REVIEW OF LITERATURE

Transcription

1 REVIEW OF LITERATURE CLINICAL FEATURES Dystrophinopathies Duchenne muscular dystrophy was first described by Meryon in 1852 and subs~qu~ntly by Duchenne (1861, 1868). DMD affects 1 in 3,300 newborn boys and is characterised by onset between age 3 and 6, progressive weakness of proximal muscles, calf hypertrophy, scoliosis, and inability to walk after the age of 20 (Emery, 1993). Cardiomyopathy and moderate mental retardation are common features of DMD. Serum creatine kinase (CPK) levels are strikingly elevated, even in preclinical stages of the disease. Muscle biopsy shows groups of necrotic and regenerating muscle fibres, proliferation of endomysia! connective tissue, and replacement of muscle fibres by connective and adipose tissue. Becker muscular dystrophy (BMD; Becker and Kiener, 1955) shares many features with DMI) but has a milder course and both are allelic qisorders caused by mutations in the dystrophin gene (Emery, 1989; and Hoffman and Kunkel, 1989). The incidence of BMD is 1 in 30,000 males (Emery, 1987; and Monaco and Kunkel, 1987). The mean age of onset of disease is 12 years. Loss of ambulation does not occur before the age of 16, as it usually occurs in the fourth decade of life. Although DMD /BMD manifests predominantly in males, a small number of females with DMD/BMD have been reported. Translocations between the X-chromosome and autosomes are found to be the major alterations observed in karyotypes of th~se patients. Female relatives of boys with DMD are heterozygous carriers of the mutated dystrophin gene. About 70% of heterozygous carriers have elevated CK levels (Moser and Vogt, 1974). The majority of (>90%) of female carriers of DMD are asymptomatic (Emery, 1967; 7

2 Moser and ~mery, 1974; and Emery, 1993) although rare carriers can present proximal muscle weakness. This latter category of heterozygous carriers have been called 1 manifesting 1 or 1 symptomatic 1 carriers (Emery 1967; and Moser and Emery, 1974). Limb-girdle muscular dystrophy The diagnostic criteria for LGMDs have been summarised by Bushby (1995) for the European Neuromuscular center-sponsered working group on LGMDs. The proposed criteria consists of predominantly proximal muscle weakness of the muscle of the trunk, with more distal weakness not occurring until later in disease progression. The facial muscle are usually spared or only minimally involved, and extraoccular muscles are usually spared. Creatine kinase (CK) activity in the serum can be normal or mildly to grossly elevated, electrophysiologic studies are myopathic, and muscle biopsy reveals myopathic to qystrophic features. Clinically this group of disorders is usually progressive, although there is remarkable variability with respect to age of onset and rate of progression within the group as a whole, as well as, within a single genetically defined entity (BOnneman et al., 1996). Certain clinical features appear to be common to the group of autosomal recessive sarcoglycan-deficient LGMDs: taken as a group, the clinical involvement and progression tend to qe more severe than in both the autosomal dominant anq the sarcoglycan-positive autosomal recessive types 2A and 2B LGMDs. In LGMD 2D again there is wide variability in the clinical severity, with a tendency for a larger number of milder cases, when compared with the other sarcoglycan disorders. Patients most commonly present with difficulty running and climbijlg stairs, l,mt as in dystrophinopathy, presentation with muscle cramps or exercise intolerance has been reported. Toe-walking was an early feature in fewer than half of the patients. The age of onset among the 20 patients reported by Eymard et al. (1997) ranged from 3 to 15 years, with a 8

3 mean of 8.5 years. Kawai et al. (1995) highlighted delayed walkingin some patients, while adult-onset cases have also been reported (Fanin et al., 1997). Most of the patients have scapular winging, often to a more pronounced degree than is seen at a similar stage in patients with dystrophinopathy. Early involvement of the deltoid was also noted, and weakness of the biceps with relative preservation of the triceps. In the lower limbs, femoral muscles are less involved than the pelvic group. The quadriceps and hamstrings are usually equally affected, as distinct from dystrophinopathy, in which quadriceps predominate (Bushby, 1999). Congenital muscular dystrophy The term 'congenital muscular dystrophy' (CMD) was first used by Howard, (1908) for a floppy infant with joint contracture. Congenital muscular dystrophies are a heterogeneous group of muscle disorders, transmitted by an autosomal recessive inheritance pattern. They constitute the most frequent cause of severe congenital hypotonia from muscular origin (Fardeau, 1992). Four different forms of CMD have been identified t9 date: classic (occidental) or 'pure' form of CMD (Banker et al., 1994); Fukuyama type CMD (Fukuyama et al 1960, 1981); Muscle-eye-brain disease (MEB) (Santavuori et al., 1977, 1989); and Walker-Warburg syndrome (Dobyns et al., 1989). Although occidental CMD is considered a 'pure' muscle disorder, a significant number of patients exhibit cerebral white matter lesions on CT or MRI, but without clinical evidence of central nervous system involvement (Philpot et al., 1995). Fukuyama type CMD exhibits muscle features similar to occidental CMD, but is associated with severe developmental central nervous system defects and profound mental retardation. This form of CMD has been identified almost excltisively in Japan (Fukuyama et al., 1960, 1981). In muscle-eye brain disease (MEB) and the Walker-W arburg syndrome, severe developmental defects of brain and eye, associated with profound weakness 9

