A molecular survey of Israeli Duchenne and Becker muscular dystrophy patients

359

A molecular survey of Israeli Duchenne and Becker muscular dystrophy patients C Legum1,2, R Shornrat’, M Glassner’, Y Shiloh2

Summary

- Duchenne (DMD) and Becker (BMD) muscular dystrophy are allelic X-linked recessive diseases caused by a mutation in the dystrophin gene located on the short arm of chromosome X (Xp21). The dystrophin gene is the largest gene known in humans, extending over 2300 kb and containing more than 70 exons coding for a 420 KD protein comprising 3685 amino acids. The gene is highly unstable, with a high percentage of deletions and rearrangements. A third of dystrophin mutations are new mutations. The frequency of DMD is I :3500 liveborn males, and that of BMD I: 10000. These dystrophies are severe. progressive, and lethal. BMD/DMD patients and 2/3 of female carriers have high levels of creatine phosphokinase (CK). During the past 5 years, 169 families with patients affected by progressive muscular dystrophy were examined and counselled. We were able to exclude the diagnosis of DMD/BMD in 49 families on the basis of clinical symptoms and signs, normal dystrophin on biopsy (I I families) and/or the absence of linkage to chromosome X by analysis of RFLP derived haplotypes. Molecular analysis was performed on I I I DMDlBMD families (five BMD and 106 DMD) with 81 available probands. This study resulted in the establishment in Israel of an integrated diagnostic protocol for DMDIBMD, employing genetic, biochemical and molecular techniques. Molecular analysis provided most of the families with new and essential information.

Duchenne

muscular

dystrophy

I dystrophin

gene

Introduction DMD and the milder type BMD are allelic diseases caused by mutations in an extremely large gene situated on the short arm of chromosome X (Xp21). DMD is progressively lethal with a mean age of onset of approximately 3.1 years. Nearly all patients are wheelchair-bound by 13 years and less than 10% survive beyond 20 years [ 151. The gene contains 2.3 Mb, with about 75 exons [ 141 and, in skeletal muscle, has a 14 Kb coding sequence [24, 291 which codes for a 427 kDa protein called Dystrophin. This plasma membrane protein contains about 3600 amino acids, and in humans is essential, in an as yet unknown manner, for maintaining the integrity of the myofibril [ 1, 19-211. The Dystrophin gene has a very high mutation rate (So-100 x 10-6) and deletion mutation is seen in over 60% of patients in most populations studied [26]. About 6% of mutations are duplications [22]. The large size and relatively

high recombination rate of approximately 11% as compared to the 2%-3% expected on the basis of gene size [31], account for the high incidence of the disease (1:3300 males). DMD, usually in a milder form affects over 3% of female carriers. These diseases, especially DMD, are severely disabling and as there is still no effective treatment, carrier detection, prenatal diagnosis and selective abortion of affected fetuses are often requested [3]. Several other autosomal recessive and X-linked muscular dystrophies mimic the symptoms of DMD/BMD thus causing occasional difficulty in precise diagnosis. Since genetic counseling to the relatives of other muscular dystrophy patients differs radically from that given to DMD/BMD families, it is essential to accurately define the disease at the clinical, biochemical and molecular levels [ 151. We present a 5-year study of genetic and molecular analysis of DMD/BMD in the Israeli population.

360 blood cells, cultured amniocytes, chorionic villous cells, Southern blotting and hybridization were performed according to previously published methods [34]. All DNA samples were tested with intragenic and flanking genomic probes (table I), and the full length cDNA probes of the dystrophin locus (table II). The molecular analysis included construction of RFLP haplotypes and a search for gene deletions and rearrangements by hybridization of the cDNA probes to Southern blots and by the use of the Polymerase Chain Reaction (PCR). The I4 PCR primer pairs used, define exons along the gene with special emphasis on two regions regarded as deletion “hot spots” according to published methods [9, 12, 131. Each family underwent genetic counselling before and after molecular analysis. Carrier risks were calculated using Bayesian analysis based on epidemiological, pedigree molecular data and on CK levels. A

Methods Letters were sent to Neurologists, Pediatricians, rehabilitation centers, the Israeli Muscular Dystrophy Association and daily newspapers announcing an inaugural public meeting and lecture. Following the lecture physicians were invited to refer families to participate in the project on a research and service basis. A register was created and first priority for molecular analysis was given to older patients. All medical records were reviewed and probands examined by a pediatrician. 2025 ml peripheral blood was drawn from each relevant family member for molecular diagnosis and CK testing. Prenatal diagnosis was carried out in suspected carriers using transcervival Chorionic Villous Sampling (CVS) and Amnioscentesis (AC). DNA isolation from nucleated

Table I. DMD/BMD

genomic

probes.

