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$: Genetic Evidence Linkage Muscular Dystrophy (Dystrophin) Gene … › 9baf › 107474d22fb728aef3... · 2017-10-05 · low fractional shortening on echocardiography. Many families$
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X-Linked Dilated CardiomyopathyMolecular Genetic Evidence of Linkage to the DuchenneMuscular Dystrophy (Dystrophin) Gene at the Xp21 Locus

Jeffrey A. Towbin, MD; J. Fielding Hejtmancik, MD, PhD; Paul Brink, MD; Bruce Gelb, MD;Xue Min Zhu, MS; Jeffrey S. Chamberlain, PhD;

Edward R.B. McCabe, MD, PhD; and Michael Swift, MD

Background. X-linked cardiomyopathy (XLCM) is a rapidly progressive primary myocardial disorderpresenting in teenage males as congestive heart failure. Manifesting female carriers have later onset (fifthdecade) and slower progression. The purpose of this study was to localize the XLCM gene locus in twofamilies using molecular genetic techniques.Methods and Results. Linkage analysis using 60 X-chromosome-specific DNA markers was performed in

a previously reported large XLCM pedigree and a smaller new pedigree. Two-point and multipoint linkagewas calculated using the LINKAGE computer program package. Deletion analysis included multiplexpolymerase chain reaction (PCR). Dystrophin protein was evaluated by Western blotting with N-terminaland C-terminal dystrophin antibody. Linkage of XLCM to the centromeric portion of the dystrophin orDuchenne muscular dystrophy (DMD) locus at Xp21 was demonstrated with combined maximumlogarithm of the scores of +4.33, 0=0 with probe XJ1.1 (DXS206) using two-point linkage and +4.81 atXJ1.1 with multipoint linkage analysis. LOD scores calculated using other proximal DMD genomic andcDNA probes and polymerase chain reaction polymorphisms supported linkage. No deletions wereobserved. Abnormalities of cardiac dystrophin were shown by Western blotting with N-terminaldystrophin antibody, whereas skeletal muscle dystrophin was normal, suggesting primary involvement ofthe DMD gene with preferential involvement of cardiac muscle.

Conclusions. XLCM is due to an abnormality within the centromeric half of the dystrophin genomicregion in heart. This abnormality could be due to 1) a point mutation in the 5' region of the DMD codingsequence preferentially affecting cardiac function, 2) a cardiac-specific promoter mutation that altersexpression in this tissue, 3) splicing abnormalities, resulting in an abnormal cardiac protein, or 4)deletion mutations undetectable by Southern and multiplex polymerase chain reaction analysis. (Circu-lation 1993;87:1854-1865)KEY WORDs * dystrophin * cardiomyopathy * Duchenne muscular dystrophy

T he cardiomyopathies represent a leading cause ofcardiovascular morbidity and mortality. Di-lated cardiomyopathy (DCM), which is more

common than either hypertrophic or restrictive forms,presents with signs and symptoms of congestive heartfailure, cardiomegaly and pulmonary edema on chest

From the Baylor College of Medicine, Departments of Pediat-rics (J.A.T., B.G., E.R.B.McC.) and Medicine (P.B.) and Institutefor Molecular Genetics (J.A.T., B.G., X.M.Z., E.R.B.McC.),Houston, Tex.; Laboratory of Mechanisms of OphthalmologicDisease (J.F.H.), National Eye Institute, National Institutes ofHealth; Department of Human Genetics (J.S.C.), University ofMichigan, Ann Arbor; and University of North Carolina (M.S.),Department of Medicine, Chapel Hill.

Supported in part by The Phoebe Willingham Muzzy PediatricMolecular Cardiology Laboratory; a Texas Affiliate Grant-in-AidAward (91R-207) from the American Heart Association; a ClinicalInvestigator Award (5-K08-HL-02485) from the National Heart,Lung, and Blood Institute; and a Research Grant (3-RO1-HD22563) from the National Institute of Child Health and HumanDevelopment.Address for reprints: Jeffrey A. Towbin, MD, Baylor College of

Medicine, Pediatric Cardiology and Molecular Genetics, OneBaylor Plaza, Room 333E, Houston, TX 77030.

Received February 24, 1992; accepted February 4, 1993.

radiography, and ventricular dilation with abnormallylow fractional shortening on echocardiography. Manyfamilies with multiple occurrences of DCM have beenreportedl-3; however, most cases occur sporadically.