4 and arthrogryposis, are obvious at birth (Williams et al., 1984; and Dubowitz and Fardeau, 1995). Affected individuals usually die within the first year of life. The CPK levels in congenital muscular dystrophy may occasionally be comparable to levels found in Duchenne muscular dystrophy (Donner et al., 1975) and muscle histology appears to be no different from Duchenne muscular dystrophy. MOLECULAR GENETICS OF DMD/BMD Mapping the dystrophin gene to chromosome band Xp21 The first indication that the gene responsible for Duchenne and Becker muscular dystrophy (DMD and BMD) is in band Xp21 in the X-chromosome short arm came from rare females with the Duchenne or Becker phenotypes. In late 1970s and early 1980s several affected girls were described (Canki et al., 1979; Enamuel et al., 1983; Jacobs et al., 1981; and Lindenbaum et al., 1979) each of whom had a de-novo X autosome translocation with a breakpoint in band Xp21. The second line of evidence placing the DMD gene at Xp21 came from family $hj.dies with the DNA probes that detect restriction length polymorphism (RFLP) on the human X-chrom.osome. The first two linked markers for DMD gene were RFLPs detected by probes RC8 (Murray et al., 1982) and L1.28 (Davies et al., 1988). The RC8 clone mapped to the distal thirct of short arm of X-chromosome while L1.28 mapped to the proximal third. In the Duchenne families both polymorphic markers were found to segregate with the DMD gene, but each displayed a recombination frequency of about 20%, placing the probes at a linkage distance of 20 em from the DMD gene. The two probes mapped 40 em apart indicating that they must flank the DMD gene to the middle of the short arm (Davies et al., 1988). 10

5 The third line of evidence placing the DMD gene at Xp21 came from a small set of patients with complex phenotypes including DMD, with one or more several X-linked phenotypes including glycerol kinase (GK) deficiency, adrenal hypoplasia (AHC), retinitis pigmentosa (RP), McLeod phenotype (XK) and chronic granulomatous disease (CGD). A syndrome of DMD coupled with AHC, GK deficiency and mental retardation (MR) had been recognised in families with 2 or more affected males, suggesting X-linked \. inheritance and possibility that the phenotype resulted from deletion of 3 or more contiguous gene (Francke et al., 1985). Cloning of PNA segment from DMD locus The first concrete evidence for deletion of contiguous genes came from study of BB, a patient with DMD, CGD, XK and RP (Francke et al., 1985). Cytogenetic analysis with high resolution chromosome banding revealed small but detectable <ieletion of part qf bands Xp21.1 and Xp21.2 The approach of Kunkel and his colleagues (Kunkel et al., 1985,) depended on the is<;>lation of ml1ltiple clones from within a smal_l region of X chromosome known to be deleted in BB. DNA from an XXXXY male (to enrich for X-c;hromosome sequences) was cleaved with the restriction endonuclease Mbol. Sheared DNA from patient BB was added in large excess in a competitive hybridisation reaction (phenol enhanced reassociation technique-pert) to compete selectively with all sequences except those from the deleted segment. Following pertreassociation, the renatured DNA was ligated to BamBIcleaved plasmi<i and the resulting library was tested clone by clone to identify those clones that mapped within the BB deletion. Among a few hunqred pert clones analysed, eight failed to hybridise to a Southern blot of DNA from BB and therefore mapped within the BB deletion (Kunkel et al., 1985; and Monaco et al., 1985). 11

6 Each of these clones was then tested for hybridisation to DNA from a series of male DMD patients. Among the eight pert clones from within the BB deletion, one clone pert87, seemed to be the closest, since DNA from 5 of the 57 DMD patients lacked the pert sequence (Monaco et al., 1985). The pert87 sequence then became the start point for a bi-directional chromosome wq.lk along a normal X-chromosome by sequential isolation of overlapping phage clones from an X chromosome enriched library (Kunkel et al., 1985; and Monaco et al., 1986,1987). The second successful approach leading to the DMP gene was that taken by Worton's group. The approach was dependent on identification of a translocated female with a rearrangement that placed the translocated segment from her X chromosome adjacent to a block of tandemly repeated genes encoding 185 and 285 ribosomal DNA (rrna) (Verellen-Dumoulin et al., 1984; Worton et al., 1984). Ribosomal DNA (rdna) probes were used to identify and clone from the patient a segment of DNA that spa,nned the translqcq.ti<;m junction (R<:ly et al., 1985). The junction clone designq.teq XJ1 contained 62Q bp of rrna at one en<i, and about 11 kb of X-chromosome sequences q.t the other. Chromosome walking from XJ1 along the normal X chromosome yielded about 120 kb of human chromosome (the DXS206 locus) derived from both sides of the junction site. Within the DX5206 locus three subclones, XJ1.1, 1.2, and 2.3 detected RFLPs that segregated in Duchenne families with DMD mutations. XJ probes showed approximately 5% recombination between the probe site and DMD mutation (Thompson et al., 1986) and detected deletions in about 6% of male patients (R~.y et al., 1985). STRUCTURE OF THE DYSTROPHIN GENE In 1987, Koenig and his co-workers completed the cloning of the dystrophin edna and showed that hybridisation of the full-length clone to human genomic DNA digested with Hindiii yielded 65 distinguishable 12

