Becker muscular dystrophy: From molecular diagnosis to gene therapy

ELSEVIER

Brain & Development

Q

1996; 18: 167- 172

Review article

Duchenne/Becker

muscular dystrophy: from molecular diagnosis to gene therapy Masafumi Matsuo

Diuision

qf Genetics,

Intrmutional

Center for Medical

Research,

Kohe Uniuersit~

*

School qf Medicine,

7-5-1, Kusunoki-cho.

Chuo-ku.

Kohr,

Hyogo 650,

Japan

Received 26 October

1995; accepted

18 December

1995

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked muscular dystrophies. The recent isolation of the defective gene in DMD/BMD and the identification of its protein product, dystrophin, have revolutionized our ability to diagnose DMD/BMD and promoted speculation regarding the application of gene therapy. The purpose of this review is to present progress made in this area of research, with particular reference to dystrophin Kobe, which is caused by exon skipping during splicing due to the presence of an intra-exon deletion. On the basis of result of moleculr analysis of dystrophin Kobe we propose a novel way of gene therapy for DMD, in which antisense oligonucleotides transform DMD into BMD phenotype by inducing exon skipping. 1. INTRODUCTION Duchenne muscular dystrophy (DMD) is a common inherited disease with a world-wide incidence of 1 in 3500 male births [I]. DMD patients appear normal until the age of 3-5 years, when they begin to experience difficulty in rising from the floor, climbing stairs, and other activities which involve the large proximal skeletal muscles. The muscular weakness is characteristically progressive with regards to both the number of muscle groups involved and the extent of their malfunction. The affected individuals are wheelchair-bound by the age of 12 and succumb to cardiac or respiratory failure in the mid to late 20s. Diagnosis of DMD is usually confirmed by the fact that serum creatine kinase levels are at least 40 times higher than normal, by electromyograms which reveal characteristic myopathy, and by histological examination of muscle biopsy which typically reveals a large range of fiber diameters, connective tissue proliferation (fibrosis), and evidence of actively degenerating and regenerating fibers. Interestingly, a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), is distinguished from DMD by delayed onset, later dependence on wheel-chair support and longer life span. The recent isolation of the gene defective in DMD/BMD and the identification of its protein product, dystrophin, have revolutionized our ability to diagnose DMD/BMD and promoted spec-

* Corresponding 0387.7604/96/$15.00 PII

author. Fax: (81) (78) 362-6064. 0 1996 Elsevier Science B.V. All rights reserved

SO387-7604(96)00007-

I

ulation regarding the application of gene therapy. The purpose of this review is to present progress made in this area of research, with particular reference to dystrophin Kobe, which is caused by exon skipping during splicing due to the presence of an intra-exon deletion [2].

2. GENETICS OF DUCHENNE/BECKER LAR DYSTROPHY

MUSCU-

Both DMD and BMD are X-linked recessive disorders. As such, the vast majority of affected individuals are male hemizygotes. Although most cases are transmitted via an unaffected carrier (heterozygote) mother, 30% of cases have no previous family history and are considered to be due to a de novo mutation in the germ line of either the mother or her parents. One of the important steps which led to the cloning of the DMD/BMD gene was the identification of the corresponding locus on the X-chromosome. This was accomplished through the analysis of rare female DMD/BMD patients who exhibited X-autosome translocation and through linkage analysis using cloned random genomic DNA fragments. Both of these analyses indicated that the DMD gene was likely to be located in the Xp 21 subregion of the short arm (Xp) of the X-chromosome. Linkage analysis of BMD families suggested that the mutant gene leading to this disorder was in a similar location, indicating that the DMD and BMD genes are either the same or closely linked [3.4].