In 1987, Berko and Swift4 reported a large kindred inwhich DCM was inherited as an X-linked dominantdisorder, with 11 affected males and five manifestingcarrier females (heterozygous females who manifest thedisease), all of whom were mothers of affected sons.Age of onset in the males ranged from late teens to early20s and the clinical course was rapidly progressive,resulting in death or transplantation within 1-2 years.Manifesting female carriers had late onset, usuallyduring the fifth decade, and had slow progression ofheart failure. No clinical findings of skeletal myopathycould be found, although elevated muscle creatinekinase (MM-CK) did occur (Table 1).

Molecular genetic techniques have been used tolocate the genes responsible for many human disorders,including the X-linked muscular dystrophies (Duchenne[DMD], Becker [BMD], and Emery-Dreifuss musculardystrophy),5-12 familial hypertrophic cardiomyopathy(HCM),13-15 Marfan syndrome,16 cystic fibrosis,17-19

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Towbin et al Linkage of X-Linked Cardiomyopathy to DMD 1855

TABLE 1. Clinical Evaluation of X-Linked Cardiomyopathy: Cardiac Studies

CardiacEcho catheter

Age Serum CK LVEDD SF LVEDP PAP CO EFSubject (years) (MM-CK) (mm) (%) (mm Hg) (mm Hg) (L/mm) (%) CourseA. XLCM-1 affected males

III-12 15 75 14 12 22/6 7.4 ... Death in 6 monthsIII-18 16 72 18 16 30/13 4.4 ... Death in 10 monthsIV-11 21 86 9 68/45 1.9 14 Death in 5 monthsIV-12 22 80 10 20 62/35 3.9 21 Transplant in 22 monthsIV-19 18 t 65 20 14 22/8 ... ... Moderate congestive

heart failureB. Carrier females

I-1 40 Death 10 years aftersymptoms

II-2 50 68 12 19 Mild congestive heartfailure over 20 years

II-5 48 60 10 19 Death 20 years afterdiagnosis

II-9 47 57 21 Mild congestive heartfailure over 10 years

III-5 45 1 55 20 Mild congestive heartfailure over 5 years

C. XLCM-2 affected malesII-2 19 t 74 12 Transplant in 1 yearII-3 18 78 10 Death in 10 monthsII-4 22 72 14 Death in 1 yearII-7 23 74 12 Transplant in 1 year

D. Carrier femalesI-1 53 60 18 Mild congestive heart

failure over 10 yearsCK, creatine kinase; LVEDD, left ventricular end-diastolic dimension; LVEDP, left ventricular end-diastolic pressure; PAP, pulmonary

artery pressure; CO, cardiac output; EF, ejection fraction; XLCM, X-linked cardiomyopathy.

neurofibromatosis,20-22 and long QT syndrome.23,24Each disease locus has been mapped to the genomeusing cosegregation analysis,2526 which measures thegenetic distance between DNA probes from knownchromosomal locations and the gene responsible for thedisease. This mapping most commonly has been per-formed by Southern blotting27 using genomic or cDNAprobes to generate restriction fragment length polymor-phisms (RFLPs),25 which are analyzed using computerprograms designed for linkage analysis to generateLOD scores (i.e., the logarithm of the odds for linkageversus the odds against linkage). In X-linked disorders,a LOD score greater than +2.0 is required for linkage,26corresponding to an odds for linkage of 102:1 or 100:1.This time-consuming process was recently aided bydevelopment of automated polymerase chain reaction(PCR), which more rapidly generates polymorphisms byamplifying specific regions of interest. These lattermethods include dinucleotide and tetranucleotide re-peat polymorphisms2.829 as well as changes in restrictionenzyme recognition sequences readily observed byPCR.303' These genetic mapping approaches were pre-viously used to identify the gene for dystrophin, thealtered or missing skeletal muscle protein in DMD andBMD. The dystrophin gene codes for the protein dys-trophin, which is 427 kd in size,6 contains 3,685 encodedamino acids separated into four distinct domains thatpredicts a 150-nm-long rod-shaped cytoskeletal pro-

tein32 and represents approximately 0.002% of totalstriated and cardiac muscle protein. The entire humancoding sequence of the dystrophin cDNA has beenshown to be 14 kb.5 DMD and BMD are the result ofmutations in this gene, and it is believed that thespectrum of disease results from disruption or mainte-nance of the translational reading frame of the mRNAleading to protein production.33-35 DCM is a commonand clinically significant finding in both of theseX-linked dystrophies,36,37 but the cause remains elusive.We report two families with X-linked DCM in whom

the genetic defect is localized to the dystrophin locuswithin chromosome region Xp21.