7 bands. As there are only five Hindiii sites within the edna sequence, this implied a minimum of 60 exons. The approximate order of these fragments, was established by edna hybridisation by Monaco et al., Large scale mapping of the gene (van-ommen et al., 1986; Kenwrick et al., 1987; Burmeister et al., 1988; and den-dunnen et al., 1987, 1989) using pulse field gel electrophoresis (PFGE) led to an estimate of 2.4 Mb for the total gene. This to date is the largest gene ever characterised, and contains the largest intron known (brain intron 1 is 400 kb in size) (Boyce et al., 1991). The study by Darras and Francke, (1988a) set forth the standard patterns of restriction fragments that are detected when normal human DNA cleaved with either Hindiii or Bglii and hybridised with seven conti~ous segm~nts comprising the entire 14 kb edna. They established normal restriction fragment patterns using 27 normal male and 39 normal female individuals of different age and ethnic origin. They observed that the seven dystrophin edna probes hybridised to a total of 66 Hindiii fragments including at least two sets of comigrating fragments that are recognised by adjacent edna probes. No RFLPs we,re~ detect~d in Hindlii digested PNA from more than 60 individuals. Seventeen Hindiii fragments were revealed by probe 1-3, ni_ne (10.5, 8.5, 8.0, 7.5, 4.6, 4.2, 3.25, :;3.2 and 3.1 kb) hybridised with prob~ 1-2a anq. nine (12.0, 1Q.5, 7.~, 9.6,?.Q, 4.0, 3.0, 2.7 and 1.7 kb) with probe 2"Q-3. The shared 10.5 kb fragment involved the exon in which the two probes overlap. Six fragments were detected with probe 4-5a (20.0, 12.0, 11.0, 7.3, 5.2, and 4.7 kb). The 11 and 12.0 kb doublet often appeared fused. Prol:>es 5b-7 and 8 hyl:>ridised to thirteen (18, 11.0, 10, 6.2, 6.0, 4.2, 4.1, 1.8, 1.5, 1.3, 0.5 and 0.45) and seven (10, 7, 3.8, 3.7, 3.1, 1.6 and 1.25 kb) Hindiii fragments respectively with the 10.0 kb fragment seen by both probes. Probes 9 and 10 revealed seven Hind III fragments each (8.8, 8.3, 7.8, 6.0, 2.3, 1.0 and 1.0 kb), (12.Q, 6.6, 6.0, 3.5, 2.8, 2.55, and 2.4 kb) respectively. Ten fragments hybridised strongly with edna (10.0, 7.8, 6.8, 6.0, 5.9, 2.1, 1.9, 1.8, 1.5 and 1.45 kb). 13

8 Prior et al. (1989) found a polymorphism that altered the exon 8 and 9 Hindiii fragments using probe 1-2a. It was shown to be a two allele polymorphism consisting of common 7.5 kb and rarer 8.3 kb alleles. They observed the polymorphism to be more frequent in African-Americans than in Caucasians. Another Hindiii polymorphism was observed by Prior et al. (1992) in African-Americans. This two allele polymorphism consisting of common exon 6, 8.0 kb and rarer 4.8 kb alleles. This RFLP was not observed in any of Caucasian chromosome. Lindor et al. (1993) examined dystrophin gene at Xp21 in African Americans by Southern blot analysis. With probe 2b-3 there was a gain of 6 kb and a loss of 5 kb band while with probe 4-Sa there was a subtle gain of a band (4-Sa/ 5.3 kb) with a decrease of intensity in 5.2 kb band. It had l?e~n demonstrated by Otto and Rottenberg. (1992) that 5.2 kb band in a Hind III digest resolves into 5.15 and 5.2 kb l)ands upon extended electrophoresis. This novel 5.3 kb band appeared to be a polymorphism involving one of the~e two normal bands. Mital et al. (1998) described a common dystrophin gene polymorphism in the Indian population with edna that altered the Hihdiii restriction site; Study by Darras and Francke, (1988a) revealed no Hindiii polymorphism with edna but study with Bgl II and Taq I revealed a 24.Q/ 28.0 kb RFLP and 1.2/1.4 kb two allele polymqrphism, respe_ctively for the same probe. Verma, (1997, M.Phil. thesis) have observed a 24.0 kb RFLP allele with Bgl II in all the 58 Indian subjects comprising DMD patients, their family members and healthy controls for the dystrophin gene. 14

9 MUTATIONAL ANALYSIS IN THE DYSTROPHIN GENE Nature of mutations The majority of Duchenne muscular dystrophy (DMD) patients as well as those suffering from milder allelic form, Becker muscular dystrophy, have been shown to carry mutations in the dystrophin gene. In 65% of patients a deletion of one or more exons of the dystrophin gene has been detected (den Dunnen et al., 1987; Forrest et al., 1987a; and Koenig et al., 1987). The deletions are not randomly distributed over the gene but are focused in two major hot-spots and appear to occur with approximately equal frequency in DMD and milder BMD (Forrest et al., 1987; and Hart et al., 1987). Another type of mutation, much less common than deletion, is intragenic duplication which has been observed in 5 % of the cases. There is duplication of an or a few exons by tandem duplication of a portion of gene, presumably by unequal crossing over between repetitive elements (Hu et al., 1988, 1990). In 30% of patients with Duchenne or Becker muscular dystrophy have no detectable deletion or duplication. It is presumed that these patients have point mutations of the dystrophin gene affecting transcription, mrna processing, translation, or protein stability (Bulman et al., 1991a; Kilimann et al., 1992; and Roberts et al., 1992). Gene deletions Deletions of the dystrophin gene in 6%-10% of individuals with DMD/BMD were first detected with the DNA probes pert87 (DXS164; Kunkel et al., 1985) and XJ (DXS206; Ray et al., 1985) (Monaco et al., 1985; Kunkel et al., 1986; and Hart et al., 1986). The use of genomic probes Jbir (DXS270) and J66-H1 ( DXS268; Monaco et al., 1987) increased the number of detectable gene deletions to 17% by Southern analysis and to more than 50% by field-inversion gel elctrophoresis (den Dunnen et al., 1987). The latter results indicated that a deletion hot spot existed in the 950 kb between probes 15

10 Jbir and J66Hl. Koenig et al. (1987) found the overall deletion frequency for the DMD ~ene to be 50% by using a series of - 1-kb dystrophin edna subclones on Hindiii digests of DNA samples from 104 patients. Eighty percentage of deletions were observed with edna probe 8 and lb. Deletions did not appear to be evenly distributed along the DMD transcript. Similar findings were reported by Darras et al. (1988) and they found an overall deletion frequency of 66% for the DMD gene. It was only in 1988, Monaco and his co-workers proposed the 'readin~ frame hypothesis'. They suggested that in mutations involving BMD, the translational frame of the messenger mrna is maintained and a smaller though functional protein is produced but in DMD cases the reading frame is disrupted, leading to misreading of the mrna and premature termination or the production of completely inactive protein. However Malhotra et al. (1988) determined exon-intron boundaries of first ten exons and analysed 29 DMD/BMD patients. A major unexpected result of the study was ~hat there were a number of BMD patients having deletion of exon 3-7 which results in disruption of translational reaqing frame. The advent of polymerase chain reaction (PCR) provided G),n opportunity to simplify the total approach to analyse gene deletions at such a large locus. Chamberlain et al. (1988) suggested a set of nine oligonucleotide primers (mutiplex PCR) to qmplify different regions of DMD locus and reported gene deletions in 37% of patients. Beggs et al. (1990) reported that about 98% of deletions in patients with DMD /BMD could be detected by using primers for nine additional exons in conjugation with those described by Chamberlain et al. (1988, 1990) in two multiplex PCR's. Testing the validity of the 'reading frame' theory in 258 independent deletions at DMD locus, Koenig et al. (1989) found a correlation between phenotype and type of deletion that was in agreement with the theory in 92% of cases and was of diagnostic and prognostic significance. Baumbach et al. 16

