Molecular genetics of Duchenne and Becker muscular dystrophy

T H E J O U R N A L OF

PEDIATRICS JULY

1990

Volume 117

N u m b e r 1, Part I

MEDICAL PROGRESS Molecular genetics of Duchenne and Becker muscular dystrophy Basil T. Darras, M D From the Departments of Pediatrics and Neurology, Division of Pediatric Neurology, Floating Hospital for infants and Children, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts

Duchenne and Becker muscular dystrophies are X-linked, recessive, neuromuscular diseases characterized by progressive muscular weakness. Duehenne muscular dystrophy is the most common X-linked disorder in man, l with an incidence of about 1 in 3500 live male births and a prevalence rate in the total population of about 3 per 100,000. The disease has one of the highest spontaneous mutation rates known in man: 1 of 10,000 gametes per generation. Up to one third of the cases have no previous family history and therefore represent de novo mutations in the germline of the mother or one of the grandparents. The onset of weakness usually occurs between 2 and 3 years of age, but it may be delayed. The child usually has difficulty with running, jumping, going up steps, and other similar activities; he has an unusual waddling gait, lumbar lordosis, and calf enlargement. Muscular weakness selectively affects proximal limb muscles before distal, and the lower limbs before the upper ones. Cardiac muscle is also affected. Patients with DMD often have varying degrees of mental retardation, although an occasional boy may have average or above-average intelligence. The affected children are usually wheel-

Supported in part by grant No. K08-NS01367-01 from the National Institutes of Health (National Institute of Neurological and Communicative Disorders and Stroke). Reprint requests: Basil T. Darras, MD, Department of Pediatrics, New England Medical Center Hospitals, NEMCH No. 309, 750 Washington St., Boston MA 02111. 9/18/21361

chair bound by the age of 12 years 2 and die in their late teens or early twenties. Becker muscular dystrophy has a similar presentation but a much milder clinical course in which the onset of symptoms is usually later, the muscle weakness is milder, and the disease progression is slower. Patients with BMD typically remain ambulatory beyond the age of 16 years and into adult life. 2 They usually survive beyond the age of 30. There is also an intermediate group of patients (with mild DMD or severe BMD) who become wheelchair bound between the ages of 12 and 16 years. The descriptions presented above BMD cDNA DMD mRNA PCR RFLP

Becker muscular dystrophy Complementary DNA Duchenne muscular dystrophy Messenger RNA Polymerase chain reaction Restriction fragment length polymorphism

support the concept of a relatively continuous spectrum of disease severity and suggest that the clinical definition of Duchenne, intermediate, and Becker phenotypes may to some extent be arbitrary. It is now known that all three phenotypes are allelic, resulting from mutations of a single gene. Until recently, the diagnosis of DMD or BMD depended on consideration of the clinical presentation, highly increased serum creatine kinase values, myopathic changes on electromyography, and myopathic pathologic changes on

2

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muscle biopsy. The main pathologic findings are variability in the size of muscle fibers, degeneration, abnormal enlargement or atrophy of individual fibers, and proliferation of endomysial and perimysial connective tissue. 2 The recent isolation of the gene defective in DMD and BMD and the identification of its protein product, dystrophin, have revolutionized our ability to address, with a high degree of accuracy, the diagnostic issues related to DMD and BMD. The purpose of this review is to present the progress made in this field of research during the past 4 or 5 years and, specifically, to discuss its practical applications in the management of patients and families with muscular dystrophy. MOLECULAR GENETICS OF THE DUCHENNE-BECKER GENE Mapping the DMD-BMD gene. The gene that, when mutated, causes the Duchenne and its allelic milder Becker phenotype has been assigned to band p21 of the short arm of the X chromosome (Xp21) on the basis of three different lines of evidence. First, as of today, there are more than 20 reported cases of DMD or BMD in girls, who have balanced X-autosomal translocations with breakpoints at Xp21 and preferential inactivation of the normal X chromosome 3, 4; the latter explains why these girls have myopathic symptoms. In these cases the translocation event presumably disrupts the D M D / B M D gene. Second, random genomic D N A segments from the short arm of the X chromosome around Xp21 were cloned by Dr. Kay Davies' group 5 (in England) and were found to be genetically linked to both DMD and BMD. This finding suggested that the D M D / BMD gene or genes was probably located in that region. The third piece of evidence, supporting the localization of the D M D / B M D locus at Xp21, came from a single patient with a small interstitial deletion in Xp21, identified with high-resolution cytogenetics and D N A analysis by Dr. Uta Francke and colleagues.6 This patient had a complex phenotype, including DMD, chronic granulomatous disease, retinitis pigmentosa, and the McLeod phenotype in his erythrocytes. Isolation of the D M D / B M D gene. Molecular analysis of genetic disorders has proceeded primarily through identification and purification of specific proteins and cloning of their respective genes. The latter usually can be accomplished by using the amino acid sequence of the purified protein or peptide. Until recently, however, as is the case with many other human genetic diseases, the biochemical basis for DMD and BMD was totally unknown. To isolate the DMD gene, a "reverse genetic" approach was employed, which is isolation of a gene on the basis of its chromosomal position. 7 Two novel approaches to isolation of the D M D / B M D

