA and 5'DYSII were assayed as described by Oudet et al. (1991) and Hugnot et al. (1991), respectively, and the 3'(CA), length polymorphism as given by Beggs and Kunkel (1990).

Results Deletions

D N A samples from the first 52 patients were assayed for deletions using both Southern analysis with c D N A probes and P C R analysis with multiplex I and multiplex II primer sets. Only the c D N A probes 1-2a, 2 b - 3 , 4 - 5 a , 5 b - 7 , 8 and 9 - 1 0 were used in Southern analyses, since no deletion had been reported to be confined within the c D N A 11-14 region.

636 Deletions were detected in 31 out of the 52 patients by the two multiplex assays. Southern analysis revealed deletions in 2 other patients. One of them lacked the 7.5kb and the 10.5-kb H i n d I I I fragments detected by the probe cDNA1-2a, and the other lacked only the 10.5-kb fragment. In the former patient's DNA sample, a junction fragment was observed, and the fragment specific to exon 8 in the multiplex I assay was amplified. Thus, it was concluded that, in both patients, the deletion in-

b

r I

I",I

m

I

I

I

60 58-59 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 40-41 38-39 37 36 35 34 30-33 29 28 26-27 22-25 21 20 19 18 17 16 14-15 13 12 10-11 8-9 7 6 5 4 3 2 1

volved only the 10.5-kb fragment, corresponding to exons 10 and 11. Twenty-nine further patients were analyzed with the multiplex assays, 14 being found to have deletions, thereby raising the number of patients with deletions to 47 (61%). Furthermore, 4 out of 11 boys referred for BMD diagnosis were observed to have deletions by the multiplex assays, raising the number of patients with deletions to 51. In 29 patients, deletions could be de-

c 3.5 6.0 8.8 1.0 2.3 8.3 7.8-1.0 7.0 3.1 3.7 1.6 1,2-3.9 10 1.5 0.5 4.1 11 4.2 6.2 6.1 1.5 1.3 0.4 1.8 18 11 4.7 5.2 20 12 7.3 3.0 12 1.7 6.0 2.7 6.6 4.0 10.5 7.5 4.6 8.0 3.1 8.5 4,2 3.25 3,2

I I ig,j~

,q,

-

I

Fig. 1. Exons deleted in patxents.R o w a eDNA probes; ro w b exon numbers; r ow c corresponding H i n d I I I fragments. B denotes BMD, J denotes junction fragments, n u m b e r s denote number of patients carrying the deletion. D a s h e d lines indicate deletion breakpoints not mapped

637 tected by both multiplex assays. In 10 patients, deletions could be detected only by multiplex I, and in another 10 patients only by multiplex II. Junction fragments were detected in 6 patients with deletions. The exons in two of the fragments could be determined by multiplex P C R assays and/or Southern analysis by using different restriction enzymes. In 3 out of the remaining 4 patients, junction fragments could be assigned to specific ends of the deletions. A duplication fragment was observed in only 1 patient. In 46 out of the 51 patients with deletions, mutations were confirmed by c D N A analysis. The breakpoints were mapped in 44 of them. The distribution of the deletions along the dystrophin gene is shown in Fig. 1. The majority of the deletions began 3' to exon 44, i.e., 39 out of 46 deletions were confined to this region of the gene. Three of the deletions detected had not been reported previously, and no junction fragment was observed in these patients by using two different restriction enzymes (HindIII and BgllI). Patient 141 was deleted in exons 47-49, patient 9.3 in exons 5-44, and patient 15.3 in exons 3-21.

Table 1. Comparison of percent frequencies of RFLP alleles in Turkish and other populations Probe

Enzyme

Allele size

Turkish Other (no. X (no. X chromosomes) chromosomes)

p754

PstI

XJ2.3

TaqI

XJI.1

TaqI

pERT87-8

TaqI

pERT87-15

XmnI

pERT87-15

TaqI

cDNA8

TaqI

cDNA8

PstI

J66

PstI

99.6

PstI

12 9 6.4 7.8 3.1 3.8 8.7 7.5 1.6, 1.2 2.8 3.1 3.3 6.8 5.7 3.4 10.8 1.50 1.45 1.35 22 13

0.64 0.36 (134) 0.54 0.46 (70) 0.60 0.40 (110) 0.67 0.33 (36) 0.64 0.36 (106) 0.66 0.34 (118) 0.95 0.05 (84) 0.91 0.09 (116) 0.60 0.27 0.12 (40) 0.58 0.42 (132)

Clinical phenotypes In patients with deletions, the clinical severity of the disorder was tested against the frameshift hypothesis (Monaco et al. 1988). Deletions that created a frameshift mutation in the protein coding region caused D M D , whereas an in-frame mutation caused BMD. Four patients were observed not to conform to the hypothesis. Deletions in these patients were mapped with high certainty, since no junction fragment was observed. Although a junction fragment too small or too large to be detectable by Southern analysis could have escaped identification, 7 out of the 8 deletion breakpoints were confirmed by multiplex PCR assays, which amplified exons adjacent to the deleted exons. Patient 9.3 and his D M D brother are deleted in exons 5-44, a previously unreported in-frame deletion. The two brothers have been nonambulatory since age nine. Patient 141 is deleted in exons 47-49, a previously unreported in-frame deletion. Symptoms began at age three. He is presently nine years old and can walk with great difficulty. Patient 104 is deleted in exons 45-47, a typical mild BMD mutation (LindlOf et al. 1989; Norman et al. 1990). Initial symptoms appeared at age four. H e is now eleven years old and can still walk. Although not typical of D M D , the symptoms began earlier than in BMD. Patient 249 is deleted in exons 45-51, an in-frame deletion previously reported to cause a BMD phenotype (Liechti-Gallati et al. 1989). The symptoms began at age three. H e is now 12 years old and walks with extreme difficulty.