Probe

Locus

D2

99-6 c7 P20

J-Bir PERT PERT PERT PERT PERT PERT XJ-2.3 XJ-1.2 XJ-I.1

87-30 87-15 87-15 87-15 87-l 87-l

RFLP

DXS43 DXS4l DXS28

Pvu II Pst I EcoRI

6.6/6 22113 137

DXS269 DXS270 DXS 164

Msp I BamH I Bgl II Xmn I BamH I Taq I Xmn I Msp 1 Taq I Bcl I Taq I

812 2115 3018 2.8/1.6 + 1.2 9.417.1 + 2.3 3.313.1 8.717.5 4/l .7 7.516.4 211.6 3.8/3. I

Pst I Bgl II

1219 6.315.6

DXS206

754 754-l 1

DXS84

Table II. cDNA probes. Probe

Insert

Exons Cloning

I-2a

The

I-II

Alleles fKb)

2b-3 4-5a 5b-7 6b

10-20 21-33 34-46

8 9-14

47-52 53-65

EcoR Xba EcoR EcoR EcoR EcoR Xba EcoR EcoR

vector

in all probes

is (pBluescript).

site

kb

I

1.5

I I I I

1.0 I.8 2.5 I.6

I I

0.8 5.8

I

I

Location Distal to the gene

Intragenic

Proximal

to the gene

written summary was subsequently sent to each family and the referring physician. In order to differentiate between DMDlBMD and other muscular dystrophies, some patients underwent muscle biopsy which was tested for the presence of dystrophin by immunohistochemical staining and by Western blotting [2]. For prenatal diagnosis, in two women non-informative for all extra and intragenic probes, and in another with an intragenic crossover, intrauterine fetal muscle biopsy was carried out by MF Golbus.

Results and Discussion One hundred and sixty-nine families were referred for genetic counseling (table III). Molecu-

361 Table III.

Families

referred

for genetic counseling. No

DMD/BMD Spinal Muscular Atrophy Limb Girdle: autosomal recessive Myotonic dystrophy Emery-Dreifuss Facioscapulohumeral Hyperthermia Congenital muscular dystrophy Vacuolar myopathy Mitochondrial myopathy Other Total

120 14 II 8 3 2 2 I I I 6 169

lar analysis was carried out in 111 families (table IV). In 24 families more than one sibling but no second-degree relative was affected. In 21 families affected children were found in several generations and in 24 other families there was only one affected child and a first degree female relative with an elevated CK level. These 69 (62%) families were considered as having transmitted mutations. In the remaining 42 families there was only one affected male, and all females had normal CK levels. The patients in these families were considered “sporadic cases”. It is of the utmost importance to define the origin of the mutant allele and to identify female carriers. In “sporadic” families this often proved to be difficult. CK levels were obtained from 240 female relatives. Amongst these women 82 belonged to “sporadic” families and 158 to transmitting families. In the latter group only 67 (42%) had raised CK levels. Of these 64, 96% were subsequently shown to be carriers by molecular analysis. In transmitting families 91 (58%) females had normal CK levels. Molecular analysis enabled us to exclude the carrier state in 69 (76%). Elevated CK levels are of particular importance in families where the patient had died before molecular

analysis had been carried out. High CK levels might help to define the mutant allele carriers in such a family [35]. These CK associations are similar to those found in other populations [ 17, 381. The contribution of molecular analysis to the identification of the genotypes in families with inherited mutations is significant and makes prenatal diagnosis feasible with a high degree of accuracy. In a sample of 129 female relatives with a calculated risk of 20%-40% of being a carrier before molecular analysis, 73 belonged to transmitting families and 56 to “sporadic” families. In the transmitting families molecular analysis significantly changed the risk in 89% (fig l), whilst in the “sporadic” families there was a significant change in only 39% (fig 2). In several families molecular analysis failed to differentiate between maternal alleles because of non informativity or crossovers. Similar results were obtained in other studies [6, 181. In families with deletion mutations the situation is clear-cut as deletions in males can be readily shown using both cDNA hybridization and PCR. These techniques detected deletions in 28 out of 81 (35%) DMD/BMD probands, three of them having a junction band. PCR detected two deletions that were not seen by cDNA hybridization because of DNA degradation (fig 3). Detection of a deletion confirms the diagnosis and enables prenatal diagnosis to be carried out with 100% certainty [38]. In the Israeli popula-

100 go-

1

60 5

70-

g

60-

;

so-

3 =

3020 10 -

Table IV.