The Journal of Pediatrics July 1990

gene were employed by two different groups. Dr. Ronald Worton's group 8 used D N A derived from a female patient with DMD and an X;21 translocation; the autosomal breakpoint was in a block of ribosomal R N A genes repeated in tandem on the short arm of chromosome 21. This group used, as probes, segments of the already cloned ribosomal R N A gene to isolate the X chromosome junction fragment (X J) at the translocation breakpoint 8 and, therefore, sequences related to the D M D / B M D gene. Another group, led by Dr. Louis Kunkel, 9, 10 used an elegant subtraction hybridization technique to isolate a number of D N A segments (pERT) missing in the D N A of the original patient with an Xp21 interstitial deletion, 6 who had DMD and other disorders. Subclones of the XJ fragment and some pERT D N A (pERT87) segments were found to detect deletions in about 6.5% of DNAs derived from DMD and BMD patients without visible cytogenetic deletions.l l This was an indication that these D N A segments were probably part of the D M D / B M D locus, which was further supported by the detection of close (95% to 96%), TM 12 although not perfect, linkage between restriction fragment length polymorphisms detected by the XJ and the pERT87 probes and the Duchenne mutations in DMD and BMD families. The 4% to 5% recombination rate of XJ and pERT87 RFLPs was initially puzzling but was later attributed to the fact that the large size of the Duchenne gene allows for intragenic recombination events. The next step was to identify messenger R N A transcribed from coding DNA segments (exons) of the Duchenne gene, in the tissue where this gene is expected to be predominantly expressed (muscle). This step proved difficult for two reasons. First, as shown later, the m R N A species encoding the Duchenne gene protein is of relatively low abundance in muscle. Second, the coding units of the Duchenne gene are very small compared with the noncoding ones and are therefore hard to identify. To this end, Dr. Kunkel's group9, l0 employed the approach of cross-species conservation; sequences conserved across species must be important and therefore most likely belong to coding units of a particular gene. In fact, one of them (pERT87-25) detected a large 14-kilobase transcript in muscle mRNA. Later on, conserved fragments (pERT87-25) and also unique sequence segments (X J10.2 and X J10.3) from the XJ clone were used successfully to clone pieces of the Duchenne gene from libraries of fetal and adult skeletal muscle complementary DNA. TM14 Subsequently the entire cDNA for the Duchenne gene was isolated. 15 It contains 14,000 nucleotides and more than 65 exons (coding DNA segments). The 3685 amino acid sequence of the Duchenne gene protein product was predicted from the nucleotide sequence of the cDNA, and the protein, named dystrophin, was immunologically characterized) 6

Volume l 17 Number l, Part t

Molecular genetics o f Duchenne and Becket muscular dystrophy

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I. Mapping•f•8DMDandt•r••BMDgenedeI•ti•nsdetect•dwithdystr•phincDNA•D••et••nshav•beenma•••d with respect to HindIlI exon-containingfragments (Darras et al.2~ Note nonrandom distribution of deletions,with major cluster near center of dystrophin gene and minor one near 5' end region. Solid bars indicate DMD deletions;broken bars indicate BMD deletions. Analysis was carried out with Southern blotting, with seven segments of 14-kilobasedystrophin eDNA ([-3, 4-5a, 5b-7, 8, 9, 10, 11-t4) used as probes. Relative approximate positionsof genomic intragenic probes (XJ, pERT87, J-Bir, J-66) used in RFLP analysis are indicated on cDNA map. Fig.

D y s t r o p h i n c D N A . The dystrophin gene is the largest gene yet identified in man. Pulsed-field gel electrophoresis data from Dr. Gert van Ommen's laboratory, 17 in the Netherlands, suggest a size of 2.3 megabases (2300 kilobases) which is 12 times the size of the coagulation factor VIII gene, one of the largest genes known. By contrast, the dystrophin eDNA is relatively small, 13,973 nucleotides long (about 14 kilobases), with the first 11,336 nucleotides coding for the dystrophin protein. The rest, about 2637 nucleotides, remain untranslated. 18 Most of the D M D / B M D mutations identified so far are intragenic deletions detected with the dystrophin eDNA in approximately 60% to 65% of the families studied.]5, 17, 19-21This incidence of deletions is unusually high. Nonetheless, when the mutation target size of the DMD gene is taken into account, it does not seem to be excessively high in comparison with other X-linked loci, 15,2~ if even distribution of the deletions along the gene is assumed. The deletions, however, are not randomly distributed; they cluster primarily near the center of the gene and secondarily near the 5' end region (Fig. 1). This suggests the presence of deletion breakpoint "hot spots" within the dystrophin gene. The clustering of deletions in two regions of the gene facilitates their detection. Although the deletion mutations seem to have meiotic and sometimes mitotic origin,2~one or more mechanisms responsible for their development remain to be determined. Partial gene duplications have also been

reported17, 21, 22 in a small percentage of patients (~5%). RFLP and duplication analysis in three families suggests that unequal sister chromatid exchange at a mitotic stage of germ cell lineage development in the proband's unaffected grandfather might be a common mechanism for duplications.23 In the remaining patients (30% to 35%) without detectable deletions or duplications, the nature of the molecular lesions leading to the DMD or BMD phenotype remains unknown. Some of the lesions could represent point mutations leading to missense, nonsense, or frame-shift defects. Because the dystrophin gene is very large, however, it is expected that many of the nondeletion mutations will represent splicing errors. Tissue-specific e x p r e s s i o n o f d y s t r o p h i n . In an effort to understand the pathophysiology of DMD and BMD, the tissue-specific expression of dystrophin has been studied extensively at both the m R N A and protein levels. Dystrophin is normally expressed in embryonic and adult skeletal muscle (all physiologic types), cardiac muscle, visceral and vascular smooth muscle, and brain. Skeletal muscle and cardiac muscle contain the highest levels of dystrophin mRNA. In smooth muscle the level of dystrophin mRNA corresponds to approximately 5% to 10% of the level in skeletal muscle.24 The levels of mRNA found in the human brain and spinal cord seem to exceed the amount of mRNA derived from its myogenic cell content. Thus the identifica-

4

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The Journal of Pediatrics July 1990

