Duchenne muscular dystrophy (DMD)Richard C. Arceo, M. D.

DESCRIPTION

Duchenne muscular dystrophy (DMD) is a severe recessive X-linked form of muscular dystrophy characterized by rapid progression of muscle degeneration, eventually leading to loss of ambulation and death.

Incidence/Prevalence

Although reliable prevalence data are lacking, the prevalence of DMD is generally estimated at 1:3,500 live male births (Emery 1991).The birth prevalence of DMD in northern England is one in 5,618 live male births.

Pathogenesis

DMD is caused by mutations in the dystrophin gene which is the largest human gene, spanning 2,200 kb on the X chromosome and occupying roughly 0.1% of the genome. The gene is composed of 79 exons and 8 tissue-specific promoters [Koenig et al., 1987]. The primary transcript measures about 2,400 kilobases and takes 16 hours to transcribe, the mature mRNA measures 14.0 kilobases. The 79 exons code for a protein of over 3500 amino acid residues.

Where is the DMD gene located?Cytogenetic Location: Xp21.2

Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. Dystrophin provides structural stability to the Dystroglycan complex (DGC), located on the cell membrane

Abnormal gene productMutations will lead to lack of dystrophin expression causing DMD, whereas those that lead to abnormal quality or quantity of dystrophin lead to BMD.

Much investigative work determined that dystrophin is involve in the release of calcium from the sarcoplasmic reticulum in muscle fibers. The lack of dystrophin causes calcium to leak into the cell, which promotes the action of an enzyme that dissolves muscle fibers.When the body attempts to repair the tissue, fibrous tissue forms, and this cuts off the blood supply so that more and more cells die.

CLINICAL FEATURESSKELETAL MUSCLEThe most distinctive feature of Duchenne muscular dystrophy is a progressive proximal muscular dystrophy with characteristic pseudohypertrophy of the calves

The first symptoms of DMD appear during preschool years. The disorder affects the legs first. A boy has trouble walking and maintaining balance. In most cases, he begins walking three to six months later than average.

As his muscles begin to weaken, he may change the way he walks. He places his legs farther apart in order to maintain balance. Walking this way produces a waddling effect that is characteristic of DMD.

Contractures usually begin at about the age of five or six.This forces a boy to walk on his tiptoes. Balance becomes more of a problem. As a result, falls and broken bones become common at this age.

By the age of nine or ten, a boy with DMD might not be able to climb stairs Or stand by himself. By age 10, braces may be required to aid in walking but most patients are wheelchair dependent by age 12.

Early common sign of muscular dystrophy

To get up from the ground, the child walks up' his thighs with his hands. This is mainly because of weak thigh muscles.

The Baskaran familySouth East of EnglandJamie Oliver

May develop a severe curve of the spine.Heart and breathing muscles also get weak. Child usually dies before age 20 from heart failure or pneumonia.

NERVOUS SYSTEMMental retardation of mild degree is a pleiotropic effect of the Duchenne gene (Zellweger and Niedermeyer, 1965) As indicated later, the finding of dystrophin mRNA in brain may bear a relationship to the mental retardation in DMD patients. In 50 DMD patients with a mean age of 11.1 years (range 3.5 to 20.3), Bresolin et al. (1994) found that 31% had a Wechsler full intelligence quotient (FIQ) lower than 75 and that only 24% had appropriate IQ levels by this index

Bushby et al. (1995) studied 74 boys with DMD, 18% of which had a full scale IQ of below 70. The authors found no significant IQ difference between the patients with promoter deletions and those without, nor did they find a relationship between the length of the deletion and full scale IQ. They found, however, that boys with distal deletions were more likely to be mentally retarded than were those with proximal deletions

CARDIAC MUSCLEMyocardial involvement appeared in a high percentage of DMD patients by about 6 years of age; it was present in 95% of cases by the last years of life. (Nigro et al., 1983).Mirabella et al. (1993) noted that electrocardiographic abnormalities had been reported in 6.6 to 16.4% of DMD heterozygous females and that in one carrier female severe cardiomyopathy had been described in association with muscle weakness. They reported 2 carriers with dilated cardiomyopathy and increased serum CK but no symptoms of muscle weakness. Heart biopsies in both patients showed absence of dystrophin in many muscle fibers

SMOOTH MUSCLENoting that in DMD functional impairment of smooth muscle in the gastrointestinal tract can cause acute gastric dilatation and intestinal pseudoobstruction that may be fatal, Barohn et al. (1988) studied gastric emptying in 11 patients with DMD. Strikingly delayed gastric emptying times were observed.

Boland et al. (1996) studied a retrospective cohort of 33 male patients born between 1953 and 1983. The mean age at DMD diagnosis was 4.6 years; wheelchair dependency had a median age of 10 years; cardiac muscle failure developed in 15% of patients with a median age of 21.5 years;smooth muscle dysfunction in the digestive or urinary tract occurred in 21% and 6% of the patients, respectively, at a median age of 15 years.In this cohort, death occurred at a median age of 17 years.