11 (1989) found deletions in 56% of affected males, a value similar to the earlier reports (Koenig et al., 1987; and Forrest et al., 1987a, band 1988) and observed no correlation between the extent of a deletion, its location and clin.ical severity of the associated disease phenotype. Liechti-Gallati et al. (1989), and Gillard et al. (1989) reported that 60-66% of the mutations in dystrophin gene were gene arrangements. They found that except for few exceptions, frameshift deletions of the gene resulted in more severe phenotype than did in-frame deletions which was well in accordance with 'reading frame' theory of Monaco et al. (1988). LindlOf et al. (1989) studied 90 unrelated DMD/BMD and found that 50% had molecular deletions of one or several of the 65 exoncontaining Hindiii fragments. They observed that qeletions were equally common in familial and sporadic cases unlike Passos-Bueno et al. (1990) who found a higher frequency of deletions in sporadic than familial cases. Deletions of the dystrophin gene in the patients suffering from BMD were Love et al. (19~0) and they observed that most of the BMD patients have intragenic deletions which leave the protein reading frame intact, which again supports the reading frame hypothesis proposed by Monaco et al. (1988). Upadhaya et al. (1990) analysed DNA from 164 unrelated DMD patients and detected deletions in :?0% of the subjects. Use of the 3 edna probes (edna 8, 6-7) detected 99% of deletions. Deletions detected were heterogeneous both in size and position. Clausters et al. (1991) studied 38 DMD/BMD patients from Southern France for intragenic deletions in the dystrophin gene. Using mtlltiplex PCR and edna probes they detected deletions in 26 patients. An apparent discrepancy of exon 51 between the Southern blot and multiplex PCR was observed in two brothers who showed deletion of exons by mpcr while, Southern hybridisation showed absence of the 3.1 kb Hindiii fragment (exon 51) detected by probe 8. 17

12 While many studies with edna on rearrangement in patients in North American and European have been reported, a few studies have been carried out among Asians. Soon et al. (1991) found partial deletions in 46% of Chinese DMD patients. Among Japanese patients Sugino et al. (1991) found deletions among 32% while Ubagai et al. (1991) found partial deletions among 69% of DMD patients. Gokgoz et al. (1993) screened 76 DMD and 5 BMD patients of Turkish origin using two separate multiplex gene amplification systems and reported deletions in 52% of the cases. The majority',of the deletions were localised within the central region of the dystrophin gene. The remaining deletions mapped tq the proximal hot spot. The efficacy of the rnpcr with that of edna analysis was compared by Katayama et al. (1993) in 30 DMD males from 27 Japanese families. Deletions were detected in 52% of 27 DMD families by PCR and in 64% of 22 families by Southern hybridisation. They concluded that where deletion was limited to a $ingle exon, the mpcr is more efficient and useful to conventional Southern blot analysi$ for detecting deletions during the prenatal and postnatal diagnosis. Nicholson et al. (1993a, b, c) ljndertook a multicj.isciplinary study to find out the variation in a large cohort of 100 patients with well defined clinical phenotypes. Deletions/ duplicationsof Xp21 gene were detected in 81.5% of. all male patients. They observed no difference in proportion of sporadic versus familial cases who had mutations (deletions or dljplications). Frequency and distribution of rearrangements in the dystrophin gene was compared by Immoto et al. (1993) between 88 Japanese DMP patients and those in, N. America, and ElJrope by Southern blot analysis. They reported that both the frequency and distribution of gene arrangements in Japanese patients were similar to those observed in N. America and Europe and suggested that there was no ethnic or racial differences in frequency and distribution of rearrangements. 18

13 Banerjee et al. (1997) studied 160 cases of DMD drawn from all parts of India, using multiplex PCR. Of these 64.4% showed intragenic deletions and most of the deletions involved exons The phenotypes of the cases with the deletion of single exons didnot differ significantly from those with deletions of multiple exons. The distribution of deletions in studies from different countries was variable but this was accounted for either by small number of cases studied, or by fewer exons analysed. It wa$ cqncludeci that there are no ethnic difference with respect to deletions in DMD gene. Mital et al. (1998) analysed dystrophin gene in 32 unrelated DMD families for the presence of deletions by mpcr for 27 exons and edna probes for the entire gene. Deletions were identified in 70% of the cases. The concordance between the clinical phenotype and 'reading frame' hypothesis was observed in 75% of cq.ses.. Correlation between phenotype and genotype of these DMO patients demonstrated that genetic studies of lymphocyte DNA may not always reflect the situation in the tissue involved (i.e. muscle tissue). Duplications Duplication m part of the DMD gene have been identified in DMD patients both with genomic probes. (Bertleson et al., 1986; den Dunnen et al., 1989; and Hu et al., 1988) and edna probes (Greenberg et al., 1988). Hq et al. (1988) studied 120 unrelated DMD/BMD patients and observed duplications in 3 patients (2 DMD and 1 BMD). According to them duplicatiqns are tandem repeats and can restj.lt in q. genetic disorder through the disruption of exon organisation. Hu et al. (1990) studied the gene duplications in 72 non deleted DMD / BMD patients. Ten patients had a duplication of part of the gene of which 6 had a novel restriction fragment that spe1nned the duplication junction. A shift of the reading frame in 4 of the 6 DMD cases anq in 1 of the 2 intermediates was also reported. Kodairo et al. (1993) smdied DNA samples from 21 nondeleted Japanese DMD pati~nts by PFGE and found, that 7 had 19