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Fig. 2. lmmunostaining of frozen biopsy sections of skeletal muscle with antidystrophin antibodies by means of indirect immunofluorescence (d-n). Parts a, b, and e illustrate sections stained by hcmatoxylin and eosin, which correspond to parts d, e, and h respectively. Note complete absence of immunofluorescence at sarcolemma in patient with DMD (c and f), compared with homogeneous staining of plasma membrane in normal human muscle (a and d). In muscle biopsy specimen from patient with BMD, note partial staining of sarcolemma (b and e). Mosaic pattern of immunostaining is observed in frozen sections of muscle derived from DMD carrier with symptomatic disease (m) and from symptom-free DMD carrier (n). Note normal staining of sarcolemma in patients with Emery-Dreifuss muscular dystrophy (g), Fukuyama congenital muscular dystrophy (h), limb-girdle muscular dystrophy (i), facioscapulohumeral muscular dystrophy ~j), myotonic dystrophy (k), and Kugelberg-Welander spinal muscular atrophy (I). (Parts a to I modified from Arahata K, Ishiura S, lshiguro T, et al. [Nature 1988;333:861-3] and part n from Arahata K, Ishihara T, Kakamura K, et al. IN Engl J Med 1989;321:398-9], by permission of the authors, Nature [copyright | 1988, Macmillan Magazines Limited], and the New England Journal of Medicine. Part m courtesy of E. Bonilla, MD, Columbia University, New York.)

tion of dystrophin expression in neuronal tissues is not related to smooth muscle contamination. Preliminary studies suggest that neurons are the predominant cells expressing dystrophin in the nervous system. Moreover, Nudel et al? 5 have shown, by using a ribonuclease I protection assay, that the first 11 amino acids of skeletal muscle dystrophin are

replaced by three amino acids in the brain form of the protein, suggesting that there may be two different tissue-specific promoters. It is conceivable that the varying degrees of mental retardation that are frequently associated with D M D may be related to alterations ofdystrophin expression in neural tissue.

Volume 117 Number l, Part t

Molecular genetics o f Duchenne and Becker muscular dystrophy

There seem to be at least eight different human dystrophin variants (isoforms) expressed as groups of two or three in different tissues, resulting from differential splicing of the mRNA in the area encoding the carboxy-terminal domain of dystrop bin'26 Thus the dystrophin protein, through its carboxyl terminus, may be interacting with different proteins in the various tissues in which it is expressed. Structure and subcellular localization of dystrophin. Dystrophin, the protein product of the D M D / B M D gene, has been characterized by cDNA sequencing and also by immunologic studies. On the basis of cDNA sequence, the protein was predicted tO contain 3685 amino acids with a total monomeric molecular weight of 427 kilodaltons. 18 The amino acids are organized in four distinct domains; the amino-terminal domain, the "extended rod" spectrin-like domain, the cysteine-rich domain, and, last, the carboxyterminal domain. The largest part of the protein is its second domain, predicted to be rod shaped and composed of 25 repeats. 27 On Western blots, dystrophin was found to have a denatured, monomerie molecular weight of about 400 kilodaltons in skeletal muscle, a size close to the 427 kilodaltons predicted by the cDNA analysis.16 With the use of anti sera, dystrophin was localized to the cytoplasmic face of the plasma membrane of muscle fibers (Fig. 2), being somewhat more concentrated at the myotendinous and neuromuscular junctions. 28-3~Immunogold electron microscopy studies of ultrathin cryosections have suggested a lattice organization of dystrophin molecules. 31 This, along with structural similarity (homology) to the cytoskeletal proteins spectrin and c~-actinin, suggests a structural role for dystrophin. Dystrophin pathology. Dystrophin can easily be detected on immunoblots of 100 #g of total muscle protein by using antidystrophin antibodies. Therefore accurate information regarding the molecular weight and relative abundance of the dystrophin contained in a patient's skeletal muscle can be obtained by using a small amount of tissue derived from a muscle biopsy specimen. Hoffman et al., 32 in Kunkel's group, carried out an extensive analysis of dystrophin in muscle biopsy specimens from patients with DMD, BMD, and other neuromuscular diseases. Th e complete or almost complete (less than 3% of normal) absence of dystrophin is very specific and characteristic of the severe Duchenne phenotype. 17 The majority of patients with BMD (~85%) have dystrophin of abnormal molecular weight, either smaller (80%) or larger (5%) in deletion or duplication cases, respectively, which is often reduced in abundance.33, 34A subgroup of patients with BMD ( ~ 15%), however, have normal-size protein of reduced abundance. These patients are expected to have nondeletion mutations, such as missense, splicing, or gene-promoter mutations. Normal levels of dystrophin of normal molecular weight

5

have been found in patients with neuromuscular diseases other than DMD and BMD. When antidystrophin antibodies have been used in combination with immunoflu0rescence, no immunostaining or only patchy immunostaining of the plasma membrane of scattered muscle fibers has been observed in frozen muscle sections from patients with DMD (Fig. 2). In BMD muscle fibers the immunostaining of the sarcolemma may appear normal or may be only partial. 283~ In the normal-appearing cases, however, discontinuities of immunostain may be observed at higher magnification. 3~ A mosaic pattern of immunostaining was observed in the sarcolemma of both cross-sectioned and longitudinally sectioned fibers in biopsy specimens derived from DMD carriers with symptoms 35 (Fig. 2). A similar pattern was also noted in four symptomfree obligate carriers of DMD with Slightly elevated creatine kinase values. 36 Furthermore, negative staining of muscle biopsy specimens for dystrophin has been documented in tw o complex glycerol kinase deficiency patients with myopathy and adrenal hypoplasia; 3' deletions of the dystrophin gene, extending into the cysteine-rich domain region, have been detected in both patients. 37

M O L E C U L A R B A S I S FOR D U C H E N N E V E R S U S BECKER M U S C U L A R DYSTROPHY lntragenic deletions of the dystrophin gene can be detected in about 60% to 65% of patients with D M D / B M D . Published studies, however, fail to reveal any apparent correlation between the size of the deletions and the severity and progression of the disease phenotype. 15, 20 Genetic information within the DNA molecule is stored in the form of triplets of nucleotide bases (codons); each codon determines the synthesis of one amino acid. The exons of a particular gene encode an open reading frame of nucleotide triplet codons, translated into protein. The open reading frame is set at the initiation codon (ATG) and proceeds until the stop codon (TAA, or TAG, or TGA) is reached, which signals the termination of protein synthesis. Exons are separated by noncoding sequences called introns, which are spliced out during the synthesis of the mature mRNA. The intron-exon borders can be defined by any of the three bases of the coding triplets for amino acids. During the splicing of introns, two contiguous exons along the gene must have, at their joining ends, codon breakpoints that maintain the correct translational open reading frame; in other words, their joining ends have to be in phase. Monaco et al. 38 proposed that dystrophin gene deletions resulting in the clinically milder BMD phenotype juxtapose exons that preserve the translational open reading frame (inframe deletions), whereas deletions that juxtapose exons which shift the translational open reading frame (out-