Molecular

analysis

of

Israeli

DMDlBMD

9

40odI

10

0

patients.

Carrier

DMD

BMD

106 30 76 27

5 0 5 1

m

Families analyzed Proband not available Probands available Proband with deletion

Pm DNA Analysis

Risk (%)

0

Post DNA Analysis

Fig 1. Distribution of a sample of 158 women inherited mutations according to their risk BMD carriers, pre and post DNA analysis.

in families with of being DMDl

362 100 90 60 -

20 loO0

Fig2. families pre and

Distribution according post DNA

5

7 20 Carrier Risk (%)

41

93 100

of a sample of X2 women in “sporadic” to their risk of being DMD/BMD carriers. analysis.

Fig 3. Size and exons‘ by cDNA hybridization

location of 23 deletions and PCR.

as determined

tion the proportion of deletions (35%) is significantly less than that observed in other western populations (65%-700/o) [S, 10, 161. This finding is in accordance with the results we obtained using genomic probes (excluding P20) which detected deletions in 3.2% of our patients as compared to 6.8% observed with genomic probes in other populations [26]. Of the 28 deletions, 72 (78%) were found between exons 44 and 52, a region known to be a major “hot spot” [ 111. This region is unstable, manifesting a high frequency of rearrangements and recombination events. The other 22% of deletions were located between exons l- 19, at another “hot spot” situated at the 5’ end of the gene. Although the distribution of the deletion mutations is similar in Eastern and Western populations, their proportion differs. Relatively low proportions of deletions have been reported in Asian populations. In Japanese families the proportion of deletions in two studies was 43% [37] and 40% [23] and in Chinese families 45% [36] and 50% [27]. However the Israeli families have the lowest proportion of deletions yet reported. No correlation was observed between the size and location of the deletions and the severity of the disease. When a deletion causes a frame shift, the disease is almost always DMD [25, 281. In our patients all probands with frame shift deletions had typical DMD. This enabled us to counsel the families more accurately with regard to the course and severity of the disease. This was especially important for younger patients whose clincial course was not yet typical of DMD or milder allelic variants. In our only BMD patient with a deletion we could not define the borders of the deletion because of a junction band. Female carriers were identified using cDNA hybridization, either by detecting a rearrangement or by estimating the intensity of the hybridization signal on Southern blots and by the intensity of PCR bands. On examination of the origin of the mutant dystrophin allele in families where three generations underwent molecular analysis we found that in eight out of 19 (42%) the mutant allele was derived from the mother’s father (“paternal origin”). In these families the carrier risk for the sisters of the patient’s mother is about 14% because of the possibility of gonadal mosaicism t5, 71. Eighty-three prenatal diagnoses were performed in this study (table V). Most of these were carried out by allele tracking using RFLP haplo-

363 Table V. Prenatal

diagnosis. 100% carrier

Women tested Chorionic villous Amniocentesis Female fetus Male fetus Affected fetus Healthy newborn

sampling

risk

48 34 I4 I3 35 19 I6

20%-d/% carrier risk

35 I2 23 21 14 2 12

types, and in families with deletions, by using cDNA and PCR analysis. In four cases of spontaneous abortion two occurred after CVS and two after AF procedures. Recombination events were seen in four male fetuses thus creating a doubt concerning their disease status. In three such pregnancies the women elected to continue with the pregnancy and all three infants, now l-2 years old, have a normal CK level and no clinical symptoms of DMD. The remaining pregnancy terminated spontaneously after fetal muscle biopsy. There were no false positives and no false negatives in this series. In 48 (58%) of these pregnancies the mother had a high risk of being a carrier. Generally, most of the high risk carriers elected to undergo CVS rather than AF (table V). There was no significant difference between the RFLPs haplotype distribution in 100 healthy individuals and 50 patients, nor between the Jewish and Arab population samples. The ethnic distribution of the DMD/BMD families was similar to that found in the genera1 Israeli population, and no linkage disequilibrium was found. The overall recombination rate within the dystrophin gene was 8.9%. This rate is high compared to the expected 2%, and probably contributes to the high new mutation rate of 33% found in the dystrophin gene [4]. Molecular analysis provided most of the families with new and essential information. This survey of Israeli DMD/BMD patients has led us to the following conclusions: The percentage of deletions in the dystrophin gene in Israeli patients is considerably less than in most other populations studied. In populations with a high proportion of deletions, considerable saving of time and expense can be achieved by initially searching for deletions by multiplex DNA amplification. In Israel we are obliged to expend more time and money as more probands require, in addition to the PCR search for deletions, full cDNA analysis and RFLPs test-