Diagnosis

1. Serum creatine phosphokinase (CK) concentration

2. Electromyography (EMG)

is useful in distinguishing a myopatic process from a neurogenic disorder. This is done by demonstrating short-duration, low-amplitude, polyphasic, rapidly recruited motor unit potentials.

3. Skeletal muscle biopsy

Muscle histology early in the disease shows nonspecific dystrophic changes, including variation in fiber size, foci of necrosis and regeneration, hyalinization, and, later in the disease, deposition of fat and connective tissue.

Findings in the Dystrophin Protein from Skeletal Muscle Biopsy

Molecular Genetic Testing

Gene: DMD is the only gene known to be associated with DMDClinical testing: Deletion/duplication Analysis 1. Multiplex PCR [Multicenter Study Group 1992], 2. Southern blotting [Darras et al 1988], and 3. FISH (with probes covering DMD exons 3-6, 8, 12, 13, 17, 19, 32-34, 43-48, 50, 51, and 60) can be used to detect deletions, which account for approximately 65% of mutations in individuals with DMD. Approximately 98% of deletions are detectable by these methodologies.

Southern blotting and quantitative PCR analysis can be used to detect duplications. Duplications may lead to in-frame or out-of-frame transcripts and account for the disease-causing mutations in approximately 6%-10% of males with DMD or BMD. In one study [Galvagni et al 1994], duplications were detected in 8.18% of individuals with DMD. In a series of individuals already screened for deletions and point mutations, duplications were detected in 87% of cases [White et al 2006].

New testing methods including single-condition amplification internal primer sequencing (SCAIP) [Flanigan et al 2003] and denaturing gradient gel electrophoresis (DGGE)-based whole-gene mutation scanning [Hofstra et al 2004] aim at detecting the remaining 30%-35% of the DMD mutations in a semiautomatic, rapid, accurate, and economical fashion.A muscle biopsy-based diagnostic approach was developed and optimized to increase the mutation detection frequency to nearly 100% [Deburgrave et al 2007].

To date, 501 deletions, 8 duplications, and 989 point mutations have been documented in the dystrophin gene (Leiden muscular dystrophy database; www.dmd.nl).5 exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions

Molecular Genetic Testing

The current methodologies used for detecting mutations in the dystrophin gene include multiplex PCR, Southern blotting [Stockley et al., 2006], multiplex ligation-dependent probe amplification (MLPA) [Gatta et al., 2005; Janssen et al., 2005; Schwartz and Duno, 2004], detection of virtually all mutations-SSCP (DOVAM- S) [Buzin et al., 2000, 2005; Liu et al., 1999], denaturing high-performance liquid chromatography (DHPLC) [Bennett et al., 2001], single condition amplification/internal primer sequencing (SCAIP) [Flanigan et al., 2003], and Sanger sequencing [Hamed and Hoffman, 2006; Stockley et al., 2006].

HUMAN MUTATION 0,1^9,2008

Signs and Symptoms in Carriers of Duchenne and Becker Muscular Dystrophy

DMD Carriers BMD Carriers None 76% 81% Muscle weakness 19% 14%Myalgia/cramps 5% 5% Left ventricle dilation 19% 16% Dilated cardiomyopathy 8% 0

From Hoogerwaard et al [1999b)

Carrier TestingA reliable and simple method based on quantitative real-time PCR detects deletions/duplications in 100% of DMD/BMD carriers [Joncourt et al 2004].Carrier testing for deletions may also be performed by FISH [Voskova-Goldman et al 1997].Carrier testing for point mutations may be performed by sequence analysis.

Genotype-Phenotype Correlations

In males with DMD, phenotypes are best correlated with the degree of expression of dystrophin, which is largely determined by the reading frame of the spliced message obtained from the deleted allele [Monaco et al 1988, Koenig et al 1989].Very large deletions may lead to absence of dystrophin expression. Mutations that disrupt the reading frame include stop mutations, some splicing mutations, and deletions or duplications. They produce a severely truncated dystrophin protein molecule that is degraded, leading to the more severe DMD phenotype.

Data suggest that dystrophin deletions involving the brain distal isoform Dp140 are associated with intellectual impairment [Felisari et al 2000

Testing StrategyEstablishing the diagnosis of DMD:

For individuals with clinical findings suggesting a dystrophinopathy and an elevated serum CK concentration, the first step in diagnosis is molecular genetic testing of the DMD gene:If a disease-causing mutation is identified, the diagnosis is established; If no DMD disease-causing mutation is identified, skeletal muscle biopsy of individuals with suspected DMD is warranted for western blot and immunohistochemistry studies of dystrophin.