6

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The Journal of Pediatrics July 1990

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Fig. 3. Molecular basis for DMD versus BMD phenotypes. Top of figure indicates Xp21 map position of DMD/BMD locus and also approximate sizes of DMD gone, its transcription product (mRNA), and dystrophin protein. Bottom of figure shows how small deletion can lead to severe DMD phenotype, in contrast to large one, which yields milder BMD phenotype. On left, deletion of single oxen (crossed-out box) disrupts translational reading frame because exons flanking deletion are out of phase (out-of-frame deletion), mRNA can be translated only in triplet codons. Therefore T - G A T . . . will be converted to TGA-T . . . . which creates nonsense mutation (TGA codon is translation stop), leading to severely truncated dystrophin protein. On right, a relatively larger deletion contains integral number of triplet codons and therefore preserves reading frame (in-frame deletion). Resulting protein is internally deleted and probably semifunctional, accounting for milder BMD phenotype. (From Hoffman EP, Kunkel LM. Neuron 1989;2:1019-29. Reprinted by permission of the authors and Cell Press; copyright 9 Cell Press.)

of-frame deletions) should result in the more severe Duchenne phenotype. Preservation of the reading frame should permit the synthesis of an internally deleted dystrophin protein, which may he at least semifunctional. By contrast, the disruption of the reading frame should lead to the synthesis of a functionless, severely truncated protein molecule (Fig. 3). This hypothesis has been supported by a number of studies. In a recent one, 39 clinical-molecular correlations proved correct in 92% of the cases. Nevertheless, deletions of ex-

ons 3 to 7 result in phenotypes of unexpected severity, 4~ whereas large in-frame deletions of more than 30 exons and deletions of the first exon result in D M D . When this latter group of deletion cases was excluded, the correlation between type of deletion mutation and phenotype was in agreement with the reading frame hypothesis in 96% of the cases. Some cases of nonconformity to the model described above can probably be accounted for by a disrupted m R N A splicing process effected by point mutations that eliminate splicing sites, generate novel splicing sites, or activate cryp-

Volume 117 Number 1, Part I

Molecular genetics o f Duchenne and Becker muscular dystrophy

tic ones. Similar splicing mutations have been described for the B-globin gene. MOLECULAR

DIAGNOSIS

The availability of genomic D N A probes flanking the Duchenne gene, intragenic genomic probes, and c D N A (dystrophin) probes has revolutionized our ability to make a precise diagnosis and therefore to provide highly accurate genetic counseling to DMD and BMD families. More recently, the dystrophin protein assay, 32 by either Western blotting or immunocytochemistry, has complemented and thereby enhanced our DNA-based diagnostic capability. These advances notwithstanding, the goal of genetic diagnosis so far, by either D N A techniques or dystrophin assay, has been simply to differentiate between the DMD or BMD phenotypes and normal phenotypes or genotypes--in other words, to confirm or predict the diagnosis of an "Xp21" muscular dystrophy. Recent studies, 21' 34, 39 however, provide enough evidence to suggest that currently available diagnostic techniques and reagents can be used to predict the severity of the disease phenotype. This section will discuss the clinical applications of old and new techniques in carrier detection, proband identification, and prenatal diagnosis of DMD and BMD. Linkage analysis. Linkage analysis uses genetic markers in the form of RFLPs detected by DNA probes with restriction endonuclease analysis. The RFLPs are naturally occurring genetic polymorphisms at the level of D N A sequence that can be used as genetic markers throughout the genome. They are seen on a Southern blot41 as restriction fragment length variations, and can be revealed by either genomic DNA (noncoding) or cDNA (coding) probes. This type of analysis does not test for the gene mutation itself but, rather, tests the segregation, in a particular family, of D N A markers physically located near (flanking)or within the dystrophin gene (intragenic). Therefore it can be used for prenatal diagnosis and carrier detection in the 3 5% of families without detectable deletions or duplications. With the use of these markers, the X chromosome region carrying or not carrying the D M D / B M D mutation can be marked and its segregation followed through the family. Given the proper family structure, this indirect approach can provide valuable information regarding the genotype and therefore the phenotype of persons at risk. However, the unavailability of D N A from an affected male or males or other key family members might make the study inconclusive or less reliable; therefore many family members need to cooperate for testing. For the study to be conclusive, key female members of the family being studied need to be "informative"--that is, to have polymorphic restriction sites in their DNA, recognized preferably by both flanking and intragenic D N A probes. Unfortunately the capacity of this