14 partial duplications spanning kb, of these 4 had duplications corresponding to the major 'hot-spot' ( kb from the 5' end of the edna) and 2 had duplications in the region about 10 kb from the 5' end of edna where causative mutations are rare. One patient however had duplication in the duplication prone region i.e., 1.0 kb from the 5' end of edna. Point mutations In one-third of DMD or BMD patients, the mutation remains unknown as it does not involve gross rearrangements of the gene. The identification of point mutations in the dystrophin gene represents a challenge because of large size and complexity of dystrophin gene. The first nonsense mutation was reported by Bulman et al. (1991a) in exon 26 of a DMD patient where immunological analysis of the truncated dystrophin from muscle biopsy material allowed prior localisation of the mutation. Other methods as singlestrand conformation polymorphism (SSCP) analysis (Orita et al., 1989) and heteroduplex analysis haye proved successful in detection of point ml,j.tations in the dystrophin gene (Nigro et al., 1992; Prior et al., 1993; and Tuffery et al., 1993). Kneepers et al. (1994) used multiplex PCR prodq.cts in SSCP for screening of point mutations in a set of 70 nondeleted DMD /BMD patients and identified 6 patients with band shift. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation while the other band shift was found to be a rare polymorphism. THE DYSTROPHIN PROTEIN Structural domains of dystrophin Limited proteolysis of muscle extract followed by immunoblotting using domain specific antibodies suggested that dystrophin folds into distinct structural domains. (Koenig and Kunkel, 1990; Yoshida et al., 1992; Cottin et al., 1992). Initial analysis of dystrophin indicated that it qelonged to spectrin 20

15 al., 1992). Initial analysis of dystrophin indicated that it belonged to spectrin family of cytosketelal proteins (Davison and Critchley, 1988; Koenig et al., 1988; and Tinsley et al., 1992). Dystrophin amino (NHz) terminus is similar in sequence and function to a large family of actin binding proteins, including P spectrin and a- actinin (Davison et al., 1989; Hammonds, 1987; Levine et al., 1990; and Hemmings et al., 1992). Biochemical analysis of the dystrophin actin binding domains has revealed that they act simply as anchors to F-actin cytoskeleton and play no role in regulating actin organisation in the cell (Winder et al., 1997). The internal region called 1 rod-region 1 of dystrophin consists of 24 homologous repeats, each averaging 109 amino acids and comprises approximately 75% of the protein (Koenig and Kunkel, 1990). By analogy to P-spectrin, this so-called 1 rod-region 1 of dystrophin probably forms a long flexible row of repeats; each repeat mostly a-helical, containing two-proline rich turns, allowing it to form a tripple helix (Speicher and Marchesi, 1984; and Koenig et al., 1988) From a functional standpoint the C-terminal domains of ciystrophin are clearly the most important, as mutations in these region have founci to cause the severest symptoms. The importance of the C-terminal region rests primarily on its interaction with the transmembrane protein P-dystroglycan (Willamson et al., 1997). The carboxyl region in dystrophin (from NHz to COOH terminus) has be~n found to comprise a WW domain (Bork and Sudol, 1994; Andre and Springael, 1994), two EF hands (Tufty and Kretsinger, 1975), a ZZ domain (Ponting et al., 1996) and two predicted parallel dimmeric coiled coils (Blake et al., 1995 ). 21

16 Function of Dystrophin and its expression Skeletal muscle represents about 30% of body mass and has a unique cellular structure. Muscle cells (myofibres) develop from thousands of individual precursor cells (myoblasts) during foetal life, and resulting cells are syncytial with thousands of nuclei sharing the same cytoplasm. The shear force$ generated by contracting myofibrils, the changes in the dis.tribution of myofibre cytoplasm during contraction, and the transduction of force from the intracellular myofibrils to the extracellular matrix (basal lamina) all combine to exert substantial stress on the lipid bilayer. The reinforcement of the plasma membrane is accomplished by a large series of proteins that together form the membrane cytoskeleton and one of them is dystrophin based membrane cytoskeleton. Dystrophin, a large protein analogous to steel girders in a high-rise building, lies on the internal face of the myofibre plasma membrane and interconnects many other proteins to impart strength anq stability on the lipid bilayer (Menke and Jockusch, 1995; and Fabbrizio et al., 1995). The protein product of the human Duchenne muscular clystrophy locus (DMD) and its mouse homologue (mdmd) were identified by Hoffman et al. (1987) by using polyclonal antibodies. The DMD protein was shown to \Je approximately 400 kda e:md was found to represent approximately 0.002% of the total stri(:1ted mlj,scle protein. This protein was also detected in smooth muscle. Muscle tissue isolated from both DMD-affected boys and mdx mice contained no detectable DMD protein, suggesting that these genetic disorders are homologous. They named the protein dystrophin because of its identification via the isolation of Duchenne muscular dystrophy locus. Immunohistochemistry provided a more definitive localisation of dystrophin at the sarcolemma (Arahata et al., 1988; Bonilla et al., 1988a; $ugita et al., 1988; and Zubrzycka-Gaarn et al., 1988). This surface localisation was subsequently confirmed in further subcellular fractionation (Salviati et al., 22