M. Matsuo/Brain

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3. IDENTIFICATION

OF THE DYSTROPHIN

& Development

GENE

Cloning of the gene itself was accomplished by two groups of researchers using two different approaches. One group cloned a portion of the gene by working with the X-autosome translocation [5,6]. The approach taken by the other group of researchers was to clone DNA from a boy with a small Xp21.2 deletion. Using an elegant substraction technique, they obtained cloned fragments of DNA that are present on a normal X chromosome but absent in the patient’s DNA. Finally a 14-kb muscle-specific transcript was cloned and shown to be encoded by 79 exons distributed over 2.5 million bp of the human X-chromosome, occupying approximately 1% of the entire chromosome or nearly 0. I % of the entire genome. Results of a recent study conducted in our laboratory have extended this gene region by more than 500 kb through the identification of a new promoter/exon sequence upstream of the known 5’ end of the gene [7]. The encoded protein, dystrophin, contains 3685 amino acids organized in four domains: actin-binding, triple helical, cysteinerich and C-terminal. The C-terminal domain is the only one which does not show sequence similarity to other proteins, suggesting that it has a dystrophin-specific function. Dystrophin is bound at or near its C-terminus to a complex of proteins and glycoproteins that co-purify with dystrophin from sarcolemal membranes [8]. Recent studies showed that some patients with clinical symptoms similar to DMD/BMD but which are inherited in an autosomal recessive manner lack one of these dystrophinassociated proteins [9,10]. At least seven promoters have been identified in the dystrophin gene, and each promoter is expressed in a tissue- or development-specific manner, giving rise to multiple isoforms of dystrophin (Table 1) [7,11,12]. Alternative splicing of introns further increases the number of dystrophin isoforms, each of which may have a specific, unique function. The diversity of forms of dystrophin make it difficult to understand its physiological role.

4. DNA AND DYSTROPHIN

STUDIES

The unusually high incidence of DMD/BMD in all human populations could be due solely to the enormous mutation target size of the gene. To identify such mutations, segments of the cDNA have been used as probes to specifically examine all of the exons by Southern blot analysis [13]. Most of the identified mutations are deletions, with over 65% of patients exhibiting the loss of one or more exons at the genomic DNA level. Two deletion hot spots have been identified near the 5’ end and in the

Table 1 Dystrophin Name L-Dystrophin C-Dystrophin M-Dystrophin P-Dystrophin R-Dystrophin S-Dystrophin G-Dystrophin

isoforms produced

by different promoters

Promoter location

Size(kb)

Site of expression

5’-region 5’-region 5’-region 5’.region intron 29 intron 55 intron 62

‘I

Lymphoblast Cortex Muscle Purkinje cells Retina Schwann cells General tissue

14 14 14 ? 6.5 5.2

1996;

18: 167-172

Normal mRNA

THEDOGCANRUNANDEAT Translation

by

tripleletters

t THE DOG CAN RUN AND EAT

Six letters Of CANRUN deletion

( in-frame deletion)

THE DOG CAN EAT One letter of C deletion

( out-frame deletion)

THE DOG ANR UNA NDE AT Fig. 1. Explanation of frame-shift theory. Normally, the 18 letters of “THEDOGCANRUNANDEAT” would be translated as “THE DOG CAN RUN AND EAT” as the triplet codons are translated into amino acid. With an in-frame deletion, the letters of “THEDOGCANEAT” would be read as ”THE DOG CAN EAT ” In the specific example of the dystrophin gene, a partially functional protein would be produced in BMD despite the presence of deletion mutation. In contrast, the letters of “THEDOGANRUNANDEAT” would be translated as “THE DOG ANR UNA NDE AT”. This corresponds to DMD.

central region of the dystrophin gene, respectively. Duplications have also been identified in a small number of patients [ 141. Although both DMD and BMD patients have been shown to have deletion mutations, the extent of the deletion does not always correlate with the severity of the disease: some BMD patients with mild symptoms have deletions encompassing numerous exons, whereas some DMD patients with severe symptoms lack only a few exons. In some cases, the long deletions resulting in BMD and the short deletions resulting in DMD may even overlap. According to Monaco et al., this apparent discrepancy can be explained by deletion-associated frame-shifting [ 151. According to this idea, the BMD patients with the long deletions might be able to produce a dystrophin mRNA that, although internally deleted for a portion of its sequence, would still direct the production of an internally truncated semi-functional protein. The shorter deletions harbored by severe DMD patients, on the other hand, would bring together exons that, when spliced, would change the translational reading frame in the mRNA (Fig. 1). This hypothesis would predict that milder BMD patients would produce a smaller protein while DMD patients would either produce a severely truncated form lacking the entire C-terminal region or would not produce a protein at all. Subsequent gene analyses have shown that over 90% of the deletion mutations that cause BMD maintain the dystrophin transcript reading frame, whereas frame shifts are usually caused by deletions resulting in DMD [ 161. Immunohistological analyses have demonstrated that dystrophin is present in muscle cell membranes. As expected, this protein is completely missing in boys with DMD, whereas muscle tissue from BMD patients contains reduced amounts of dystrophin. [17]. Thus, DMD and BMD represent examples of

M. Matsuo/Brain

& Deoelopment

allelic heterogeneity. Western blot analyses using dystrophin antibody have revealed a band corresponding to 427 kd, close to the predicted size of dystrophin, in extracts of normal muscle tissues. Shorter proteins can sometimes be detected in extracts of tissues from patients with BMD [ 181.