DNA polymorphisms D M D mothers were assayed for 10 different RFLPs using eight probes. The probes and the restriction enzymes used were 754/PstI, XJ2.3/TaqI, XJ1.1/TaqI, pERT87-8/ TaqI, pERT87-15/XmnI, pERT87-15/TaqI, cDNA8/

0.62a 0.38 0.70a 0.30 0.72a 0.28 (130) 0.69a 0.31 (105) 0.68a 0.32 (75) 0.67~ 0.33 (75) 0.90b 0.10 (62) 0.88c 0.12 (52) 0.60a 0.30 0.10 (29) 0.71a 0.29 (26)

a Pearson et al. (1987) b Laing et al. (1988) c Darras and Francke (1988)

Fig. 2. Family analysis for the (CA)n repeat polymorphism 5'DYSII on a NuSieve-agarose gel demonstrating 3 different alleles and a crossing-over in one of the patients

TaqI, cDNA8/PstI, J66/PstI and 99.6/PstI. A comparison of the frequencies is shown in Table 1. Except for the two polymorphisms detectable by cDNA8, the Turkish population exhibited a high degree of heterozygosity. Mothers were also highly polymorphic for the (CA)n length polymorphism 5'DYSII described by Hugnot et al. (1991) and Oudet et al. (1991). Out of 35 mothers, 24 were found to be heterozygous upon analysis on 3% NuSieve-l% agarose gels; assay on acrylamide gels revealed another 6 heterozygotes. The results of a family analysis on a NuSieve-agarose gel are shown in Fig. 2. The other two length polymorphisms were assayed on acrylamide gels. Only 3 alleles were detected at each locus. Heterozygosity was observed to be more infrequent than reported for other populations (Table 2).

638 Table 2. Comparison of percent heterozygosity for length polymorphisms in Turkish and other populations Locus name

Turkish

Other

Reference

5' DYSII 5'DYS MSA 3' CA

88 27 28

82 57 50 37

Feener et al. (1991) Oudet et al. (1991) Beggs and Kunkel (1990) Oudet et al. (1990)

Discussion The distribution of the deletions in Turkish D M D patients were different from those in other populations in two respects: deletions were confined to exons 3-60, and the great majority began 3' to exon 44. Two deletions not previously reported were observed; the larger one involved exons 5-44 and the smaller involved exons 4 7 49. Both were in-frame deletions resulting in D M D , thereby causing clinical phenotypes not conforming to the frame-shift hypothesis. Because of the large size of the deletion and the involvement of exons 5-7, which are essential parts of the gene (Malhotra et al. 1988), the observation that the large in-frame deletion (exons 5-44) caused D M D was no surprise. The most intriguing nonconforming deletion was the one involving exons 47-49. The facts that this deletion was within the region where most D M D deletions have been observed in all populations, but that it has not previously been reported, raised the question of whether it usually resulted in a normal or very mild disease phenotype, as has been reported for two other deletions (England et al. 1990; Nordenskj61d et al. 1990). This could not be tested in our seemingly sporadic case, since the patient had no brothers. Moreover, the mother did not seem to carry the deletion in her leukocytes, as judged by the intensity of the autoradiographic bands. One of her 3 brothers was analyzed and found not to carry the deletion. Multiplex ! and multiplex II P C R assays detected 31 out of 33 deletions in 52 patients. The only uncertainty with these assays involved fragment 9 of multiplex II. This fragment was specific to exon 52, and failed to amplify efficiently in two patients, although it was shown not to have been deleted via Southern analysis. A common problem of fragment f of multiplex I failing to amplifying efficiently was resolved when the modified buffer system (without dimethyl sulfoxide) of Abbs et al. (1991) was adopted. The two P C R assay systems were equally efficient in detecting deletions independently, and 29 out of 49 deletions were commonly detected by the two assays. D M D mothers were assayed for 10 different RFLPs. All RFLPs, except one, involved the restriction enzymes PstI or TaqI, to render the analyses cost-effective and less labor-intensive. The polymorphisms detected by the probes XJI.1 and XJ2.3 also exhibited linkage equilibrium in this population. (CA)n polymorphisms were also shown to be suitable for heterozygote detection in the population, although heteorzygosity was much lower at 5 ' D Y S MSA and 3 ' C A loci than reported for other populations. The efficiency of our assays in detecting the

polymorphic alleles at the 3 ' C A locus was verified, using the heterozygote D N A samples kindly supplied by Dr. A . H . B e g g s ; therefore, the low degree of informativeness could not be attributed to the assay system. In the 5 ' D Y S MSA locus, the predominant allele of about 80bp was also the most common in our population. However, other than the common allele, only alleles 10 and 5 were observed, the latter being the most infrequent. As a result, these two polymorphisms were not as informative as most of the RFLPs studied; nevertheless, as these polymorphisms were close to the gene, they were useful in informative families. Moreover, the two 5'loci are within 40kb of each other, and exhibit linkage equilibirum. In all 81 families studied, carrier status in women could be determined with 95% confidence, assuming about 12% recombination probability along the gene locus (Abbs et al. 1990). Thus, the D N A polymorphisms used in this study are sufficient for linkage analysis in Turkish D M D families.

Acknowledgments. We are grateful to Peter Ray and Jean-Claude Kaplan for their support and discussions. This work was supported by Association Fran~aise contre les Myopathies and the Bo~azi~i University Research Fund.

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