MethodsTwo families were evaluated after obtaining informed

consent. The first family was previously described andclinically evaluated at the University of North Carolina,Chapel Hill, by Berko and Swift4 (Figure 1, top). Thesecond, smaller family (Figure 1, bottom) was studied atBaylor College of Medicine and the University of TexasHealth Science Center-Houston. All members wereevaluated by physical examination, ECG, and echocar-diography (two-dimensional, M-mode, and Doppler).DCM was diagnosed in patients with left ventricularend-diastolic dimensions (LVEDD) measured to be>60 mm in adults 255 kg or >2 SDs above the mean inchildren or adults <55 kg based on weight/size, in



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1856 Circulation Vol 87, No 6 June 1993

Probe Order

754 (DXS84)64-10 (DXS142)XJ1.1 (DXS206)PERT87.30 (DXS164)

1.

2 2112

2 1

25

2

XLCM - 2

o Female Unaffected |* Female Affected

ill

FIGURE 1. Top panel: X-linkedcardiomyopathy (XLCM)-J pedi-gree, the truncated four-genera-tion famil initially described by

Berko and Swift.4 Bottom panel:XLCM-2 pedigree, the three-gen-eration family evaluated locally.Affected males, manifesting fe-male carriers, and unaffected in-dividuals are noted Patient num-bers are shown above eachindividual. Haplotypes for theprobes 754(DXS84), pERT 84-lO(DXS142), XJI.I(DXS206),and pERT 87.30 (DXSJ64) are

shown below each individual

addition to a shortening fraction (SF) <24%. The frac-tional shortening was measured using the formulaSF=(LVEDD-LVESD)/LVEDD. Blood studies to ruleout other causes of cardiomyopathy included thyroidfunction studies, carnitine, iron, total iron-binding capac-ity, viral and toxoplasma titers, and creatine kinase (CK).From 10 to 20 mL of blood anticoagulated with acidcitrate dextrose solution or sodium heparin was obtained,and DNA extraction was performed using an AppliedBiosystems model 340A automated DNA extractor. Fivemilliliters of blood was used for lymphoblastoid cell lineimmortalization with Epstein-Barr virus.38 The DNA wasdigested overnight with single restriction endonucleasesas suggested by the supplier (Boehringer-MannheimBiochemicals). Agarose gel electrophoresis (0.6-1.2%)(FMC Bioproducts) was performed in Tris acetate/EDTA (TAE). Southern blotting27 was performed usingZeta probe membranes (Bio-Rad Laboratories) and al-kaline transfer as described previously.39

Sixty X-chromosome-specific DNA probes identify-ing known RFLPs and having known map positions onthe X chromosome were used ("Appendix"). DNAprobes were labeled to >109 cpm/lg with [a-32PldCTP(Amersham) by the method of Feinberg and Vo-gelstein40 and hybridized as described by Church andGilbert4' at 650C overnight using random primer label-ing kits (Boehringer-Mannheim Biochemicals). All hy-bridized blots were washed and autoradiographed as

described previously.39 Linkage analysis was performedusing the LINKAGE (V5.03) program package.42 Two-point LOD scores were calculated for all pairs of loci byusing the MLINK program.42 Multipoint linkage analyseswas performed using the LINKMAP program.42 Three

investigators analyzed the RFLP pattern identified byeach specific DNA probe, independently and in ablinded fashion. The mode of inheritance was assumedto be X linked. Penetrance was considered to be 100%,but analysis was performed at values of 90% and 95%for stringency. The disease prevalence was assumed to

TABLE 2. Clinical Evaluation of X-Linked Cardiomyopathy:Blood and Tissue Analysis

XLCM-1 XLCM-2

Viral titers Normal NormalToxoplasmosis titers Normal NormalThyroid function studies Normal NormalSerum iron Normal NormalTotal iron-binding capacity Normal NormalSerum carnitine Normal NormalFree muscle carnitine NA IncreasedTotal muscle carnitine NA IncreasedGlycogen content NA Increaseda-Glucosidase NA NormalDebrancher enzyme NA NormalPhosphorylase b kinase NA NormalPhosphorylase NA NormalPhosphofructokinase NA NormalMitochondrial content Normal NormalComplete blood count Normal NormalSedimentation rate Normal NormalAntinuclear antibody Normal NormalRheumatoid factor Normal Normal

XLCM, X-linked cardiomyopathy; NA, not assessed.