Management

Evaluations Following Initial DiagnosisTo establish the extent of disease in an individual diagnosed with a dystrophinopathy, the following evaluations are recommended:Physical therapy assessmentDevelopmental evaluation before entering elementary school for the purpose of designing an individualized educational plan, as necessaryIf the individual is older than age ten years at diagnosis, evaluation for cardiomyopathy by electrocardiography, chest radiography, cardiac echocardiography, pulmonary function studies, and/or MRI [Towbin 2003]

MedicationsPrednisone. Studies have shown that prednisone improves the strength and function of individuals with DMD. It is hypothesized that prednisone has a stabilizing effect on membranes and perhaps an anti-inflammatory effect.Whether the improvement is the result of an immunosuppressive effect remains unclear, as individuals treated with azathioprine did not have a beneficial effect.

In a randomized double-blind six-month trial, prednisone administered at a dose of either 0.75 mg/kg/day or 1.5 mg/kg/day increased strength and reduced the rate of decline in males with DMD [Mendell et al 1989]. The improvement begins within ten days of starting the treatment, requires a single dose of 0.75 mg/kg/day of prednisone for maximal improvement, reaches a plateau after three months, and can be sustained for as long as three years in those children maintained on doses of 0.5 and 0.6 mg/kg/day [Fenichel et al 1991]. One open-label study suggested that therapy with prednisone could prolong ambulation by two years.

Side effects include weight gain (>20% of baseline) (40%), hypertension, behavioral changes, growth retardation, cushingoid appearance (50%), and cataracts [Mendell et al 1989, Griggs et al 1993].

Pulmonary:Baseline pulmonary function testing before confinement to a wheelchair (usually age ~9-10 years)Evaluation by a pediatric pulmonologist twice yearly after any one of the following: confinement to a wheelchair, reduction in vital capacity below 80% predicted, and/or age 12 years [Finder et al 2004]

Deflazacort:Deflazacort, a synthetic derivative of prednisolone used in Europe but not currently available in the US, is thought to have fewer side effects than prednisone, particularly with regard to weight gain [Angelini 2007]. A larger study comparing deflazacort to prednisone, carried out in Europe, showed that the two medications were similarly or equally effective in slowing the decline of muscle strength in DMD. Another European multicenter, double-blind, randomized trial of deflazacort versus prednisone in DMD showed equal efficacy in improving motor function and functional performance [Bonifati et al 2000]. A more recent study of deflazacort treatment showed efficacy in preserving pulmonary function as well as gross motor function [Biggar et al 2006].

In a comparison of two different protocols of deflazacort treatment in DMD, a 0.9-mg/kg/day dose was more effective than a dose of 0.6 mg/kg/day for the first 20 days of the month and no deflazacort for the remainder of the month [Biggar et al 2004]; 30% of children on the highest dose developed asymptomatic cataracts that required no treatment. A systematic review and meta-analysis of 15 studies showed that deflazacort improves strength and motor function more than placebo; whether it has a benefit over prednisone on similar outcomes remains unclear [Campbell & Jacob 2003].

Therapies Under Investigation

Aminoglycosides. Up to 15% of individuals with DMD exhibit the gene mutation known as a premature stop codon. Suppression of stop codons has been demonstrated with aminoglycoside treatment of cultured cells; the treatment creates misreading of RNA and thereby allows alternative amino acids to be inserted at the site of the mutated stop codon. In the mdx mouse, in vivo gentamicin therapy resulted in dystrophin expression at 10%-20% of that detected in normal muscle [Barton-Davis et al 1999], a level that provided some degree of functional protection against contraction-induced damage.

Aminoglycoside therapy has been suggested as an alternative to gene therapy but could be aimed only at individuals with premature stop codons. In a preliminary study in which gentamicin (7.5 mg/kg/day) was administered to four individuals for two weeks, full-length dystrophin did not appear in the muscles of the treated individuals [Wagner et al 2001]. Some authors, unable to reproduce the results previously published for the mouse model of DMD, have called for more preclinical investigation of this potential therapy [Dunant et al 2003]. In an in vitro study [Kimura et al 2005], dystrophin expression was detected in myotubes of males with DMD using gentamicin; however, the treatment was more effective in persons with the nonsense mutation TGA than TAA or TAG.

PTC124 is a new, orally administered non-antibiotic drug that appears to promote ribosomal read-through of nonsense (stop) mutations. Preclinical efficacy studies in mdx mice have yielded encouraging results [Barton et al 2005, Welch et al 2007]. A Phase I multiple-dose safety trial is ongoing [Hirawat et al 2005].Morpholino antisense oligonucleotides mediate exon skipping [Aartsma-Rus et al 2006a] and have improved the mdx mouse model of DMD [Wilton & Fletcher 2005, Alter et al 2006].