5. DELETION SCREENING CHAIN REACTION

USING

POLYMERASE

The advent of polymerase chain reaction (PCR) technology has revolutionized our approach to DNA analysis of the dystrophin gene by providing a powerful alternative to Southern hybridization analyses for the detection of deletion mutations. PCR can amplify specific regions of any gene up to a million-fold. Since most deletions are clustered in two regions of the dystrophin gene [ 131,examination of only a subset of the 79 exons is sufficient to detect the majority of deletions. Indeed, 98% of deletions of the dystrophin gene were detected by amplifying only 18 selected exons [ 191. Furthermore, PCR can now be used as the initial step in the molecular diagnosis of dystrophinopathy [20]. We have screened Japanese DMD/BMD patients by PCR and found that the incidence of deletion mutations is lower than in Caucasians [21]. Although the deletions were found in the same regions of the gene in Japanese and Caucasian patients, Philippino DMD/BMD patients showed a significantly higher incidence of deletions in the 5’ region than in the central region compared to either of these two population groups [22]. This predisposition to deletions in the 5’ region might be due to an unknown factor specific to Philippinos.

6. FINE MUTATIONS

OF THE DYSTROPHIN

GENE

As already mentioned, two thirds of patients with DMD/BMD carry a large deletion in the dystrophin gene. Many of the patients for whom this is not the case probably have small mutations which can not be detected by Southern blotting or PCR methods. However, only a limited number of such mutations have been reported because the dystrophin gene is too large and difficult to examine for minor nucleotide changes. The identification of new mutations in the dystrophin gene has important clinical implications, however. For example, once a mutation has been identified, highly accurate, direct DNA testing can be employed for carrier and prenatal diagnosis. Thus, many kinds of analytical methods have been tested in an attempt to facilitate the identification of point mutations in the dystrophin gene. At present, a protein truncation test (PTT) seems to be the most powerful method to identify nonsense mutations in DMD [23,24]. PTT is based on the fact that nonsense mutations cause the production of prematurely terminated polypeptides. In PTT analysis of dystrophin, the cDNA is divided into 10 fragments, each of which is translated so that the size of synthesized polypeptide can be determined by Western blotting. Once an abnormally short peptide has been identified, the location of nonsense mutation can be predicted and the corresponding genomic DNA region sequenced. This method has accelerated the identification of nonsense mutations, about 100 of which have recently been reported [23-251. These mutations occur in several different exons and seem to be unique to single patients. How-

1996; 18: 167-172

169

ever, regions around exons 19 and 70 seem to be point mutation hot spots because 5 and 4 mutations. respectively, have been identified in these regions. We identified the first Japanese case of a point mutation in the dystrophin gene by analyzing the dystrophin transcript using reverse-transcription PCR [26]. Since the incidence of DMD/BMD is the same throughout the world, we anticipate that more examples of point mutations will be identified in Japanese patients.

7. GENE THERAPY The treatment of DMD/BMD is, of course, one of the primary goals of all DMD/BMD research. Even with the newly acquired knowledge, however, the biological role of dystrophin remains speculative, and our understanding of the disease remain incomplete. Consequently, the progression of the disease can not yet be slowed by therapeutic treatment. Myoblast transfer or gene therapy have been proposed as alternatives for effective drug therapy. Since the clinical application of myoblast transfer has several limitations, gene replacement therapy is now the most plausible candidate for effective therapy of DMD. A new gene could be introduced into muscle tissue by direct injection of DNA. Of course, it would be impossible to transfer the complete dystrophin gene because of its large size. Instead. the main aim in the field of gene therapy is to establish a way to inject constructed dystrophin minigenes consisting of a partial or full-length cDNA joined to an appropriate promoter [27-321. Although much progress has been made in this field of study, we still seem to be a long way from achieving a clinically significant result.