XLCM- 1



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Towbin et al Linkage of X-Linked Cardiomyopathy to DMD

FIGURE 2. Approach utilized for linkage ofX-linked cardiomyopathy (XLCM) (shown for XLCM-1). Panel A: AutoradiogramofXLCM-1 digested with the restriction enzyme TaqI and hybridized to XJ1.1 (DXS206). Allele 1 (Al) =3.8 kb; allele 2 (A2) =3.1kb. Note that all affected patients (male and female) carry the A2 allele. Alleles obtained in both families are shown in Figure 1.Panel B.: Dinucleotide repeatpolymorphism, intron 44. This (CA) repeatpolymorphism generates five alleles. The largest allele isdesignated as "0, " and the others denote the number ofCA units smaller than allele "0. "Note that allele -3 comigrates with affectedstatus. Panel C: Dinucleotide repeatpolymorphism, intron 45. This (CA) repeatpolymorphism generates six alleles, with the largestallele ("0") cosegregating with the affected status. Panel D: MaeIII digestedpolymerase chain reaction (PCR) polymorphism. ThisPCR polymorphism lies in the 5' region of the dystrophin gene at the pERT 84 locus, within 30 kb of the promoter region ofdystrophin. The largest band, 841Q-, a 236-bp fragment, comigrates with the diagnosis of XLCM. 841Q+ =128 bp+108 bpfragments.

be 1:10,000 population. Allele frequency for each probeis shown in the "Appendix."

Multiplex PCR was performed in an automated ther-mal cycler (Perkin-Elmer Cetus) for 25 cycles using themethod described by Chamberlain et al.43 Polymor-phisms were generated by PCR in the dystrophin geneusing the protocols described by Roberts et a131 forMaeIII restriction site polymorphisms near the dystro-phin promoter, by Oudet et a130 for 3' DMD polymor-phisms, and Weber and May28 for other dinucleotiderepeat polymorphisms. The intron 44 and 45 microsat-ellite repeats within the dystrophin gene" were ampli-fied by PCR using 75 ng genomic DNA template per 15,uL reaction. The intron 45(CA)n was internally labeledwith radioactive dCTP. Final nucleotide concentrationswere 200 ,uM dATP, dTTP, and dGTP and 5 gM colddCTP plus 0.66 lCi a-dCTP32 per 15 iLL reaction. TheCA strand of the DMD intron 45 microsatellite wasselectively labeled by adding yATP end-labeled primerto the reaction at a concentration of 0.2 uM. The finalconcentration of both primers was 0.25 ,uM. LabeledPCR products were run on a 6% denaturing polyacryl-amide gel, fixed in 7% acetate acid and methanol, anddried on a BioRad gel dryer before being exposed tofilm for 48 hours at -700C.

Immunoblotting with antibody to dystrophin was per-formed by the method of Hoffman et al6,45 and Bulman

et a146 using 6% polyacrylamide gels. Total protein andmyosin concentration in each lane was evaluated usingPonceau-S (Sigma Chemical) staining of the immuno-blots as described previously.46 Dystrophin antibodyraised in sheep to a TrpE-dystrophin fusion protein(exons 4-16), detecting the N-terminal portion of dys-trophin or C-terminal antibody recognizing the final 17amino acids of dystrophin,46 was incubated with West-ern blots47 of total protein obtained from 50 mg ofcardiac and 50 mg of skeletal tissue from individualsaffected with XLCM as well as from control patientswith HCM, DCM, and DMD. Paraffin blocks of cardiacand skeletal muscle from affected individuals were usedwhen no other samples were available. An estimate ofthe relative proportions of different bands was obtainedby scanning immunoblots with an LKB Ultrascan lasermicrodensitometer.46 The entire blot was scanned, andeach immunoreactive dystrophin band was analyzed inthree dimensions (X, Y, density). Volume integrationwas performed after first subtracting background sur-rounding the band to be quantitated. Standard curves ofWestern blots containing a dilution series (10-50 gg ofnormal skeletal muscle protein) were analyzed to en-sure linearity of densitometric measurements. The ab-sorbance of the dystrophin band from the normalcontrol was compared with that of each patient on thesame blot. The relative abundance of patient dystrophin