ing using a minimal number of genomic probes (one informative intragenic and two informative flanking extragenic probes). The exclusion of non DMD/BMD families by dystrophin immunohistochemistry staining of the proband’s initial muscle biopsy would save the time and expense of molecular analysis at the dystrophin locus in the non DMD/BMD families. Although CK serum levels in transmitting families is a better predictor of carrier status than in “sporadic” families, the degree of probability obtained using Bayesian analysis based on molecular studies, CK testing and pedigree analysis is often insufficiently accurate to satisfy the needs of the counselee. The application of point mutation detection techniques [32, 331 may solve this problem on a routine basis in the future.

Acknowledgments We are grateful to Dr L Kunkel for providing us with the genomic probes and full length cDNA probes, to Dr GJB Van Ommen for probes J-66 and P20, to Dr MF Golbus for obtaining in utero fetal muscle biopsies on two of our patients, to Doctors G Yedwab, A Yaffo, R Amster and Y Sagi for obtaining amniotic fluid and chorionic villous samples, and to M Haran. T Erlich and R Lazar for technical assistance. We also thank the many muscular dystrophy patients, their families and their physicians for their willing co-operation.

References Arahata K, Soichi I. Sugita H. lmmunostaining of skeletal and cardiac muscle surface against Duchenne muscular 1988;333:861-3

membrane dystrophy

with peptide.

antibody Nature

Arahata K. Hoffman EP, Kunkel LM, Ishiura S et al. Dystrophin diagnosis: comparison of dystrophin abnormalities by immunofluorescent and immunoloblot analysis. Proc Nat1 Acad Sci USA 1989;86:7154-8 Bakker E. Bonten EJ, de Lange LF, Veenema H et al. DNA probe analysis for carrier detection and prenatal diagnosis of Duchenne muscular dystrophy: a standard diagnostic procedure. J Med Genef 1986a;23:573-80 Bakker E. Pearson PL. Mutation of Duchenne muscular dystrophy gene associated with meiotic recombination. C/in Gene1 1986b;30:347-9 Bakker E. van Broeckhoven C, Bonten EJ, van de Vooren MJ. Germline mosaicism and Duchenne muscular dystrophy mutations. Narure 1987;329:554-6 Bakker E, Bonten EJ. Veenema H. den Dunnen JT, Grootscholten PM, van Ommen GJB. Pearson PL. Prenatal diagnosis of Duchenne muscular dystrophy, a threeyear experience in a rapidly evolving field. J inherit Mefab Dis 1989a; 12: 174-90

364 7 Bakker E, Veenema H. den Dunnen JT. van Broeckhoven C. Germinal mosaicism increases the recurrence risk for “new” Duchenne muscular dystrophy mutations. J Med Genet 1989b;26:553-9 8 Baumbach LL, Chamberlain JS. Ward MS, Farwell NJ, Caskey CT. Molecular and clinical correletion of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 1989;39:465-74 9 Beggs AH, Koenig M, Boyes FM, Kunkel LM. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990;86:45 10 Blonden LAG, Den Dunnen JT. Van Passen HMB, Wapenaar MC et al. High resolution deletion breakpoints mapping in the DMD gene by whole cosmid hybridization. Nucleic Acid Res 1989;17:561 l-21 I I Blonden LAG, Grootscholten PM, Den Dunnen JI, Bakker E et al. 242 Breakpoints in the 200 Kb deletion-prone P20 region of the DMD gene are widely spread. Genomics 1991;10:631-9 12 Chamberlain JS, Gibbs RA. Ranier RA, Nguyen PM, Caskey CT. Deletion screening of DMD locus via multiplex DNA amplification. Nucleic Acid Res 1988;16:1 114156 13 Chamberlain JS, Gibbs RA, Ranier JE, Cascey CT. Multiplex PCR for the diagnosis of DMD. In: PCR Protocols. New York: Academic Press, 1990;277-81 14 Den Dunnen JT, Grootscholten PM, Bakker E, Blonden LAJ et al. Topography of the Duchenne muscular dystrophy (DMD) gene: FUGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835-47 15 Emery AEH. Duchenne Muscular Dystrophy 2nd edition. Oxford University Press: New York, 1988 16 Gilgenkrantz H, Chelly J, Lambert M. Recan D et al. Analysis of molecular deletions with cDNA probes in patients with Duchenne and Becker muscular dystrophies. Genomics 1989;5:574-80 17 Harper PS. The muscular dystrophies. In: Striver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. New York: McGraw-Hill Co, 6th edn 1989;2869-2902 18 Hejtmanick JF, Harris SG, Tsao CC et al. Carrier diagnosis of Duchenne muscular dystrophy using RPLFs. Neurology 1986~36: 1552-3 19 Hoffman EP, Brown RH. Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919-28 20 Hoffman EP, Hudecki M, Rosenberg P, Pollina C. Kunkel LM. Cell and fiber-type distribution of dystrophin. Neuron 1988;411-20 21 Hoffman EP, Kunkel LM. Dystrophin abnormalities in Duchenne and Becker muscular dystrophy. Neuron 1989;2:1019-29 22 Hu X. Ray PN. Murphy EG, Thompson MW, Worton RG. Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution origin and phenotype genotype correlation. Am J Hum Genet 1990;46:682-95 23 Kitoh Y, Matsuo M. Nishio H. Takumi T et al. Amplification of ten deletion-rich exons of the dystrophin gene by polymerase chain reaction shows deletions in 36 of 90