7

indirect approach to detect reliably new mutation events is limited. Figures 4 and 5 exemplify the way linkage analysis is used for prenatal diagnosis and detection of carriers. 42 The D N A used in linkage analysis is derived from leukocytes (5 to 10 ml of ethylenediaminetetraacetic acid-anticoagulated blood). Fetal DNA can be isolated from either cultured amniocytes or a chorionic villus biopsy sample. Deletion analysis. The high incidence of deletion mutations in patients with DMD and BMD that can be identified by means of the dystrophin cDNA probes (Fig. 1) has provided a direct method for genetic diagnosis that permits a high degree of diagnostic accuracy in more than 65% of the families studied. 43 The cDNA probes detect the site of the mutation itself, so meiotic recombination events become irrelevant. Therefore the chance of a diagnostic error is essentially zero. Figure 6 illustrates the first dystrophin cDNA-based prenatal diagnosis that I made in Dr. Uta Francke's laboratory, using cDNA probes kindly provided by L. M. Kunkel, PhD. In families with detectable deletions, diagnosis of carriers can be accomplished as shown in Fig. 6. For accurate carrier detection, however, and because of the presence of one normal copy of the dystrophin gene in a carrier, gene dosage analysis with densitometry may become necessary in the area of the deletion. Deletion detection with the Southern blot technique, in spite of its advantages over linkage analysis, is still a tedious, expensive, time-consuming, and sometimes difficult-tointerpret method because of the complexity of the obtained restriction patterns. 44 Chamberlain et al.,45 in Dr. Thomas Caskey's group, have successfully applied the polymerase chain reaction technique to detect deletions in male patients. Because of the clustering of deletions in two "hot spot" areas, 70% of the deletions detectable by cDNA probes can be detected in a single reaction (multiplex PCR) by amplifying six regions of the gene. The detection rate can be increased to more than 98% of cDNA-detectable deletions by t~sing more recently developed primer sets (Alan Beggs, PhD: personal communication, Jan. 15, 1990). Caskey's group has also shown that the same technique can be applied to prenatal diagnosis with amniotic fluid cells, With chorionic villus sampling, t h e P C R technique remains usable as long as the level of contamination with maternal D N A remains less than 3% to 5%. 45 The PCR technique is very sensitive (only 0.5 m! of blood is needed), eliminates the use of radioisotopes, and also provides reliable results in 1 or 2 days, in compariso n with an average of 1 to 2 weeks needed for deletion detection with the Southern blot technique. Nevertheless, until more primers are developed to cover the entire dystrophin gene by PCR, Southern blotting will remain the method of choice for evaluating the effect of deletions on the reading frame.

8

Darras

The Journal of Pediatrics July 1990

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Although the incidence of partial gene duplications is relatively small ( ~ 5 % ) , they can be used, like deletions, to confirm the clinical diagnosis of DMD or BMD and to allow for prenatal diagnosis and detection of carriers. Dystrophin assay by Western blot technique. A minimum of 5 mg of frozen muscle tissue (stored at - 7 0 ~ C) is needed for the dystrophin assay by Western blot. If dystrophin,

which appears as a 400-kilodalton protein, is normal in both quantity and quality, then the diagnosis of DMD or BMD can be excluded with a high degree of certainty. With the use of polyclonal antidystrophin antibodies, more than 99% of patients with DMD have complete or almost complete absence of dystrophin in skeletal muscle tissue. 32 A single reported sporadic DMD patient, with apparently normal

Volume l 17 Number l, Part t

Molecular genetics o f Duchenne and Becker muscular dystrophy

muscle dystrophin levels, has probably been misdiagnosed. 27 In the second " D M D " case, in which dystrophin was normal, the patient had a history of consanguinity, suggesting autosomal recessive inheritance. 46 In patients with BMD, dystrophin of either altered molecular weight or decreased abundance, or both, is identified on immunoblots. Of two BMD patients with no detectable dystrophin on Western blot, both had large deletions encompassing the segment of the dystrophin protein used to produce antibodies. This finding suggests that multiple antisera, raised against different segments of the dystrophin molecule, might have to be used in some patients. 47 The test is very specific because, as of this writing, no patient with an unambiguously established clinical diagnosis of a neuromuscular disease unrelated to DMD and BMD has been found to have dystrophin deficiency. In addition, the test can be extremely useful in cases with no clear-cut X-linked family history of myopathy, which can easily be misdiagnosed as an autosomal recessive disease such as limb-girdle dystrophy or even spinal muscular atrophy. The test also holds great promise in the area of precisely predicting the severity of the evolving phenotype. So far, it appears that the quantity rather than the size of dystrophin determines the severity of the phenotype. Patients with D M D have less than 3% of the normal quantity of dystrophin. Regardless of the protein size, dystrophin levels between 3% to 10% of normal correlate with an intermediate phenotype (mild DMD, severe BMD), whereas levels greater than 20% correlate with a mild or moderate BMD phenotype. 34 It seems that the already described differences, at the protein level, between DMD and BMD might be delineated further by studying larger patient populations and also by utilizing domain-specific antidystrophin antibodies. 47 At present, however, the test does not appear

9

suitable for the detection of symptom-free D M D carriers or for prenatal diagnosis, which can be reliably performed with D N A analysis. The D M D carriers who have symptoms might have low levels of dystrophin detectable as abnormal by the dystrophin assay, but this has to be confirmed. Moreover, some BMD carriers can be identified on Western blot by their double-band pattern, which consists of dystrophin of both normal and altered sizes. 48 lmmunohistoehemical studies. The clinical diagnosis of D M D or BMD can also be confirmed by immunostaining of frozen sections of skeletal muscle biopsy specimens with antidystrophin antiserum, with the use of indirect immunofluorescence. As discussed in the section on dystrophin pathology, above, in muscle biopsy specimens from patients with DMD, there is either complete or almost complete absence of immunofluorescence in the sarcolemma of individual fibers 28, 30 (Fig. 2). ~n patients with BMD, either normal or partial staining of the sarcolemma is observed. As expected, in patients with other neuromuscular diseases there is homogeneous staining of the surface membrane. Carriers of D M D with symptomatic disease and also those who are symptom free have a distinct mosaic pattern of dystrophin immunostaining related to X-chromosome inactivation (Fig. 2, m and n). Carrier status, however, cannot be excluded by a normal pattern of immunostaining; the latter may be related to nonrandom X-chromosome inactivation, to selective preponderance of dystrophin-positive fibers, or even to sampling error. Furthermore, the immunohistochemical analysis cannot be used reliably for identification of BMD carriers. 48 PITFALLS

IN MOLECULAR

DIAGNOSIS

The D N A techniques described above have greatly enhanced our diagnostic capability in families with DMD