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TABLE 3. Pairwise LOD Scores Between XLCM and DNA Markers (X Chromosomal)

00 0.001 0.01 0.02 0.03 0.04 0.05 0.10 0.20 0.30 0.40

XLCM-1 versus probe99-6 (DXS41) -99.00 -2.36 +0.35 +0.65 +0.76 +0.85 +0.91 +1.03 +0.92 +0.63 +0.26

P20 (DXS269) -99.00 -0.02 +0.94 +1.19 +1.31 +1.38 +1.43 +1.45 +1.17 +0.74 +0.27

Intron 45 PCR +1.64 +1.22 +1.04 +0.83 +0.67 +0.52 +0.42 +0.33 +0.30 +0.27 +0.16Intron 44 PCR +1.98 1.58 +1.32 +1.17 +1.09 +0.94 +0.88 +0.73 +0.68 +0.59 +0.45

pERT 87-30 -99.00 -1.83 -0.84 -0.56 -0.40 -0.30 -0.22 -0.02 +0.07 +0.05 +0.02

XJ2.3 (DXS206) +2.68 +2.67 +2.63 +2.55 +2.50 +2.45 +2.34 +2.28 +1.70 +1.08 +0.50XJ1.1 (DXS206) +2.91 +2.91 +2.77 +2.64 +2.60 +2.57 +2.48 +2.23 +1.88 +1.22 +0.55XJ1.2 (DXS206) +2.61 +2.60 +2.57 +2.52 +2.48 +2.43 +2.39 +2.15 +1.63 +1.04 +0.43

pERT84-10 (DXS142) +2.48 +2.48 +2.46 +2.44 +2.40 +2.35 +2.31 +2.07 +1.55 +0.98 +0.41

9-7 (DMD cDNA 1-2a) +2.05 +1.98 +1.86 +1.62 +1.31 +1.14 +0.85 +0.68 +0.43 +0.27 +0.09MaeIII +2.18 +2.09 +1.92 +1.60 +1.36 +1.17 +1.03 +0.81 +0.67 +0.59 +0.38754 (DXS84) -99.00 -2.72 -1.86 -1.52 -1.32 -1.17 -1.05 -0.66 -0.28 -0.10 -0.01

OTC (HU731) -99.00 -5.25 -3.15 -2.47 -2.06 -1.77 -1.54 -0.86 -0.28 -0.06 +0.01

XLCM-2 versus probe99-6 -99.00 -1.66 -0.42 -0.13 -0.05 +0.11 +0.14 +0.17 +0.12 +0.10 +0.06P20 -99.00 -0.57 -0.20 -0.04 +0.34 -0.39 -0.43 -0.52 +0.48 +0.26 +0.11

Intron 45 PCR +0.62 +0.80 +0.78 +0.74 +0.70 +0.64 +0.58 +0.50 +0.39 +0.21 +0.08Intron 44 PCR +0.86 +0.82 +0.78 +0.74 +0.72 +0.68 +0.61 +0.56 +0.45 +0.30 +0.18pERT 87-30 -99.00 -1.27 -0.93 -0.81 -0.66 -0.45 -0.41 -0.28 -0.22 -0.19 -0.03XJ2.3 +1.18 +1.16 +1.15 +1.12 +1.09 +0.95 +0.92 +0.85 +0.79 +0.64 +0.50XJ1.1 +1.42 +1.40 +1.35 +1.28 +1.22 +1.17 +1.15 +1.06 +0.98 +0.79 +0.68XJ1.2 +1.16 +1.10 +1.08 +1.04 +0.98 +0.92 +0.86 +0.81 +0.76 +0.64 +0.48pERT 84-10 +1.04 +0.98 +0.94 +0.89 +0.82 +0.77 +0.70 +0.63 +0.58 +0.49 +0.409-7 +0.83 +0.78 +0.72 +0.67 +0.60 +0.51 +0.48 +0.39 +0.32 +0.25 +0.18MaeIII PCR +0.80 +0.76 +0.72 +0.66 +0.58 +0.52 +0.48 +0.36 +0.30 +0.22 +0.16754 -99.00 -1.70 -0.95 -0.80 -0.64 -0.38 -0.25 -0.17 -0.09 -0.05 -0.01OTC -99.00 -2.01 -1.65 -1.28 -1.17 -1.05 -0.88 -0.77 -0.59 -0.37 -0.08

XLCM, X-linked cardiomyopathy; PCR, polymerase chain reaction.

was recorded as a percentage of the absorbance in thenormal control.