8. TRANSFORMATION

LAR DYSTROPHY DYSTROPHY

OF DUCHENNE MUSCUINTO BECKER MUSCULAR

As the injection of dystrophin minigenes into muscle will not be feasible for some time, an alternative strategy for DMD treatment might be to retard progression of the clinical symptoms, i.e., to convert DMD into BMD phenotype. Theoretically, this therapy can be done by changing a frame-shift mutation causing DMD into an in-frame mutation characteristic of BMD by modifying the dystrophin mRNA. It might be possible to modify the editing of the mRNA for this purpose, but the exact mechanism of mRNA splicing is not clear and thus can not yet be employed for therapy. It might be possible to control intron splicing during mRNA maturation, however. The cis-acting elements implicated in pre-mRNA splicing, the 5’ and 3’ splice site sequences, branch point sequences and their locations [33], are well conserved in exon and intron boundaries. Splicing errors are often due to mutations that affect one of these elements. In one particular dystrophin gene mutation named dystrophin Kobe. we found that exon skipping during splicing was induced by the presence of intra-exon deletion mutation in the genome. although all of the consensus sequences known to be required for splicing were unaffected. The deletion was detected by PCR analysis, which revealed that the product amplified from the exon 19 encompassing region from the DMD case in question was smaller than normal This result suggested the presence of a

M. Matsuo/Brain

170

& Development 1996; 18: 167-172 52 bp deletion

Exon

3

5

Meseenger RNA precursor

Splicing

Intro”++ b

Mature messenger RNA

I

17

12

20

Fig. 2. Schematic representation of exon 19 of dystrophin Kobe and the splicing error which occurs. In the mRNA precursor sequence (upper line), 52 bp of the 88 bp exon 19 of the dystrophin (shaded box) gene are deleted in dystrophin Kobe. This truncated exon 19 is spliced out together with the in&on to produce mature mRNA lacking all of exon 19.

In v&o splicing of dystrophln pre-mRNA

exonw

exonl9

Mutant(antisense) •~~~

K-

\

\ynt

novel mutation within the amplified region. Sequence analysis confirmed that this was the case by showing that 52 bp out of 88 bp of exon 19 were deleted from 2-3 bp upstream from the splice donor site [34]. This 52 bp deletion was considered to result in a frameshift mutation that would cause DMD. The dystrophin transcript of dystrophin Kobe was then analyzed using reverse-transcription PCR (RT-PCR). Surprisingly enough, the

s:I:I::::::::m

A A A 0 1 0 1 0 1 (hr)

exonl819 2’0Me-antisense

RNA

I

I

0

1

10 lOO(pM)

123456

Fig. 3. In vitro splicing of dystrophin pre-mRNAs from minigenes. Pre-mRNA (A) with a normal exon sequence was spliced to produce mature mRNA containing exons 18 and 19 (lanes 3 and 4). However, pre-mRNA (B) with the same exon 19 sequence as dystrophin Kobe was not spliced (lanes 5 and 6). This inhibition of splicing was slightly suppressed by inserting an inverted sequence into dystrophin Kobe (Cl (lanes 1 and 2). The open and filled boxes and lines represent the exons, the vectors and introns, respectively. In minigene A, sequences of two exons are completely normal but the size of intron 18 is reduced to 158 bp without altering the general consensus sequences for splicing. The shaded area indicates the region that has been deleted in dystrophin Kobe. In minigene B, exon 19 is replaced by that of dystrophin Kobe gene. The deleted region of dystrophin Kobe is reinserted in reverse orientation in minigene C. The arrow in minigene C shows the direction of the inserted exon 19 sequence.

1

2

3

4

Fig. 4. Effect of an antisense 2’-O-Me RNA on dystrophin splicing. In vitro splicing of pre-mRNAs from minigenes consisting of exon 18, intron 18 and exon 19 sequences (lanes I-4) was performed for 1 h in the presence of an antisense 2’0Me RNA. The concentration of the antisense 2’-O-Me RNA added is indicated above each lane. The composition of the corresponding to the RNA products is shown schematically at the right and left. Mature mRNA was not produced in the presence of high concentrations of antisense 2’-@Me RNA (lanes 3 and 4).

M. Matsuo/

exon 18

Brain & Dewlopment

exon 19

1996; 18: 167-172

exofl21

exotl20

i

I

I

I

.