Oxandrolone, an anabolic (androgenic) steroid with a powerful anabolic effect on skeletal muscle myosin synthesis [Balagopal et al 2006], was shown in a pilot study to have effects similar to prednisone, with fewer side effects [Fenichel et al 1997]. A randomized, prospective, controlled trial showed that oxandrolone did not produce a significant change in the average manual muscle strength score of males with DMD, as compared with placebo; however, the mean change in quantitative muscle strength was significant [Fenichel et al 2001]. The investigators conducting this study felt that oxandrolone may be useful before initiating therapy with corticosteroid because it is safe in the short term, accelerates linear growth, and may be beneficial in slowing the progression of weakness. However, the long-term effects of oxandrolone in the treatment of DMD have not been studied.

Gene Therapy: Experimental gene therapies are currently under investigation [Gregorevic & Chamberlain 2003, Tidball & Spencer 2003, van Deutekom & van Ommen 2003, Nowak & Davies 2004].

A mouse model for DMD exists and is proving useful for furthering our understanding on both the normal function of dystrophin and the pathology of the disease. In particular, initial experiments that increase the production of utrophin, a dystrophin relative, in order to compensate for the loss of dystrophin in the mouse are promising and may lead to the development of effective therapies for this devastating disease.

Stem cell therapy: is under investigation but remains experimental [Gussoni et al 1997, Gussoni et al 1999, Gussoni et al 2002, Skuk et al 2004].

PM R. 2009 Jun;1(6):547-59. Mesenchymal stem cells: emerging therapy for duchenne muscular dystrophy. Markert CD, Atala A, Cann JK, Christ G, Furth M, Ambrosio F, Childers MK. Department of Neurology, School of Medicine, and Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC(dagger).

Other:Immunosuppression with azathioprine is not beneficial.Myoblast transfer has been inefficient.Creatine monohydrate has been studied as potential treatment in muscular dystrophies and neuromuscular disorders [Tarnopolsky & Martin 1999, Walter et al 2000, Louis et al 2003]. In a recent randomized, controlled, cross-over trial, 30 boys with DMD were given creatine (~0.1 g/kg/day) for four months and placebo for four months [Tarnopolsky et al 2004]. Treatment with creatine resulted in improved grip strength of the dominant hand and increased fat-free mass when compared to placebo; however, no functional improvement was noted. Given the limited data and modest benefit, treatment with creatine monohydrate cannot be recommended for treatment of DMD.

Cyclosporin was reported to improve clinical function in children with DMD who received the medication for eight weeks. Nevertheless, because of the rare reports of cyclosporin-induced myopathy in individuals receiving the medication for other reasons, the use of cyclosporin in treating DMD remains controversial.Histone deacetylase inhibitors have improved the mdx mouse by inducing the expression of the myostatin inhibitor follistatin [Minetti et al 2006].

Genetic CounselingMode of Inheritance: The dystrophinopathies are inherited in an X-linked manner.Genetic counseling is advised for people with a family history of the disorder. Duchenne muscular dystrophy can be detected with about 95% accuracy by genetic studies performed during pregnancy.

Carrier females have a 50% chance of transmitting the DMD mutation in each pregnancy. Sons who inherit the mutation will be affected;daughters who inherit the mutation are carriers and may or may not develop cardiomyopathy.

Prenatal Testing

Prenatal testing is possible for pregnancies of women who are carriers if the DMD mutation has been identified in a family member or if linkage has been established. The usual procedure is to determine fetal sex by karyotype or specialized studies to identify the sex chromosomes from cells obtained by chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation or by amniocentesis usually performed at approximately 15-18 weeks' gestation.

If the karyotype is 46,XY, DNA extracted from fetal cells can be analyzed for the known disease-causing mutation or using the linkage previously established.Preimplantation genetic diagnosis (PGD)Preimplantation genetic diagnosis may be available for families in which the disease-causing mutation has been identified.

Preimplantation genetic diagnosis (PGD) is a new alternative to conventional prenatal diagnosis particularly for those couples for whom termination of pregnancy is not acceptable.PGD is currently available for a wide range of single gene disorders including many X-linked disorders, cystic fibrosis, and -thalassaemia (Handyside et al., 1989 , 1992 ; Cui et al., 1995 ; Coonen et al., 1996 ; Ray et al., 1996a ; Kuliev et al., 1998 ).

Sexing of embryos for PGD (Handyside et al., 1989 ) has allowed the transfer of healthy female embryos where embryos are at risk of X-linked diseases such as DMD. However, with a gender-only selection strategy, all male embryos will be discarded even though half of these are not affected and female embryos are transferred regardless of carrier status.

SummaryWhat is Duchenne muscular dystrophy?Duchenne muscular dystrophy (DMD) is a rapidly progressive form of muscular dystrophy that occurs primarily in boys. It is caused by a mutation in a gene, called the DMD gene that can be inherited in families in an X-linked recessive fashion, but it often occurs in people from families without a known family history of the condition.

Molecular Genetic Testing

There is no known cure for Duchenne muscular dystrophy, although recent stem-cell research is showing promising vectors that may replace damaged muscle tissue.

