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

Moser and ~mery, 1974; and Emery, 1993) although rare carriers can present

proximal muscle weakness. This latter category of heterozygous carriers have

been called 1manifesting1 or 1symptomatic1 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

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

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

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 BamBI­

cleaved 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).

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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

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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., 1987. 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 11-14 (10.0, 7.8, 6.8, 6.0, 5.9, 2.1, 1.9, 1.8, 1.5 and 1.45 kb).

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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 11-14 that altered the Hihdiii restriction

site; Study by Darras and Francke, (1988a) revealed no Hindiii polymorphism

with eDNA 11-14 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

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

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

(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 exon­

containing 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 48-50 by mPCR

while, Southern hybridisation showed absence of the 3.1 kb Hindiii fragment

(exon 51) detected by probe 8.

17

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

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 45-51. 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

partial duplications spanning 50-400 kb, of these 4 had duplications

corresponding to the major 'hot-spot' (7.5-8.5 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 single­

strand 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

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 1rod-region1 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 1rod-region1 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

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

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

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 14-42. 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

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 NHz­

terminal 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

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 co­

workers (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

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 13-47 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

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 1adhal1

, meaning muscle (Roberds et al., 1993). The carboxyl terminus of this

protein lies within the muscle cell. The protein has a long extracellular

28

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 dystrophin­

associated 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'in­

frame' 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

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 dystrophin­

positive 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

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

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

Somatic delivery of this vector by intra muscular injection, in 6 day old mice

resulted in the expression of full length recombinant dystrophin at the muscle

membrane. Dystrophin associated proteins were restored in muscle fibres

expressing recombinant dystrophin. Feasibility of myoblast mediated ex-vivo

gene transfer of full length dystrophin in mdx mouse was checked by Floyd

et al. (1998). They infected myoblasts which were not capable of producing

dystrophin with an adenoviral vector lacking all the viral genes but

expressing full length dystrophin and P-galactosidase. Subsequently those

transduced myoblasts were injected into mdx muscle, where the injected cells

restored dysb:ophin, as well as dystrophin-associated proteins.

LIMB-GIRDLE MUSCULAR DYSTROPHY (LGMD)

Autosomal recessi'.re limb-girdle muscular dystrophies (AR-LGMDs) are a

heterogenous group of genetic disorders in which there is a progresive

weakness of pelvic and shoulder girdle musculature.

Eight genes have been already mapped for autosomal recessive-LGMDs

(AR-LGMDs) which are: LGMD2A at 15q (Beckmann et al., 1991), LGMD2B

at 2p (Bashir et al., 1994), LGMD2C at 13q (Noguchi et al., 1995), LGMD2D at

17q (Roberds et al., 1994), LGMD2E at 4q (Lim et al., 1995; B()nnema11Il et al.,

1995) LGMD2F at 5q (Passos-Bueno et al., 1996b) LGMD2G at 17q (Moreira et

al., 1997) and LGMD2H at 9q (Weiler et al., 1998). Of these four diseases,

LGMD2D, LGMD2E, LGMD2C, and LGMD2F are caused by defects of genes

for rx.-, P-, y-, and o-sarcoglycan respectively. A primary mutation in any one

of these genes (a-, p-, y-, and o-sarcoglycans) may lead to total or partial loss

of that sarcoglycan as well as secondary deficiency of other sarcoglycans, and

sometimes reduction of dystrophin labelling in muscle as well (Vainzof et al.,

1996; Bushby et al., 1997; and Jones et al., 1998). With any primary

sarcoglycan involvement, however, this, pattern of secondary deficiencies can

strikingly variable, though total loss of complex is most commnly seen with

33

mutations of~- or o-sarcoglycan. When the mutation is in a- or y-sarcoglycan,

the pattern of sarcoglycan loss may be much more restricted and in fact some

sarcoglycans may appear to be expressed normally (Vainzof et al., 1996;

Bonnemann et al., 1996; Bushby et al., 1997; Jones et al., 1998; and Ozawa et

al., 1998). The extent of interrealtionships of these complex members means

that a precise diagnosis of the type of sarcoglycanopathy cannot be reached

without the use of a range of antibodies in muscle, followed by mutation

detection, though the detection of all the mutations may be problematic

(Duggan et al., 1997a, b).

In 1992, Matsumura et al. reported adhalin deficiency in muscle biopsy

specimens from Algeria and Lebanese patients presenting with a severe

childhood autosomal recessive muscular dystrophy (SCARMD). The clinical

phenotype of this disease was close to Duchenne (DMD) and Becker (BMD)

muscular dystrophies as reported by Ben Hamida et al. (1983). Presence of

dystrophin in the muscle biopsy specimens (Jelloun-Dellagi et al., 1990) led to

a serach for deficiency in one of the dystrophin-associated glycoproteins

(DAG)/ dystrophin-associated proteins (DAP) and to the discovery of adhalin

deficiency (Matsumura et al., 1992). Several authors have subsequently

reported adhalin deficiency in European (Fardeau et al., 1993; Romero et al., ·

1994; and Morandi et al., 1996), Brazilian (Passos Bueno et al.,1993; 1995) and

Asian patients (Sewry et al., 1994b; Kawai et al., 1995; and Hayashi et al.,

1995) with SCARMD or milder muscular dystrophies.