ResultsPedigreesXLCM-1, the truncated 63-member pedigree with

X-linked DCM initially described by Berko and Swift,4is shown in Figure 1 (top). Affected males and mani-festing female carriers are denoted. Total CK andMM-CK were elevated in all affected males and somemanifesting carrier females as previously reported4 (Ta-ble 1). XLCM-2, the second pedigree evaluated (Figure1, bottom), had clinical findings similar to the largerfamily, XLCM-1. All males presented in their late teensor early 20s with signs of congestive heart failure andechocardiograms consistent with DCM. Manifestingcarrier females had later onset and less severe symp-toms. All affected males and obligate carrier females inXLCM-2 had CK measured; each had abnormally ele-vated MM-CK (Table 1). No abnormalities were foundin any other blood studies in either family, as seen inTable 2. All phenotypic criteria for affected males andcarrier females were described before establishing geno-types, and no phenotypes were changed after genotypeswere obtained.

Southern Analysis and PCR PolymorphismsAll probes used have previously been mapped, and

their order on the X-chromosome was known. The

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LOCUS (Distance In Morgans)

FIGURE 3. Plot of LOD score versus locus (in Morgans):multipoint analysis. Plot of LOD scores for the multipointanalysis ofX-linked cardiomyopathy versus probes 754, pERT84-10, XJ1.2, XJJ.1, and pERT 87-30. The peak LOD scoreof +4.81 is seen at XMi.] (DXS206) in the 5' portion of thedystrophin gene.



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FIGURE 4. Xp21 region map including Duchenne muscular dystrophy (DMD) locus, flanking markers, and LOD scoresgenerated for pedigrees X-linked cardiomyopathy (XLCM)-1 and XLCM-2. Top: Scale of the DMD gene, -2 mb. Middle:Genomic region of Xp21 with DMD and flanking markers, exon positions of several probes, and LOD scores obtained withtwo-point and multipoint analyses ofXLCM-1 and XLCM-2. Bottom: 14-kb DMD cDNA, the portions used as probes, andLODscores generated with DMD cDNA probes.

intronic probe XJ1.1 (DXS2O6) located in the Xp21.2region of the short arm of the X chromosomes89 withinthe proximal (5') portion of the DMD gene between

exon 7 and exon 8 provided evidence of linkage with acombined LOD score of +4.33 at 0=0 using two-pointlinkage. In both families, autoradiographs of the XJ1.1

FIGURES. Multiplexpolymerase chain reaction (PCR) analysis ofpatients with X-linked cardiomyopathy (XLCM). Left: Nine-primerpair multiplex method ofChamberlain et a143 demonstrates no deletions in three affected males (IV-11, IV-12, III-12), three manifestingfemale carriers (II-2, 1-5, 11-9), one unaffected male (III-2), and one unaffected female (II-6). An unaffected control individualdemonstrates the expected bands, and a patient with glycerol kinase deficiency (GKD) demonstrates deletion of exon 51. Right: Mapof the nine-primer pair multiplex primers and the dystrophin fragment sizes that are expected to be amplified.

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FIGURE 6. Multiplexpolymerase chain reaction (PCR) ofpatients with X-linked cardiomyopathy (XLCM). Left: Five-primerpairmultiplex PCR method of Beggs et als5 using the same patients as in Figure 5. No deletions are seen in any XLCM-i patients orcontrol individuals. The patient with glycerol kinase deficiency (GKD) is deleted in exons 50 and 52. Right: Map of the five-primerpair multiplex primers and the dystrophin fragment sizes expected to be amplified.