I

\ Deletion of exon 20

Resdlno frame: 242

bp / 3 = 80 --- 2

-)

DMD

Dslation of exons 19 and 20 RssdlnO *me:

(

88 bp t 242 bp )/ 3 = 110 --- 0

BMD

Fig. 5. Theoretical use of antisense oligonucleotide for treatment of DMD. The DMD phenotype of the patient with an exon 20 deletion would be transformed into a BMD phenotype by inducing the exclusion of exon 19 from the matured transcript in the presence of antisense 2’-@Me RNA.

product amplified from the region encompassing exon 19 was smaller than predicted according to the results of the genomic DNA

analysis.

Sequence

analysis

indicated

that

the

whole

of

exon 19 was missing from the dystrophin cDNA, causing an out-of-frame mutation. In particular, this indicated that the deletion mutation within an exon sequence could induce a splicing error during maturation of messenger RNA, even though the known consensus sequences at the 5’ and 3’ splice sites of exon 19 were maintained [2] (Fig. 2). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19 (Fig. 3) [35]. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is missing in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of a polypurine track within the downstream exon 19. More recently, several exons have been shown to include purine-rich regions, called exon recognition sequences or ERSs, which are necessary for splicing of the upstream intron [36]. ERSs are likely targets for splicing factors that identify exon sequences and promote their inclusion in the mature mRNA. The possibility that oligonucleotides could be used as modulators of gene expression, and hence as chemotherapeutic agents, is currently under intense investigation. The modulation of splicing by antisense oligonucleotides has recently attracted much attention [37.38]. Dominski and Kole recently described an elegant experiment in which aberrant splicing induced by a thalassemia mutation was corrected by an antisense 2’-O-methylribonucleotide (2’0Me RNA) [38]. This prompted us to test whether splicing of dystrophin pre-mRNA could also be modulated by an

antisense RNA as the first step towards evaluating the potential therapeutic use of antisense RNA to correct aberrant splicing reactions in patients with DMD. An antisense 3 1 mer 2’-O-methylribonucleotide complementary to the 5’ half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wildtype pre-mRNA in a dose- and time-dependent manner (Fig. 4) [35]. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy. For example, the same antisense nucleotide will be used to treat a DMD case with a 242 nucleotide deletion of exon 20 (Fig. 5). If we are able to induce exon 19 (88bp) skipping in vivo, the dystrophin transcript will lack both exons 19 and 20 but the translational reading frame will be restored. As a result, this modulation of splicing should transform DMD into BMD. More extensive studies are required before clinical trials can begin; for example, we need to confirm modulation of splicing by antisense oligonucleotide in an in vivo experiment. We also need to develop an efficient method to deliver antisense oligonucleotides to the nucleus.

REFERENCES I. Emery AEH. Duchenne muscular dystroph!. Oxford: Oxford University Press, 1993: 392. 2. Matsuo M, Masumura T, Nishio H, et al. Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy Kobe. J C/in kwest 1991: 87: 2127-31. 3. Hoffman EP, Kunkel LM. Dyhtrophin abnormalities in Duchenne/Becker muscular dystrophy. Neuron 1989: 2: 1019-29. 4. Rojas C, Hoffman E. Recent advances in dystrophin research. Curr @in Neurobiol 1991; 1: 420-29. 5. Hoffman E, Brown R, Kunkel L. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51: 919-28. 6. Burghea AHM, Logan C, Hu X. et al. A cDNA clone from the Duchenne/Becker muscular dystrophy gene. Nature 1987: 328: 434-7. I. Nishio H, Takeshima Y, Narita N, et al. Identification of a novel first exon in the human dystrophin gene and of a new promoter located

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Matsuo/ Brain & Decelopment 1996: 18: 167-172