17 1989). Hoffman et al. (1989) assessed the quantity (relative cellular abundance) and quality (approximate molecular weight) of dystrophin in muscle biopsies from DMD /BMD patients. They suggested that Duchenne/ Becker dystrophy can be divided into 4 clinically useful categories. Duchenne dystrophy (wheelchair at about age 11 years; dystrophin quantity < 3% of normal); severe Becker dystrophy (wheelchair age 13 to 20 years, dystrophin 3% to 10%); and moderate/mild Becker dystrophy (wheelchair >20 years; dystrophin quantity ~20% ). Immunoblot characterisation and immunofloresence localisation of dystrophin was undertaken by Arahata et al. (1989) for 76 human patients with various neuromuscular disorders They found that biochemical abnormalities of dystrophin (either lower or higher molecular weight dystrophin) resulted in patchy, discontinuous immunostaining of the muscle fibres in l3md patients. No detectable dystrophin was present in DMD patients while unrelated diseases showed normal dystrophin. England et al. (1990) described a deletion in the dystrophin gene which removed a central part of the dystrophin gene (exon 17-48) in a family segregating for a very mild BMD. Immunological analysis of muscle from one of the patients demonstrated that the mutation resulted in the production of a truncated polypeptide localised correctly in muscle cell. They concluded that the findings are meanigful in context of gene therapy which would be facilitated by replacement of a very large dystrophin gene with a more manipulative min-gene construct. Biochemical and immunohistochemical analysis of dystrophin in 226 patients with various neuromuscular disorders was reported by Nicholson et al. (1990) with monoclonal antibody against the rod domain of the dystrophin. Approximately 40% of biopsies obtained from patients diagnosed having DMD showed isolated clearly positive fibres and a further 20% had a very weak labelling on a large number of fibres. Biopsies from patients with BMD showed inter- and intra fibre variation in labelling 23

18 intensity. Overall the estimated abundance of dystrophin correlated well with the clinical assessment of the disease severity expressed in the patients. Angelini et al. (1990) described a patient with an in-frame duplication that spanned exons Molecular weight of the dystrophin protein detected in the patient's muscle was approximately 600 kda. Despite the gene alteration, the patient had relatively mild clinical progression compatible with the diagnosis of Becker muscular dystrophy. Bulman et al. (1991 b) used antibodies directed against the amino- and carboxyl terminal regions of dystrophin to characterise 25 DMD, 2 intermediate patients, and 2 BMD patients. Western blot analysis revealed an altered size (truncated) immunoreactive dystrophin band in 11 of 25 DMD patients, in 1 of the 2 intermediate patients, and in both BMD patients, when immunostained with antiserum raised against the amino terminus of dystrophin. None of the DMD or intermediate patients demonstrated an immunoreactive dystrophin band when immunostained with an antiserum specific for the C-termina1 of the protein. In contrast, dystrophin was detected in both BMD patients by the antiserum specific.for the carboxyl terminus. Their results suggested that a differential diagnosis between DMD and BMD would benefit from examination of both N-terminal and C-terminal of the protein, in addition to measurements of relative abundance of the protein. In hopes of shedding light on the molecular basis for the extreme variability seen among patients with BMD, Beggs et al. (1991) correlated DNA and protein data on 68 patients with detectable but abnormal dystrophin. They found that deletions within the amino-terminal domain I tended to result in low levels of dystrophin and a more severe phenotype. The phenotypes of patients with deletions or duplications in the central rod domain was more variable. In contrast, deletions and duplications in the proximal region of rod domain tended to cause severe cramps and myalgia. Loss of the middle of rod region caused a mild phenotype. 24

19 Helliwell et al. (1992) described a DMD patient having a frameshift deletion of exons 42 and 43. A 225 kda protein was detected by western blotting with N-terminal antibodies only. The result suggested that an NHzterminal truncated dystrophin fragment encoded by exons 1-41 is able to associate with the muscle cell membrane. Recan et al. (1992) examined muscle from a one-year-old patient with a large deletion that removed the cysteine rich and C-terminal domains, and extended beyond the glycerol kinase and congenital adrenal hypoplasia genes. Immunological analysis of muscle dystrophin showed that the deletion resulted in the production of a truncated, but stable, polypeptide correctly localised at the sarcolemma. Their data indicated that neither the cysteine rich domain, nor the C-termi.nal domain, are necessary for the appearance of normal dystrophin sarcolemmal localisation similar to the findings of Helliwell et al. (1990). Bushby et al. (1993a) correlated a detailed clinical assessment of 67 patients with proven BMD with results from genetic and protein analysis. They found that the size of deletions were inversely proportional to the size of the protein produced but there was no such relationship between size of the deletion and abundance of dystrophin produced. A multidisciplinary study was undertaken by Nicholson et al. (1993) to record relationships between clinical severity and dystrophin gene and protein. Dystrophin in higher proportion of DMD patients was detected compared to other laboratories (Hoffman et al., 1988; Beggs et al., 1991; Bulman et al., 1991) and at a hisher abundance. The size of dystrophin detected in DMD patients was compatible with synthesis from mrna in which the reading frame had been restored. Dystrophin abundance among majority of BMP patients was above 30%. Comi et al. (1994) investigated 59 BMD patients, to test the hypothesis of predictability of muscle dystrophin expression and clinical phenotype based on location of dystrophin gene mutations. They reported that Domain I 25

20 deleted patients tended to have a worse phenotype, with earlier presentation, faster progression rate and lower dystrophin expression, while distal rod domain deleted patients showed a more classic BMD phenotype.. Their data confirmed that different BMD gene in-frame mutations have different effects on dystrophin expression and clinical severity, indicating several functional roles of dystrophin domains. Correlation of phenotype, genotype with protein abnormaliti~s It has been found that mutations in the amino-terminal actin-binding domain (domain I) of dystrophin generally lead to a severe BMD phenotype (Koenig et al., 1989; Beggs et al., 1991; and Comi et al., 1994) but does not cause the severest DMD phenotype. Takeshima et al. (1994) reported a Japanese boy with large in-frame deletion of exons 3-41 anq whose clinical symptoms were intermediate between Duchenne and.becker muscular dystrophy. Immunocytochemical analysis of the skeletal m~scle showed that dy$trophin was detectable with antibody directed against C-terminal part but not with antibodies directed against the amino-terminal part. They suggested that in situations where amino terminal actin-binding qomain is qeleted, there may be sufficient actin binding property in the remaining coiled-coil region to prevent the manifestation of the severe DMD phenotype. However, there is one mutation that does not fit this hypothesis. Prior anq his coworkers (1993) described a point mutation (leucine to Arginine at conserved residue 54) in the amino terminus of dystrophin that led to DMD phenotype. At the other end of actin-binding domain, mutations i:n the proximal roq domain (domain II) (in-frame deletions) give rise to mildest 13MD phenotype (Koenig et al., 1989), or are even asymptomatic from the point of day to qay life of the affected individual. A family with a large deletion that removed 46% of dystrophin had extremely mild BMD, with one family member still ambulant at the age of 61 (England et al., 1990). Deletions or duplications in 26