polymorphism demonstrated that the 3.1-kb band (al-lele 2) was observed in all affected males and manifest-ing carrier females, whereas no unaffected males pos-sessed this allele (Figure 2A). Other probes in the XJ(DXS206) series (XJ1.2, XJ2.3) also gave LOD scores>2.0 at 0=0 (Table 3). Further support for linkage wasprovided by the proximal 5' DMD cDNA 1-2a9-7 andpERT 84-10 (DXS142) (Table 3). Multipoint linkageusing probes 754 (DXS84), pERT 84-10, XJ1.1, XJ1.2,and pERT 87-30 (DXS164) demonstrated a combinedmaximum LOD score of +4.81 (Table 3 and Figure 3).The only recombinations noted with markers in thedystrophin region were with pERT 87-30 (DXS164) andmarkers distal, including P20 (DXS269) and J66H1(DXS268), suggesting that the mutation causing XLCMlies centromeric to these markers. It is well describedthat the DXS164 region of the dystrophin gene is a hotspot for recombination48 and therefore this is not asurprising finding. (CA)n polymorphisms lying approx-imately 12 kb 3' of dystrophin exon 44 or 1 kb 3' of exon45 yielded LOD scores of > +2.0 for intron 44 andintron 45 at 0=0 (Table 3). The intron 44 microsatelliterepeats displayed five alleles in this family that differ bytwo nucleotides each (Figure 2B). Intron 45 microsat-ellite repeats displayed six alleles differing by twonucleotides each (Figure 2C). The LOD scores gener-ated by an MaeIII polymorphism localized near thedystrophin gene promoter, within 30 kb of this region at

Xp21.2, also supported linkage (Figure 2D and Table3). Probes flanking the dystrophin region and thoselocated at other portions of the short arm (Xp) or longarm (Xq) of the X chromosome were excluded fromlinkage. A summary of the linkage data and the relationof the probes to the dystrophin region are shown inFigure 4.

Multiplex PCRUsing the nine-primer pair PCR method described

by Chamberlain et al,43 which amplifies areas of thedystrophin gene likely to contain mutations, no dele-tions were demonstrated (Figure 5). The primers usedin this method detect approximately 85% of all dele-tions in the dystrophin gene.49 The addition of thefive-primer pairs described by Beggs et al50 allows fordescription of approximately 98% of all known dele-tions; no deletions occurred (Figure 6) using all 14primer pairs, therefore ruling out a known deletion asthe cause of disease.

Western BlotImmunoblots were stained for total protein in each

sample lane using Ponceau-S. Total protein and myosinstaining was compared in all lanes, with equal concen-trations of protein and myosin shown (Figures 7 and 8).Utilizing the N-terminal dystrophin antibody, low abun-dance or absence of dystrophin was shown in cardiac



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Towbin et al Linkage of X-Linked Cardiomyopathy to DMD

FIGURE 7. Western blot of total cardiac protein obtained from members of both X-linked cardiomyopathy (XLCM)-1 andXLCM-2 utilizing C-terminal and N-terminal dystrophin antibody and Ponceau-S staining of total protein and myosin. Panel A:XLCM cardiac muscle versus C-terminal dystrophin antibody (which recognizes the final 17 amino acids of dystrophin). Thenormal 427-kd dystrophin band is seen in normal mouse control as well as in control patients with dilated cardiomyopathy(DCM), and hypertrophic cardiomyopathy (HCM). Deleted dystrophin protein bands are seen in the mdx mouse andpatient withDuchenne muscular dystrophy (DMD). A normal dystrophin band is seen in the XLCMpatients from both families (XLCM-1,IV-12; XLCM-2, 1I-2, and II-7). Myosin staining is shown to be equivalent in all lanes. Immunoreactive bands between dystrophinand myosin probably represent degradation products ofdystrophin. Panel B.: XLCM cardiac muscle versus N-terminal dystrophinantibody raised to the region including exon 4-16 of dystrophin. Using the same patients shown in panel A, the normal 427-kddystrophin band is again seen in normal mouse and control individuals. Low abundance or absent staining is seen in XLCMpatients from both families (XLCM-1-IV-12; XLCM-2, II-2 and II-7), and absent staining is seen in mdx mouse. Myosin stainingis again shown in all lanes. Immunoreactive bands between dystrophin and myosin probably represent degradation products ofdystrophin.

tissue from patients with XLCM, whereas the unrelatedcontrol samples demonstrated the normal 427-kd dys-trophin protein bands (Figure 7). Skeletal muscle sam-ples from the same individuals demonstrated normalstaining of the 427-kd dystrophin band in affected andcontrol samples. Negative control human DMD andmdx mouse skeletal muscle had no staining with thisantibody. C-terminal antibody immunoblotted to car-diac and skeletal muscle protein demonstrated normalstaining of the 427-kd dystrophin band in normal con-trol and XLCM individuals; mdx mouse skeletal musclehad no staining with this antibody (Figure 8). Theseresults were reproducible using tissue from members ofboth XLCM-1 and XLCM-2. Densitometry confirmedthese results and demonstrated equal protein loading inall lanes. Although frozen tissue is preferred, fixedtissue gave identical results.