Fig. 4. Prenatal diagnosis of DMD and BMD by means of linkage analysis. A, Map position on short arm of X chromosome of set of intragenic (pERT84, X J1.1, pERT87, J-Bir, J-66) and proximal and distal flanking DNA markers used for genetic linkage studies in families with DMD or BMD. OTC, Ornithine transcarbamylase gene. B, Pedigree of family A. Black symbols indicate three affected males (deceased), Pregnant woman (III-2) was probable carrier because of her two affected brothers and also because of her elevated creatine kinase (CPK)levels. Her mother (I1-2) was an obligate DMD carrier. Therefore, if III-2 were indeed a carrier, her maternally derived X chromosome (stippled bar, in panel D) would have been the one carrying dystrophin gene mutation. C, DNA fragments of variable length (alleles) detected by Southern blotting in four members of family A and control female DNA (C). Their DNA was restricted with restriction endonucleases EcoRV, Bgl II, and Taq I, blotted, and hybridized with DNA probes C7. pERT87-30, pERT87-15, and XJ-I.1, all of which revealed RFLPs in DNA derived from Ill-2 (two fragments, each one marking one of her X chromosomes). Male fetus (IV-l) received all four fragments derived from his maternal grandfather (1I-1). kb, Kilobase. D, Xp21 hap[otypes of members of family A, constructed from Southern blotting results. Upper and lower case letters correspond, respectively, to larger and smaller DNA fragments (alleles) shown in panel C. Set of alleles constitutes haplotype. White, stippled, and hatched bars designate distinct haplotypes. Fetus received grandpaternally derived Xp21 haplotype (hatched bar) without crossover. Because DMD mutation in nucIear family (D) has grandmaternat (11-2) origin, fetus was predicted to be unaffected with more than 95% probability. His normal status was confirmed postnatally. CPK, Creatine kinase. (From Darras BT, Harper JF, Francke U. N Engl J Med 1987;316:985-92. Reprinted by permission of the New England Journal of Medicine.)

10

Darras

The Journal of Pediatrics July 1990

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Fig. 5. Prenatal and carrier diagnosis by means of linkage analysis. RFLP alleles have been designated as explained in Fig. 4. Female fetus (III-1) received mutation-carring Xp21 haplotype (black bar) and was therefore predicted to be a carrier with more than 99% probability, which was supported postnatally by elevated serum creatine kinase (CPK) values. Mutation-carrying haplotype (black bar) is partially shared by two affected brothers (111-2 and 11I-3) from proximally flanking marker 754 to distal marker C7, and has grandpaternal (I-1) origin. OTC alleles are different in two affected brothers because of crossover event. Grandfather (I-1) was unaffected, so mother's (1I-2) X-chromosome mutation must represent a new one. Various types of bars designate distinct haplotypes. (From Darras BT, Harper JF, Francke U. N Eugl J Med 1987;316:985-92. Reprinted by permission of the New England Journal of Medicine.)

and BMD. Unfortunately, there are a number of potential pitfalls that are inherent in D M D genetics and that are related to possible autosomal forms of muscular dystrophy, intragenic recombination events, exceptions to the "reading frame model" for predicting the severity of the D M D / B M D phenotype, and also the high incidence of germline mosaicism. Autosomal recessive muscular dystrophy. Several recent reports have provided clinical and molecular evidence for the existence of an autosomal recessive form of muscular dystrophy that is clinically indistinguishable from D M D and BMD. Initially the evidence was clinical, derived from consanguineous matings and inbred populations in central and northern Africa, 49, 50 as well as from those in other countries. 51 The first molecular genetic evidence was published recently. 52 R F L P analysis with genomic and c D N A

probes, deletion analysis with the dystrophin cDNA, and dystrophin testing by Western blot showed that the dystrophin gene was not involved in two of three unrelated families with affected brother-sister pairs. In one of the families, the affected sister had a random, instead of the expected nonrandom, X-chromosome inactivation pattern; in addition, her affected brother's muscle biopsy specimen showed dystrophin of normal size and quantity. In the other family the affected male and his unaffected brother were concordant for their dystrophin c D N A haplotypes, which suggested that both had inherited the same dystrophin gene from their mother. In the absence of detectable deletions, these results provide sufficient evidence for an autosomal gene involvement in these two families. It is conceivable that counseling results can be erroneous if based solely on R F L P analysis in families without an ob-

Volume 117 Number 1, Part I

Molecular genetics o f Duchenne and Becker muscular dystrophy

vious X-linked family history or without a firm clinical diagnosis of DMD or BMD. If a dystrophin gene deletion cannot be detected at the DNA level, a muscle biopsy should be performed in an affected family member for dystrophin testing. If immunoblotting shows dystrophin of normal size and abundance, an autosomal recessive mutation will need to be considered. The proportion of autosomal recessive muscular dystrophy in the 35% of families with no demonstrable gene deletion is now unknown. Therefore, unless X-linked inheritance is obvious, diagnosis of DMD or BMD should be confirmed with dystrophin testing before one attempts RFLPbased counseling of muscular dystrophy families. Intragenie recombination. In affected families without a detectable deletion or duplication, carrier detection and prenatal diagnosis will depend on RFLP linkage analysis. The large size of the dystrophin gene, however, allows for intragenic recombination events with meiotic crossovers within the gene itself. 12,42 The site of the mutation is usually unknown, so the 4% to 5% intragenic recombination frequency can lead to diagnostic errors if only a small number of markers are used. This difficulty can be overcome by concurrently using a set of intragenic and flanking DNA markers to develop Xp21 haplotypes, 53 as shown in Figs. 4 and 5. Although this approach can decrease the chance of an erroneous result to less than 1%, it is both labor-intensive and time-consuming. Multiplex PCR-based strategies in combination with recently discovered highly informative (CA)n block genetic markers may simplify and greatly expedite RFLP typing of the families without detectable deletions. Exceptions to the reading frame model. One of the most rewarding benefits of the described research will be the ability to prognosticate the severity of the muscular dystrophy phenotype by means of the dystrophin assay and, potentially, the translational reading frame rule. As described above, however, the clinical molecular correlations39proved correct in 92% of the cases with deletions analyzed at the reading frame level. Therefore, in the absence of dystrophin analysis results and until the mechanisms that underlie the exceptions to the reading frame rule are delineated, caution should be exercised when deletion analysis data are used to inform families with young patients about the rate of progression and severity of the disease. For the time being, dystrophin testing remains the method of choice for prognosticating the severity of the disease, because it is technically simpler and more reliable.48 Germline mosaicism. Of major importance for the genetic counseling of families in which there are sporadic cases of DMD or BMD is the very well described phenomenon of germline mosaicism. Because of a mutation at a mitotic stage of germ cell lineage development, a dystrophin dele-