21 the proximal rod region caused only severe cramps and myalgia (mild phenotype) (Beggs et al., 1991; and Comi et al., 1994). However, Bushby et al. (1993) found a patient with a deletion of exons but who was severly affected and became wheelchair bound at the age of 12 years. Deletions in mid rod domain (central part of domain II) was found to either cause very mild phenotypes or the patient presented was asymptomatic (Beggs et al., 1991; and Comi et al., 1994). Deletions in the distal rod domain cause typical BMD, however, phenotypic variability among patients with similar mutation suggests that epigenetic and/ or environmental factors play an important role in determining the clinical progression (Beggs et al., 1991). Bushby et al. (1993) reported that patients with deletion in this region had mild progression of the disease and Comi et al. (1994) observed partial clinical and biochemical heterogenity in the patients with deletions in distal domain II. Pathologically mutations in the carboxyl-terminal region of the dystrophin are most important (Koenig et al., 1989; Beggs et al., 1991). Patients with deletion in these domains and the cysteine rich region particular have the most severe DMD phenotypes, often despite the presence of substantial amount of altered amount of dystrophin protein (McCabe et al., 1989; and Bies et al., 1992). Carboxyl-terminal domain (cysteine-rich) is crucial to the interaction of dystrophin with dystrophin associated protein complex, particularly: ~-dystroglycan (Suzuki et al., 1992, 1994, 1995) while deletions of the carboxyl terminus distal to cysteine-rich region produces a BMD phenotype (Koenig et al., 1989; and Beggs et al., 1991). THE DYSTROPHIN-GL YCOPROTEIN COMPLEX (DGC) The mode of interaction of dystrophin with the sarcolemma was unclear until biochemical experiments demonstrated that dystrophin is tightly associated with membrane glycoproteins, called dystrophin-associated 27

22 proteins (DAPs) (Campbell and Kahl, 1989; Jorgensen et al., 1990; Yoshida and Ozawa, 1990; Yuan et al., 1990; Ervasti et al., 1990, 1991a; and Ervasti and Campbell, 1991). The DAPs are now classified into three groups (Ozawa et al., 1995; Matsumura et al., 1997; Lim and Campbell, 1998; and Ozawa et al., 1998). One group is comprised of the members of the syntrophin family, q._-, J31- and J32 syntrophins, with molecular masses of around 60 kda. Both other groups are comprised of sarcolemmal glycoproteins and form two distinct subcomplexes in DCC. One is the dystroglycan complex (Ibraghimov-"6eskrovnaya et al., 1992). comprised of a- and!3-dystroglycans with molecular masses of 156 and 43 kda, respectively.!3-dystroglycan is a transmembrane glycoprotein, while a-dystroglycan is a heavily glycosated extrinsic peripheral membrane that can bind basal lamina protein agrin, laminin and merosin (Ibraghimov Beskrovnaya et al., 1992; Gee et al., 1993, 1994; Sugiyama et al., 1994; and Bowe et al., 1994) while!3-dystroglycan binds intracellularly to the cysteine rich domain and the first half of the C-terminal domain of dystrophin (Suzuki et al., 1992, 1994, 1995; and Jung et al., 1995). In Oq.chenne muscular ciystrophy, mutations in the dystrophin lead to the loss of this complex from the membrane (Ahn and Kunkel, 1993). The disruption of this link, concomitant loss of the sarcoglycans, have been proposed to cause a malfunction in the sarcolemma that eventually leads to cell death (B()nnemann et al., 1995; and Noguchi et al., 1995). The other group has been named the sarcoglycan complex, after it was shown that its members could be separated from other proteins by using special detergent conditions (Yoshida et al., 1994). The sarcoglyc~n proteins are designated as a-,!3-, y-, and 8-sarcoglycans, which, respectively, are 50, 43, 35, and 35 kda. a-sarcoglycan was originally named adhalin, from the arabic 1 adhal 1, meaning muscle (Roberds et al., 1993). The carboxyl terminus of this protein lies within the muscle cell. The protein has a long extracellular 28

23 domain with both glycosylation sites and five cysteines, four of which are close to the membrane surface (McNallyet al., 1994; and Roberds et al., 1994). The absence of dystrophin causes the drastic reduction of dystrophinassociated proteins (DAPs) in the sarcolemma and the loss of linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy. Ohlendiek et al. (1993) investigated the status of dystrophin associated proteins (DAPs) in skeletal muscle from 17 DMD patients of various ages. A dramatic reduction in all of the dystrophin associated proteins in the sarcolemma of DMD muscle was observed when compared with normal muscle and muscle from a variety of other neuromuscular diseases. The results indicated that absence of dystrophin leads to loss in all of the DAPs, which renders DMD muscle fibres susceptible to necrosis. Similarly, Matsumura et al. (1993a) reported that all of the DAPs were drastically reduced in the sarcolemma of 3 DMD patients in whom dystrophin was lacking in the COOH-terminal domains but reported mild to moderate reduction in all of DAPs in BMD patients with huge or small'inframe' deletions in the rod domain of dystrophin and a moderate reduction of DAPs in patients with huge deletions that involve both the NH2-terminal and rod-domains of dystrophin. However the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminals domains of dystrophin. (Matsumura et al. 1993b, 1994). MANIFESTING CARRIERS Mothers of affected boys can be divided arbitrarily into three categories: definite carriers having an affected son and a previous affected male on the maternal side of the family history, probable carriers with two or more affected child without family history (Thompson et al., 1967; Smith et al., 1979) and possible carriers who have abnormal karyotype but are assumed to a carry a mutation on one X-chromosome and exhibit a skewed in-activation 29