more than 500 kb upstream of the nearest known promoter. / Clin Inoest 1994; 94: 1037-42. 8. Yamamoto H, Hagiwara Y, Mizuno Y, Yoshida M, Ozawa, E. Heterogeneity of dystrophin-associated proteins. J Biochem 1993; 114: 132-9. 9. Matsumura K, Tome F, Collin H, et al. Deficiency of the SOK dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature 1992; 359: 320-2. 10. Piccolo F, Roberds SL, Jeanpierre M, et al. Primary adhalinopathy: a common cause of autosomal recessive muscular dystrophy of variable severity. Nat Genet 1995; 10: 243-5. 11. Ahn AH, Kunkel LM. The structural and functional diversity of dystrophin. Nat Genet 1993; 3: 283-9 I. 12. D’Souza VN, thi Man N, Morris GE, et al. A novel dystrophin isoform is required for normal retinal electrophysiology. Hum Mel Genet 1995; 4: 837-42. 13. Koenig M, Hoffman EP, Bertelson CJ, et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50: 509-17. 14. Hu X, Ray P, Murphy E, Thompson M, Worton R. Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotype genotype correlation. Am J Hum Genet 1990; 46: 682-95. 15. Monaco AP, Bertelson C, Liechti-Gallati S, Moser H, Kunkel L. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90-5. 16. Gillard EF, Chamberlain JS, Murphy EG, et al. Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene. Am J Hum Genet 1989; 45: 507-20. 17. Arahata K, Ishiura S, Ishiguro T, et al. Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988; 333: 861-3. of 18. Hoffman EP, Fischbeck KH, Brown RH, et al. Characterization dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N Engl J Med 1988; 318: 1363-g. 19. Beggs AH, Koenig M, Boyce FM, Kunkel LM. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990; 86: 45-8. 20. Ishigaki C, Patria SY, Nishio H, Yabe M, Matsuo M. A Japanese boy with myalgia and cramps and a novel in-frame deletion of the dystrophin gene. Neurology, in press. 21. Kitoh Y, Matsuo M, Nishio H, et al. Amplification of ten deletion-rich exons of the dystrophin gene by polymerase chain reaction shows deletions in 36 of 90 Japanese patients with Duchenne muscular dystrophy. Am J Med Genet 1992; 42: 453-l. 22. Cutiongco EM, Padilla CD, Takenaka K, et al. More deletions in the 5’ region than in the central region of the dystrophin gene were identified among Filipino Duchenne and Becker muscular dystrophy patients. Am J Med Genet, 1995 59: 266-7.

23. Gardner RJ, Bobrow M, Roberts RG. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test. Am J Hum Genet 1995; 57: 31 l-20. 24. Hogervorest FBL, Cornelis RS, Bout N, et al. Rapid detection of BRCAl mutations by the protein truncated test. Nature Genet 1995: 10: 208-12. 25. Roberts RG. Gardner RJ, Bobrow M. Searching for the I in 2,400,OOO: a review of dystrophin gne point mutations. Hum Mutut 1994; 4: I-II. 26. Hagiwara Y, Nishio H, Kitoh Y, et al. A novel point mutation (G-’ to T) in a 5’ splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy. Am .I Hnm Genet 1994; 54: 53-61. 27. Acsadi G, Dickson G, Love D, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nutwe 1991; 352: 815-18. 28. Wells D, Wells K, Walsh F. et al. Human dystrophin expression corrects the myopathic phenotype in transgenic mdx mice. Hum Mel Genet 1992; 1: 35-40. 29. Cox G, Phelps S, Chapman V, Chamberlain J. New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin. Nat Genet 1993; 4: 87-93. 30 Dunckley M, Wells D, Walsh F, Dickson G. Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo. Hum Mel Genet 1993; 2: 717-23. 31 Ragot T, Vincent N, Chafey P, et al. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice. Nature 1993; 361: 647-50. 32 Vincent N, Ragot T, Gilgenkrantz H, et al. Long-term correction of mouse dystrophic degeneration by adenovirus-mediated transfer of a minidystrophin gene. Nat Genet 1993: 5: 130-4. 33 Green MR. Pre-mRNA splicing. Annu ReLl Genet 1986; 20: 671-708. 34 Matsuo M, Masumura T, Nakajima T, et al. A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene. Biochem Biophys Res Commun 1990; 170: 963-7. 35. Takeshima Y. Nishio H, Sakamoto H, Nakamura H, Matsuo M. Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe. J C/in Inrest 1995; 95: 515-20. 36 Tanaka K, Watakabe A. Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. MO/ Cell Biol 1994; 14: 1347-54. 37. Barabino SML, Sproat BS, Lamond AI. Antisense probes targeted to an internal domain in U2 snRNP specifically inhibit the second step of pre-mRNA splicing. Nucleic Acids Res 1992; 20: 4457-64. 38. Dominski Z, Kole R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotidea. Proc Nat1 Acad Sci USA 1993: 90: 8673-7.