Japanese families 1992:42:453-7

with

DMD.

Am

J

Mrd

Genet

24 Koenig M, Hoffman EP, Bertelson CJ. Monaco AP. Feener C, Kunkel LM. Complete cloning of the Duchennc muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987:50:509-17 25 Koenig M. Beggs AH. Moyer M et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum &net 1989;45:498-506 26

Kunkel patients Nature

LM et al. Analysis of deletion in the DNA of with Becker and Duchenne muscular dystrophy. 1986;322:73-5

27

Lao YL. Srivastava G, Wong V. Liu YT et al. Deletion, duplication and novel restriction fragment length polymorphism in Duchenne and Becker muscular dystrophy. C/in Genet 1992;41:252-8 28 Malhotra S, Hart K, Klamont H. Thomas N et nl. Frame shift deletion with Duchenne and Becker muscular dystrophy. Science 1989;242:755-9

29

Monaco AP, Neve RL, Colleti-Feener C, Bertelson CJ et a/. Isolation of a candidate cDNA for portion of the Duchenne muscular dystrophy gene. Nature 1986;323:64650

30

Moser aspects

A. Duchenne muscular dystrophy: and genetic prevention. Hum Gene,

pathogenic 1984;66: 17-40

3 I Oudet C, Heilig R, Hanauer A, Mandel JL. Nonradioactive assay for new microsatellite polymorphisms at the 5’ end of the Dystrophin gene. and estimation of intragenic recombination. Am J Hum Genet 1991;49:31 l-19 32

Roberts RG, Montandon AJ, Bobrow M, Bentley DK. tection of novel genetic markers by mismatch analysis, cleic Acids Res 1991;17:5961-71

DeNu-

33 Roest PAM, Robert RG. Sugino S, Van-Ommen GJB. den Dunnen JT. Protein truncation test (PTT) for rapid detection of translation-terminating mutation. Hum Molec Genet 1993;2(10):1719-21 34 Sambrook EF, Fritsch Y, Maniatis T. MolecuIar Cloning: a Laboratory Manual. Cold Spring Harbor : Cold Spring Harbor Laboratory Press, 1989 35

Shomrat R, Driks N, Legum C. Shiloh Y. Use of cystrophin genomic and cDNA probes for solving difficulties in carrier detection and prenatal diagnosis of DMD. Atrr J Med Genet 1992;42:28 I-7

36 Soong BW, Tsai TF, Su Ch, Kao KP, Hsiao KJ, Su S. DNA polymorphism and deletion analysis of the Duchenne-Becker muscular dystrophy gene in the Chinese. Am J Med Genet 1991;38:593-600 37 Sugino S, Fujishita S, Kamimura N et al. Moleculargenetic study of Duchenne and Becker muscular dystrophies: deletion analysis of 45 Japanese patients and segregation analyses in their families with RFLPs based on the data from normal Japanese families. Am J Med Genet 1989;34:555-61 38 Ward PA, Hejtmancik JF. Witkowski JA et al. Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis. Am J Hum Genet 1989;44:270-81