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Fig. 6. Prenatal diagnosisof DMD by dystrophin cDNA. DNAs derived from key family members were digested with restriction endonucleasesHindIII and Bgl II, subjected to Southern blot assay, and hybridized with the seven eDNA probes (see Fig. 1) covering entire dystrophin gene. In affected subject (III-1), some of HindIII and Bgl II restriction fragments detected by cDNA probe 8 (Fig. 1) are deleted (large arrows). These fragments are present in DNA derived from male fetus (III-2) and also in DNA from male normal control subject (C). Therefore fetus was predicted to be unaffected with almost 100% probability, which was confirmed postnatally. DNA derived from mother of fetus (II-2) showed twocopy intensity for deleted fragments, compared with one-copy intensity (one X chromosome) of signal in normal males I-l, III-2, and C. This result suggested two intact X chromosomes in the mother (II-2), which, in assumed absence of germline mosaicism, excluded carrier status in her. Arrowheads indicate size in kilobases (kb) of exon-containingHindIII and Bgl II restriction fragments. (From Darras BT, Koenig M, Kunkel LM, Francke U. Am J Med Genet 1988;29:713-26. Reprinted by permission of Alan R. Liss, Inc.)

tion mutation can be observed in more than one offspring of a male or female family member whose somatic cells lack the mutation.54, 55 Figure 7 illustrates the case of a male germline mosaic. If it is assumed that the deletion or any other type of mutation occurs very early in postzygotic embryo development, male or female germline-plus-somatic mosaicism may occur which can be manifested with mod-

12

Darras

The Journal of Pediatrics July 1990

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Q Fig. 7. Male germline mosaicism for dystrophin gene deletion mutation. DNA RFLPs shown are designated as explained in Fig. 4. Dystrophin gene deletion is indicated by symbol 9 del. Sets of alleles (haplotypes) are illustrated by specific shading or hatching. White areas for D2 and 99-6 alleles indicate uncertainty about their chromosomal origin, because of homozygosity in respective mothers. Clinically unaffected male (I-2), whose Xp21-22 haplotype is indicated by black bar, transmitted deleted X chromosome to two (lI-2, II-4) of his five daughters, and same chromosome without deletion to other three daughters (1t-5, II-6, ll-7). Ones who received deletion (lI-2, II-4) became carriers (white circle with center dot) and transmitted mutation to subsequent generations, including fetus IV-I, who was aborted. Therefore 1-2 had germline mosaicism for the deletion; in this ease however, possibility of mosaicism involving germ cells and also somatic tissues cannot be excluded. Various types of bars designate distinct haplotypes. (From Darras BT, Francke U. Nature 1987;329:5568. Reprinted by permission of Nature [copyright | 1987, Macmillan Magazines Limited].)

estly elevated creatine kinase levels in the affected persons, regardless of gender. If a woman without a family history of D M D or B M D has a single affected son, and if she herself is found not to be a carrier by deletion analysis of D N A derived from her peripheral blood, her risk of having another child with D M D or B M D is not negligible. Because of the possibility of germline mosaicism, her risk may be as high as 7%. 33 Therefore this i m p o r t a n t occurrence needs to be considered

in the counseling of families with sporadic cases of D M D or B M D caused by new mutations. RECOMMENDED PROTOCOL FOR MOLECULAR DIAGNOSIS OF DMD BMD

OR

T h e diagnosis of D M D or B M D can be made clinically on the basis of the symptoms and signs at presentation, the increased serum creatine kinase values, and the myopathic

Volume 117 Number 1, Part 1

Molecular genetics o f Duchenne and Becker muscular dystrophy

Male patient with ] probable DMD/BMD and negative family history (sporadic case) l Muscle biopsy

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13

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Fig. 8. Algorithm for molecular diagnosis of DMD and BMD. In sporadic cases, Western blot assay can confirm clinical diagnosis of DMD or BMD and can be used to predict severity of disease phenotype. After diagnosis is established, deletion-duplication (del/dupl) DNA analysis or RFLP linkage analysis can be performed for prenatal diagnosis and detection of carriers. If muscle biopsy specimen is not available, or if severity of disease is predictable or obvious at presentation, detection of mutation by DNA analysis will confirm diagnosis of DMD or BMD. If RFLP-deletion studies are uninformative, muscle biopsy will need to be considered, in potential carrier females, for dystrophin immunohistochemistrystudy. Because intrafamilial variability in phenotypic expression of DMD and BMD is relatively uncommon, muscle biopsy and dystropbin assay for prediction of severity of disease are not needed in familial cases, if diagnosis of DMD or BMD has already been confirmed in another affected family member by analysis of dystrophin or DNA or both. If diagnosis has not been confirmed, molecular diagnosis of DMD or BMD can be accomplished in 60% to 65% of such familial Cases by PCR or Southern blot screening or both. Nevertheless, muscle biopsy for dystrophin testing might have to be considered in familial cases without detectable deletion or duplication, particularly if (1) clinical presentation is atypical, (2) inheritance does not have clear-cut X-linked pattern, and (3) there are affected male and female siblings suggestive of autosomal recessive form of muscular dystrophy. NM, Neuromuscular.