Gene Therapy: Experimental gene therapies are currently under investigation [Gregorevic & Chamberlain 2003, Tidball & Spencer 2003, van Deutekom & van Ommen 2003, Nowak & Davies 2004].

Treatment is generally aimed at controlling the onset of symptoms to maximize the quality of life.

The Baskaran familySouth East of EnglandParents, Ben and Debby, have four sons:Jason, 19, Jamie, 13, Oliver, 12 and Konnor, aged 10Both Oliver and Jamie have Duchenne muscular dystrophy. Thank You

Normal allelic variants. The DMD gene spans 2.4 Mb of DNA and comprises 79 exons. It has at least four promoters. It is the largest known human gene. Innumerable intragenic variants have been described, many of which are useful as markers for genetic linkage analysis.Pathologic allelic variants. Disease-causing alleles are highly variable, including deletion of the entire gene, deletion or duplication of one or more exons, and small deletions, insertions, or single-base changes. In both DMD and BMD, partial deletions and duplications cluster in two recombination hot spots, one proximal at the 5' end of the gene, comprising exons 2-20 (30%), and one more distal, comprising exons 44-53 (70%) [Den Dunnen et al 1989]. Duplications cluster near the 5' end of the gene, with duplication of exon 2 being the single most common duplication identified [White et al 2006]. More than 4,700 mutations have been identified [Aartsma-Rus et al 2006b].

CYTOGENETICSGreenstein et al. (1977) found DMD in a 16-year-old girl with a reciprocal X;11 translocation. The mother was thought not to be a carrier. Possibly the break at Xp21 caused a null mutation; the normal X chromosome was inactivated. Verellen et al. (1978) reported the same situation with X;21 translocation and break at Xp21. Canki et al. (1979) described similar findings in a girl with X;3 translocation with break at Xp21. The mother was thought to be heterozygous. Zneimer et al. (1993) used a combination of conventional and molecular cytogenetic techniques to investigate the twins first reported by Richards et al. (1990). The twins carried a deletion of approximately 300 kb within the dystrophin gene on one X chromosome. A unique DNA fragment generated from an exon within the deletion was hybridized in situ to metaphase chromosomes of both twins, a probe that would presumably hybridize only to the normal X chromosome and not to the X chromosome carrying the deletion. The chromosomes were identified by reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine in culture to distinguish early and late replicating X chromosomes, corresponding to active and inactive X chromosomes, respectively. The experiment showed predominant inactivation of the normal X chromosome in the twin with DMD. With an improved method of high resolution R-banding, Werner and Spiegler (1988) showed deletion of Xp21.13 in an 8-year-old boy with normal intelligence and no disorder other than DMD. His healthy mother was heterozygous for the deletion, which was subject to random X inactivation in lymphocytes.

Zneimer et al. (1993) used a combination of conventional and molecular cytogenetic techniques to investigate the twins first reported by Richards et al. (1990). The twins carried a deletion of approximately 300 kb within the dystrophin gene on one X chromosome. A unique DNA fragment generated from an exon within the deletion was hybridized in situ to metaphase chromosomes of both twins, a probe that would presumably hybridize only to the normal X chromosome and not to the X chromosome carrying the deletion.The chromosomes were identified by reverse-banding (R-banding) and by the addition of 5-bromodeoxyuridine in culture to distinguish early and late replicating X chromosomes, corresponding to active and inactive X chromosomes, respectively. The experiment showed predominant inactivation of the normal X chromosome in the twin with DMD. With an improved method of high resolution R-banding, Werner and Spiegler (1988) showed deletion of Xp21.13 in an 8-year-old boy with normal intelligence and no disorder other than DMD. His healthy mother was heterozygous for the deletion, which was subject to random X inactivation in lymphocytes.

MAPPING Duchenne muscular dystrophy is not linked to colorblindness or G6PD (Emery et al., 1969; Zatz et al., 1974). No linkage with Xg has been found; total lod scores were -14.6 and -2.4 for theta of 0.10 and 0.30, respectively (Race and Sanger, 1975).Lindenbaum et al. (1979) found DMD with X-1 translocation and suggested that the DMD locus is at Xp1106 or Xp2107. A number of females with X-autosome translocations with the breakpoint in the Xp21 band have shown Duchenne muscular dystrophy. One interpretation is that the gene locus is in that region and that the locus on the normal X is inactivated. Murray et al. (1982) found linkage of DMD with a restriction enzyme polymorphism at a distance of about 10 cM. The cloned DNA sequence bearing the polymorphism (lambda RC8) was assigned to Xp22.3-p21 by study of somatic cell hybrids. Spowart et al. (1982) outlined reasons for doubting the location of the DMD gene at Xp21. Wieacker et al. (1983) studied the linkage between the restriction fragment length polymorphism defined by the cloned DNA sequence RC8 and X-linked ichthyosis. At least 2 crossovers were found among 9 meioses in an informative family, suggesting that RC8 and STS may be about 25 cM apart. Since STS is 15 cM proximal to the Xg locus and since the RC8 and Duchenne muscular dystrophy are closely linked, DMD may be 50 cM or more from Xg. Worton et al. (1984) studied a female with DMD and an X;21 translocation which split the block of genes encoding ribosomal RNA on 21p. Thus, ribosomal RNA gene probes can be used to identify a junction fragment from the translocation site and to clone segments of the X at or near the DMD locus.