24 pattern which results in this X-chromosome being the active one in most cells. In addition, a number of fully manifesting females have been described with DMD or BMD secondary to translocation between an X chromosome and an autosome (Boyd et al., 1986). Approximately 8% of carriers have some clinical manifestation, ranging from pseudohypertrophy of the calves to marked proximal muscle wasting (Dubowitz, 1982). The wide variation in symptoms expressed by carriers is usually explained in terms of random X inactivation according to the Lyon hypothesis (Vogel and Motulsky, 1986). With cloning of the DMD gene and identification of dystrophin it became possible to indirectly visualise X-inactivation in muscle biopsies of carriers by a mosaic pattern of dystrophin immunostaining with both dystrophinpositive and dystrophin-negative fibres, as seen by immunoflourscence (Bonilla et al., 1988b; and Arahata et al., 1989b). This mosaic pattern was found to be diagnostic of, and specific for, female carriers of DMD (Clerk et al., 1991). With increased utilisation of protein analysis of muscle biopsies for molecular diagnosis, many female myopathy patients with no previous family history of any neuromuscular disease have been found to have a mosaic dystrophin immunostaining pattern on muscle biopsy (Minetti et al., 1991). In a large follow-up study of 505 muscle biopsies from female myopathy patients, Hoffman etal. (1992) found that about 10% of the women with hyperckemia, myopathic pattern by muscle biopsy, and no family history of DMD could be identified as carriers of DMD when tested with dystrophin immunofluorescence assay. On the basis of biochemical findings on muscle biopsy, it has been hypothesised that all female dystrophinopathy patients -show skewed X-inactivation where the X-chromosome ~hat has the normal gene is preferentially inactivated, leaving the dystrophin gene 30

25 mutation carrying X-chromosome active (Minetti et al., 1991; Hoffman et al., 1992). Sewry et al. (1993) studied ten females presenting with muscle weakness and a raised serum creatinekinase. Their results showed that analysis of dystrophin expression is useful for the differential diagnosis of carriers of Xp21 dystrophy and autosomal muscular dystrophy, but that dystrophin expression does not correlate directly with the degre~ of clinical weakness. Bushby et al. (1993b) analysed the results of clinical assessment, X inactivation status, deletion screening and dystrophin analysis in manifesting carriers of DMD and BMD. They found that dystrophin analysis seems to be reliable in identifying manifesting carriers of DMD and BMD but the relationship between X-inactivation, dystrophin analysis ancl phenotype is not simple. The expression of the DAPs, ~-dystroglycan, a.-sarcoglycan, y-sarcoglycan, and syntrophin as well as utrophin was investigatecl by DiBlasi et al. (1996) in the muscles of DMD/l?MD carriers. DAPs were highly reduced in all fibres lacking dystrophin in the DMD carriers, but were almost normal in fibres of BMD carriers with highly truncated dystrophin. In the l?md carriers with nearly normal dystrophin, the few fibres with reduced or patchy dystrophin immunostaining also showed reduced DAP expression. Immunoblot for ~ dystroglycan and a.-sarcoglycan confirmed the immunohistochemica.l findings. GENE THERAPY IN DUCHENNE MUSCULAR DYSTROPHY Gene transfer methocls that have l:>een tried include naked plasmid DNA, retroviral and adenoviral vectors (Ascadi et al., 1991; Vincent et al., 1993; and Dunckley et al., 1993). For gene therapy to be successful in DMD, the large dystrophin gene must be inserted into at least 10% of muscle cells and be distributed to both 31

26 proximal and distal muscles (Jiao et al., 1994). Direct injection of a 12 kb human dystrophin edna (Dickson et al., 1991) or the 6.3 kb Becker like gene in an expression vector gave rise to approximately 1% dystrophin positive fibres (Acsadi et al., 1991), but only skeletal and cardiac muscle seemed to be receptive to this technique. The advantage of direct,gene therapy is that there is no danger of virus infection or cancer which might occur with virus based delivery system (Feigner and Rhodes, 1991) and the naked DNA seems to elicit little pathogenic immune response. Another means of introducing a missing or defective gene into skeletal muscle is by viral vectors. Retroviral vectors containing reporter gene have shown that it is possible to get expression of reporter gene following direct injection of the virus into rodent skeletal muscle (Thomason and Booth, 1991). It has been shown that 6.3 kb dystrophin minigene in retroviral vector, p:eabe neo, can be transduced into cultured mdx mononucleated muscle precursor cells (mpc) in vitro. It gave rise to truncated dystrophin protein in the sarcolemma of the resultant myotubes (Dunckley et al., 1992; and Dicksa,n and Dunckley, 1993). Injection of this construct in the presence of polybrene into adult mdx quadriceps and tibialis anterior muscle gave rise to substantial numbers of dystrophin positive muscle fibres in which 43-kDa dystrophin associated glycoprotein (DAG) was also restored (Dunckley et al., 1993) but retroviral mediated transfer of dystrophin into dystrophin deficient skeletal muscle is reliant on the presence of dividing host satellite cells, and only a small number of which may be mitotically active in a muscle at a given time. Intravenous injection of a recombinant adenoviral vector into mice resulted in expression of the reporter gene in variot:j.s tissues including skeletal and cardiac muscle, upto 12 months after the injection of the virus (Stratford-Perricaudet et al., 1992). Clemens et al. (1996) generated a adenoviral vector that contained no viral genes' and encoded full length dystrophin edna with muscle creatine kinase and LacZ marker gene. 32