Genetic studies indicated a heterogeneity of adhalin deficiencies. Ben

Othmane et al. (1992) mapped the defective gene, responsible for the form of

severe childhood autosomal recessive muscular dystrophy (SCARMD)

prevalent in North Africa, to chromosome 13q12 and this was confirmed by

Azibi et al. (1993). Passos-Beuo et al. (1993) reported exclusion of this locus by

linkage analysis in three Brazilian families and Romero et al. (1994) reported

similar results in one French family, all affected by adhalin gene.

34

Subsequently the adhalin gene was mapped to chromosome 17q12-q21.33 and

Roberds et al. (1994) identified the first missense mutations in a previously

reported French family (Romero et al., 1994). Different groups subsequently

reported other cases of fiprimaryi adhalinopathy (Sewry et al., 1994; Kawai et

al., 1995; Piccolo et al., 1995; and Ljunggren et al., 1995). Secondary adhalin

deficiencies are due to mutations affecting ~-, y-, or 8-sarcoglycan genes,

which are localizied to 4q12 (Lim et al., 1995; Bonneman et al., 1995), 13q12

(Noguchi et al., 1995), or 5q33 (Nigro et al., 1996a, b, c; and Jung et al., 1996).

CONGENITAL MUSCULAR DYSTROPHY

Congenital muscular dystrophies (CMD) are usually clinically apparent at

birth or in the first few months of life with hyoptonia, weakness, and variable

degrees of arthrogryposis (multiple contractures) (Banker, 1994). CK is often

elevated but can be normal also. The muscle biopsy shows changes consistent

with a muscular dystrophy, frequently with impressive connective tissue

prolification.

In general two groups can be distinguished by virtue of the presence or

absence of abnormalities of brain formation evident on neuroimaging studies

or on autopsy examination of the brain. The first group, children with what

appears to be 'pure' CMD without clinically apparent brain dysfunction or

malformations of the brain, has l;>een referred to as 'classical' CMP. This

group is clearly heterogenous. The most important subclassifying feature here

is the presence or absence of the laminin a2 chain (merosin) by

immunohistochemistry on the muscle biopsy. In the second group, affected

individuals have varying degrees of mental retardation, evidence of diverse

malformations of the brain, and often clinical involvement of eyes as well.

This group includes Fukuyama muscular dystrophy (F-CMD) (Fukuyama et

al., 1961, 1981), Walker-Warburg syndrome (WWS) (Dobyns et al., 1989), and

muscle-eye-brain disease (F-MEB-D) (Santavuori et al., 1989).

35

Laminin a. 2 chain-deficient congenital muscular dystrophy

Tt.rough the application of neuroimaging techniques it became apparent

that there were patients with classic CMD who have evidence for an

abnormality of the white matter resembling leukodystrophy on neuroimaging

studies. These neuroimaging findings were concordant among siblings in a

given family with CMD (Philpot et al., 1995). Independently, Tome et al. 1994

recognized that by immunohistochemistry a significant number of patients

with classic CMD had a deficiency of a. 2 chain on muscles biopsy. A

concomitant increased staining for the 0.1 chain, a component of laminin-1,

was observed in these cases (Tome et al., 1994; and Sewry et al., 1995). It

became evident that the laminin 0.2 chain deficiency correlated with abnormal

findings of the white matter on T2 - weighted cranial magnetic resonance

imaging (MRI) studies, thus defining a distinct subgroup among the patients

with CMD.

Linkage analysis by homozygosity mapping in consanguineous pedigrees

with laminin 0.2 chain deficient CMO mapped the gene to chromosome 6q22-

23 (Hillaire et al., 1994), the same region where the human laminin 0.2 chain

gene LAMA2 resides (Vuolteenaha et al., 1994). Subsequently, the first

mutations in the human gene were reported in two families (Helbling-Leclerc

et al., 1995). Nissinen et al. (1996) found missense mlJ.tation in the gene

causing a dimnished, but not absent, pattern of staining for laminin 0.2 chain

on muscle biopsy.

By subdividing the group of classic CMDs into those that are laminin q_2

chain deficient versus those that are laminin 0.2 chain positive, clinical

differences between the two have become evident (Philpot et al., 1994;

Vainzof et al., 1995; Connolly et al., 1996; and North et al., 1996). In the

laminin a.z chain deficient patients, the initial neonatal presentation was more

severe, with a higher percentage of infants presenting with arthogryposis,

36

and these patients also had a far worse prognosis for ambulation. In fact,

none of the a2 chain deficient children achieved independent ambulation,

whereas almost all the positive patients eventually did, albeit sometimes with

assisting devises. In addition, serum CK values on the whole appear to be

higher in laminin a2 chain deficient group, usually around Qr above 1000.

There are other clinical phenotypes associated with CMD, sometimes

restricted to a single family, that do not fit easily into the classification system

outlined. No completely satisfactory classifcation system exists at moment for

CMDs.

37