Kingston et al. (1983, 1984) found linkage of BMD with the cloned sequence L1.28 (designated DXS7 by the seventh Human Gene Mapping Workshop in Los Angeles; D = DNA, X = X chromosome, S = segment, 7 = sequence of delineation). The interval was estimated to be about 16 cM, which is also the approximate interval between DXS7 and DMD. DXS7 is located between Xp11.0 and Xp11.3. Thus, these 2 forms of X-linked muscular dystrophy appeared to be allelic, a possibility also supported by the finding of both severe and mild disease (Duchenne and Becker, if you will) in females with X-autosome translocations. Contrary to reports of others, Kingston et al. (1984) found no evidence of linkage of BMD to colorblindness; Xg also showed no linkage. Francke et al. (1985) studied a male patient with 3 X-linked disorders: chronic granulomatous disease with cytochrome b(-245) deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa. A very subtle interstitial deletion of part of Xp21 was demonstrated as the presumed basis of this 'contiguous gene syndrome.' That this was a deletion and not a translocation was demonstrated by the absence of 1 DNA probe from the genome of the patient. Since this probe (called 754) was clearly very close to DMD and recognized a RFLP of high frequency, it proved highly useful for linkage studies of DMD. The close clustering of CGD, DMD, and RP suggested by these findings was inconsistent with separate linkage data, which indicated that McLeod and CGD were close to Xg and that DMD and RP are far away (perhaps at least 55 cM) and as much as 15 cM from each other. At least 4 possible explanations of the discrepancy were proposed by Francke et al. (1985). One suggestion was that the deletion contained a single defect affecting perhaps a cell membrane component with the several disorders following thereon. Mulley et al. (1988) reported the recombination frequencies between DMD and intragenic markers from 8 informative families containing 30 informative meioses. No recombinants were observed. The authors commented that the average theta between intragenic markers and DMD may be 1 to 2%. Grimm et al.(1989) reported a recombination rate of 4% between 2 subclones of the DNA segment DXS164 within the dystrophin locus, indicating a hotspot for recombination.

Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells

Nicole D. Hussey1,6, Hu Donggui1,3, David A.H. Froiland1, Damian J. Hussey2, Eric A. Haan4, Colin D. Matthews1 and Jamie E. Craig5 1 Department of Obstetrics and Gynaecology and 2 Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville 5011, South Australia, Australia, 3 Institute of Obstetrics and Gynaecology, The 2nd People's Hospital, Guangzhou, 510150, People's Republic of China and 4 South Australian Clinical Genetics Service, The Women's and Children's Hospital, North Adelaide, 5006, South Australia Abstract We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 4649 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 04.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.

Abstract We have developed five conventional duplex polymerase chain reaction (PCR) protocols on single lymphocytes and blastomeres from embryos, in order to analyse five exons commonly deleted in deletion-type Duchenne muscular dystrophy (DMD). The five DMD gene exons (17, 19, 44, 45 and 48) can be analysed in separate duplex PCR reactions together with the sex-determining region Y (SRY) gene which enables simultaneous gender assignment. We present here PCR amplification results from single lymphocytes isolated from a normal male (220 cells), a normal female (24 cells) and a male DMD patient (40 cells) carrying a deletion of exons 4649 within the DMD gene. The method failed to produce a PCR signal for the SRY gene in 8/220 normal male cells (3.6%) and for a DMD exon in 04.5% of normal male cells. One negative control out of 112 was positive. When this method was used to analyse two blastomeres from each of five embryos, concordant results were obtained for each pair of blastomeres. All embryos produced signals for the DMD exon tested with four of the embryos found to be male and one female. This method is therefore suitable for preimplantation genetic diagnosis and will allow the transfer of healthy embryos (both male and female) in families carrying DMD gene deletions involving at least one of the five exons 17, 19, 44, 45 and 48.