findings on electromyography or muscle biopsy, or both. Furthermore, a family history positive for an X-linked muscular dystrophy of the Duchenne or Becker type, in conjunction with the findings described above, can also suggest the diagnosis of D M D or BMD. Figure 8 outlines an algorithm for the molecular diagnosis of DMD and BMD. When th e family history is negative for DMD and BMD (sporadic), the Western blot assay of protein derived from a muscle biopsy specimen can confirm the clinical diagnosis of DMD or BMD and can be used to predict the severity

of the disease. If the dystrophin assay result is abnormal, D N A analysis should be performed, preferably by PCR, and then by Southern blot if PCR failed to detect a deletion. Detection of a dystrophin gone deletion or duplication will greatly facilitate carrier detection and prenatal diagnosis in the proband's family. In the event that no deletion or duplication is found, R F L P linkage analysis can be attempted fo r prenatal diagnosis and detection of carriers. If a muscle biopsy specimen is not available, or if the severity Of the disease is predictable or apparent at presentation, P C R or Southern blot testing of peripheral blood D N A can be per-

14

Darras

The Journal of Pediatrics July 1990

formed to confirm the diagnosis of D M D or BMD. This can be accomplished in more than 60% to 65% of the cases. However, failure to detect a deletion or duplication will not exclude the diagnosis of D M D or BMD. In new cases of typical D M D or BMD, with a family history of X-linked D M D or B M D muscular dystrophy, molecular diagnosis may not be necessary if the clinical diagnosis has been confirmed in another affected family member by analysis of dystrophiu or D N A or both. In such familial cases the clinical course in an affected family member rather adequately, although not always, predicts the severity of the evolving phenotype for other members of the same family. If the diagnosis has not been confirmed with analysis of dystrophin or D N A in other members of the proband's family, the less invasive P C R , with or without Southern blot testing, should be attempted first. If D N A analysis fails to detect an Xp21 mutation, a muscle biopsy for dystrophin assay will need to be considered, particularly in clinically atypical cases, in families without a clear-cut X-linked pattern of inheritance, and also in families with affected male and female siblings suggesting an autosomal recesslve form of muscular dystrophy.

PROSPECTS

FOR T H E R A P Y

The high incidence of new mutations in the dystrophin gene accounts for about one third of the cases, and thus the disease will not be eliminated by voluntary termination of pregnancies found by D N A testing to be at risk. Therefore one of the major objectives of research in muscular dystrophy today is the design of efficient therapies by exploiting the recently acquired knowledge of the dystrophin gene and its protein product. One of the therapeutic approaches being explored is the transplantation of normal myoblasts. In a recent study, 56 myoblasts taken from a normal mouse and injected into the muscle of a dystrophic (mdx) host mouse fused with the dystrophic fibers and produced dystrophin. This experiment has raised great hope in patients and families and has stimulated much interest in human muscle transplant studies. In spite of the potential problems, such as immune rejection, the need for a large number of myoblasts, and also the necessity for closely spaced multiple injections, this approach is being pursued vigorously by different groups in this country. Other approaches to dystrophin replacement therapy can be investigated as well. Thus the possibility of finding some way to slow, or even arrest, the progress of the disease is no longer unrealistic, and the development of effective therapy is a reasonable expectation for the future. I am most grateful to Dr. Uta Francke for introducing me to the wonderful world of genetics and for helping me to understand the basic principles of this exciting discipline. 1 would also like to thank Drs. L. M. Kunkel, E. P. Hoffman, K. Arahata, and H. Sugita for providing permission to reprint figures; Dr. E. Bonilla for kindly

providing figures illustrating dystrophin immunostaining of muscle biopsies; and Drs. U. Franckc and N. P. Rosman for critical reading of this manuscript.

REFERENCES 1. Moser H. Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Hum, Genet 1984;66:17-40. 2. Dubowitz V. Muscle biopsy: a practical approach. London: Baitli~re Tindatl, 1985:289-339. 3. Greenstein RM, Reardon MP, Chan TS, et al. An (X;ll) translocation in a girl with Duchenne muscular dystrophy. Cytogenet Cell Genet 1980;27:268. 4. Boyd Y, Buckle V, Holt S, Munro E, Hunter D, Craig 1. Muscular dystrophy in girls with X; autosome translocations. J Med Genet 1986;23:484-90. 5. Davies KE, Pearson PL, Harper PS, et al. Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome. Nucleic Acids Res 1983;I1:2303-12. 6. Francke U, Ochs HD, de Martinville B, et al. Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet 1985;37:250-67. 7. Orkin SH. Reverse genetics and human disease. Cell 1986;47:845-50. 8. Ray PN, Belfall B, DuffC, et al. Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy. Nature 1985;318:672-5. 9. Kunkel LM, Monaco AP, Middlesworth W, Ochs, HD, Latt SA. Specific cloning of DNA fragments absent form the DNA of a male patient with an X chromosome deletion. Proc Natl Acad Sci USA 1985;82:4778-82. 10. Monaco AP, Bertelson C J, Middlesworth W, et al. Detection of deletions spanning the Duchenne muscular dystrophy locus using a tightly linked DNA segment. Nature 1985;316:842-5. 1I. Kunkel LM, Hejtmancik JF, Caskey CT, et al. Analysis of deletions in DNA from patients with Becket and Duchenne muscular dystrophy. Nature 1986;322:73-7. 12. Fischbeck KH, Ritter AW, Tirschwell DL, et al. Recombination with pERT87 (DXSI64) in families with X-linked muscular dystrophy [Letter]. Lancet 1986;2:104, 13. Monaco AP, Neve RL, Colletti-Feener C, et al. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature 1986;323:646-50. 14, Burghes AHM, Logan C, Hu X, Betlfall B, Worton RG, Ray PN. A cDNA clone from the Duchenne/Becker muscular dystrophy gene. Nature 1987;328:434-7. 15. Koenig M, Hoffman EP, Bertelson C J, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50:509-17. 16. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919-28. 17. Den Dunnen JT, Grootscholten PM, Bakker E, et al. Topography of the Duchenne muscular dystrophy (DMD) gene: F1GE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835-47. 18. Koenig M, Monaco AP, Kunkel LM. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988;53:219-28.

Volume 117 Number 1, Part I

Molecular genetics o f Duchenne and Becker muscular dystrophy

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