Introduction Duchenne or Becker muscular dystrophy (DMD/BMD) is one of the most common X-linked lethal genetic diseases with a worldwide frequency of one in 3500 live male births (Harper, 1989 ). Since no effective therapy exists thus far, most patients die at ~20 years of age. Mutations in the DMD gene can be divided into three different catagories of deletions, duplications and point mutations. Deletions within the 79 exon DMD gene account for ~60% of all DMD cases, 98% of which can be detected by two sets of multiplex polymerase chain reaction (PCR) reactions (Beggs et al., 1990 ; Chamberlain et al., 1990 ). Prenatal diagnosis using these two multiplex PCR protocols can determine whether a male pregnancy is affected when the deletion mutation for the family is known (Abbs, 1996 ). Preimplantation genetic diagnosis (PGD) is a new alternative to conventional prenatal diagnosis particularly for those couples for whom termination of pregnancy is not acceptable. PGD is currently available for a wide range of single gene disorders including many X-linked disorders, cystic fibrosis, and -thalassaemia (Handyside et al., 1989 , 1992 ; Cui et al., 1995 ; Coonen et al., 1996 ; Ray et al., 1996a ; Kuliev et al., 1998 ). Sexing of embryos for PGD (Handyside et al., 1989 ) has allowed the transfer of healthy female embryos where embryos are at risk of X-linked diseases such as DMD. However, with a gender-only selection strategy, all male embryos will be discarded even though half of these are not affected and female embryos are transferred regardless of carrier status.

Pleiotropy: Gene that affects more than one characteristic of an individualExample:Sickle cell diseaseCystic fibrosis

Dystroglycan Complex: In muscles, a complex of transmembrane glycoproteins links a network of dystrophin and actin filaments on the inside of the plasma membrane to two proteins of the extracellular basal lamina, alpha2 laminin and agrin.These protein associations stabilize the muscle plasma membrane from inside and outside. This muscle membrane skeleton resembles in concept and function the actin-spectrin network or red blood cells. Genetic defects or deficiencies in dystrophin, transmembrane linker proteins of the dystroglycan/sarcoglycan complex, or alpha laminin cause muscular dystrophy in humans, most likely due to the mechanical instability of the membrane leading to cellular damage and eventual atrophy of the muscle.

Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes[1]. It includes routine analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH).

Advent of banding techniques In the late 1960s Caspersson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Deletions within one chromosome could also now be more specifically named and understood. Deletion syndromes such as DiGeorge syndrome, Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid, and elongation techniques for all culture types that allow for higher resolution banding.

Human Male Karyotype

Beginnings of molecular cytogeneticsIn the 1980s advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969, movement was now made in using fluorescently labeled probes. Hybridizing them to chromosomes preparations made using existing techniques came to be known as fluorescent in situ hybridization (FISH). This change significantly increased the usage of probing techniques as fluorescently labeled probes are safer and can be used almost indefinitely. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated, cloned and studied in ever greater detail.

MosaicismPresence of 2 kinds of chromosome constitution in the same individual (zygote)

Routine analysisRoutine chromosome analysis refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing.Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (R-banding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes.Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes.High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.

KaryotypeIs the use of nomenclature to describe the normal or abnormal, constitutional or acquired, chromosome complement of an individual, tissue or cell line46,XX or 46,XY47,XX,+21KaryogramA systematized array of the chromosomes prepared either by drawing, digitized imaging or by photographyIdeogramDiagrammatic representation of a karyotype/ chromosome

Chromosome Designation9q34.2Chrom numberChrom armChrom regionChrom bandSub-band

Linkage Map/Chromosome MapGene linkage: the existence of several genes on the same chromosome. The genes on the same chromosome form a linkage group because these genes tend to be inherited together.Linkage Map/Chromosome Map: Tells the order of gene loci on chromosomes. To construct a chromosome map, investigators can sometimes rely on crossing over. Crossing-over occurs between nonsister chromatids when homologous pair of chromosomes pair prior to separation during meiosis. During crossing-over, the nonsister chromatids exchange genetic materials and therefore genes. Following crossing-over, recombinant chromosomes occur. Recombinant chromosomes contribute to recombinant gametes. Recombinant means a new combination of alleles.All the genes on one chromosome form a linkage group that tends to stay together, except when crossing-over occurs.

Blotting: Transfer step method to detect molecules separated by gel electrophoresis. Specific proteins are often detected with antibodies. Typically proteins are transferred electrophoretically from the polyacyrlamide gel to a sheet of nitrocellulose or nylon before reaction with antibodies.

A deletion is a mutation in which a part of a chromosome or a sequence of DNA is missing. Deletion is the loss of genetic material. Any number of nucleotides can be deleted, from a single base to an entire piece of chromosome. Deletions can be caused by errors in chromosomal crossover during meiosis. This causes several serious genetic diseases.

The three major single chromosome mutations; deletion (1), duplication (2) and inversion (3).

The two major two chromosome mutations; insertion (1) and translocation (2).

Numerical abnormalitiesWhen an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (trisomy, tetrasomy, etc). An example of a condition caused by a numerical anomaly is Down Syndrome, also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21, rather than two). Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome, an X.Structural abnormalitiesWhen the chromosome's structure is altered. This can take several forms:Deletions: A portion of the chromosome is missing or deleted. Known disorders include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4; and Jacobsen syndrome, also called the terminal 11q deletion disorder. Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. Known disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17.

Gene duplication (or chromosomal duplication or gene amplification) is any duplication of a region of DNA that contains a gene; it may occur as an error in homologous recombination, a retrotransposition event, or duplication of an entire chromosome. The second copy of the gene is often free from selective pressure that is, mutations of it has no deleterious effects to its host organism. Thus it mutates faster than a functional single-copy gene, over generations of organisms.A duplication is the opposite of a deletion. Duplications arise from an event termed unequal crossing-over that occurs during meiosis between misaligned homologous chromosomes. The chance of this happening is a function of the degree of sharing of repetitive elements between two chromosomes. The product of this recombination are a duplication at the site of the exchange and a reciprocal deletion.[2]

Gene duplication as amplificationGene duplication doesn't necessarily constitute a lasting change in a species' genome. In fact, such changes often don't last past the initial host organism. From the perspective of molecular genetics, amplification is one of many ways in which a gene can be overexpressed. Genetic amplification can occur artificially, as with the use of the polymerase chain reaction technique to amplify short strands of DNA in vitro using enzymes, or it can occur naturally, as described above. If it's a natural duplication, it can still take place in a somatic cell, rather than a germline cell (which would be necessary for a lasting evolutionary change).Also, in either event, duplications can be and often are marginally or severely detrimental. For instance, duplications of oncogenes are a common cause of many types of cancer, as is the case with P70-S6 Kinase 1 amplification and breast cancer.[8] In such cases the genetic duplication occurs in a somatic cell and affects only the genome of the cancer cells themselves, not the entire organism, much less any subsequent offspring. Genomic microarrays detect DuplicationsTechnologies such as genomic microarrays, also called array comparative genomic hybridization (array CGH), are used to detect chromosomal abnormalities, such as microduplications, in a high throughput fashion from genomic DNA samples. In particular, DNA microarray technology can simultaneously monitor the expression levels of thousands of genes across many treatments or experimental conditions, greatly facilitating the evolutionary studies of gene regulation after gene duplication or speciation [9][10].

Translocations: When a portion of one chromosome is transferred to another chromosome. There are two main types of translocations. In a reciprocal translocation, segments from two different chromosomes have been exchanged. In a Robertsonian translocation, an entire chromosome has attached to another at the centromere; these only occur with chromosomes 13, 14, 15, 21 and 22. Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted. Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material. Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. They often lead to an increased tendency to develop certain types of malignancies.

InheritanceMost chromosome anomalies occur as an accident in the egg or sperm, and are therefore not inherited. Therefore, the anomaly is present in every cell of the body. Some anomalies, however, can happen after conception, resulting in mosaicism (where some cells have the anomaly and some do not). Chromosome anomalies can be inherited from a parent or be "de novo". This is why chromosome studies are often performed on parents when a child is found to have an anomaly.

G-banding is technique used in cytogenetics to produce a visible karyotype by staining condensed chromosomes.The metaphase chromosomes are treated with trypsin (to partially digest the protein) and stained with Giemsa. Dark bands that take up the stain are strongly A,T rich (gene poor). The reverse of G-bands is obtained in R-banding. Banding can be used to identify chromosomal abnormalities, such as translocations, because there is a unique pattern of light and dark bands for each chromosome.It is difficult to identify and group chromosomes based on simple staining because the uniform color of the structures makes it difficult to differentiate between the different chromosomes. Therefore, techniques like G-banding were developed that made 'bands' appear on the chromosomes. These bands were same in appearance on the homologous chromosomes, thus, identification became easier and more accurate. The acid/saline/giemsa protocol reveals G-bands.

When the proband's DMD mutation is not known. Linkage analysis can be offered to at-risk females to determine carrier status in families with more than one affected male with the unequivocal diagnosis of DMD/BMD/DMD-associated DCM. Linkage studies are based on accurate clinical diagnosis of DMD/BMD/DMD-associated DCM in the affected family members and accurate understanding of the genetic relationships in the family. Linkage analysis relies on the availability and willingness of family members to be tested. The markers used for linkage in DMD/BMD/DMD-associated DCM are highly polymorphic and informative, and lie both within and flanking the DMD locus; thus, they can be used in most families with DMD/BMD/DMD-associated DCM [Kim et al 2002]. Note: The large size of the DMD gene leads to an appreciable risk of recombination. It has been estimated that the gene itself spans a genetic distance of 12 centimorgans [Abbs et al 1990]; thus, multiple recombination events among different members of a family may complicate the interpretation of a linkage study. Linkage testing is not available to families in which there is a single affected male.

Immunosuppressive therapy: The following recommendations for immunosuppressive therapy are in accordance with the national practice parameters regarding corticosteroid therapy developed by the American Academy of Neurology and the Child Neurology Society [Moxley et al 2005].

Boys with DMD who are older than age five years should be offered treatment with prednisone (0.75/mg/kg/day). Prior to the initiation of therapy, the potential benefits and risks of corticosteroid treatment should be carefully discussed with each individual.