DiscussionThe gene for XLCM has been mapped in this report

to the 5' portion of the dystrophin gene at the Xp21.2region in two families (Figure 4). Dystrophin has beenshown to have significantly decreased concentration incardiac muscle of affected individuals in these families,

but it is present in normal quantity in their skeletalmuscle. Dystrophin, the muscle protein that is altered orabsent in patients with DMD and BMD, is normallydistributed in equal abundance in skeletal and cardiactissue.6 In addition to skeletal muscle weakness, a DCMis found consistently in patients with these musculardystrophies. In patients with XLCM, however, no weak-ness attributable to, and no histological evidence of,skeletal muscle dystrophy is seen. Elevations in CK-MMhas been seen in affected males and manifesting femalecarriers with XLCM and probably is evidence of abiochemical abnormality of the skeletal muscle in thesepatients.The cardiac involvement seen in DMD, which primar-

ily targets the posterobasal and contiguous left ventric-ular wall, generally manifests late in the course of thedisease and usually is life threatening.36 Although themajority of affected patients with DMD or BMD aremales, between 5% and 10% of known female carriersof a gene for DMD and BMD show signs of skeletalmuscle weakness.51Because the disease phenotype in XLCM differs

from that seen in either DMD or BMD (i.e., cardio-myopathy with no skeletal disease), it most likely is

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FIGURE 8. Western blot of total skeletal protein obtained from members of both X-linked cardiomyopathy (XLCM)-1 andXLCM-2 utilizing C-terminal and N-terminal dystrophin antibody and Ponceau-S staining of total protein and myosin. Panel A:XLCM skeletal muscle versus C-terminal dystrophin antibody. The 427-kd dystrophin band is seen in control lanes (dilatedcardiomyopathy [DCM], hypertrophic cardiomyopathy [HCMJ) and is in low abundance or absent in Duchenne musculardystrophy (DMD) and mdx mouse lanes. Normal staining is seen in the XLCM patients (XLCM-1, IV-12; XLCM-2, II-2, andII-7). Myosin staining is equivalent in all lanes. Immunoreactive bands between dystrophin and myosin probably representdegradation products of dystrophin. Panel B: XLCM skeletal muscle versus N-terminal dystrophin antibody. Normal staining ofthe 427-kd dystrophin band in the same XLCMpatients as seen in panelA and in control lanes are demonstrated. The dystrophinband is absent in theDMD and mdx mouse lanes. Equivalent staining ofmyosin is seen in all lanes. Immunoreactive bands betweendystrophin and myosin probably represent degradation products of dystrophin.

that a specific defect is required to produce thisdisorder. Patients with BMD typically have mild skel-etal myopathy but may have severe, even lethal, car-diomyopathy.52 Occasionally, these patients undergocardiac transplantation53 and continue to do well aftersurgery with only slowly progressive skeletal muscledisease. In many of the BMD patients, abnormalitiesof the 5' end of the dystrophin gene have beendemonstrated.53-55These two families with XLCM demonstrate the

mildest skeletal muscle form of the BMD-DMD pheno-type spectrum seen thus far. They exhibit no clinicalskeletal muscle involvement and mild biochemical fea-tures of skeletal disease while having marked cardiomy-opathy. This disorder could be due to 1) a 5' mutationin the coding sequence of the dystrophin gene that, inturn, affects cardiac muscle function preferentially; 2) aspecific mutation in an unknown cardiac promoter thatcauses this preferential cardiac muscle involvementwithout skeletal muscle dysfunction; 3) alternative splic-ing in the 5' region of dystrophin, which creates anabnormal protein product leading to cardiac dysfunc-tion; or 4) a rare or new deletion in a critical cardiacregion of the dystrophin gene not readily evaluated bythe analyses described, which creates a disease-causingprotein abnormality. Another possibility, that a DCM-causing gene is present in a large intron at the 5' end of

the DMD region or in the 5' upstream sequence,appears to be unlikely, based on our dystrophin data.Interestingly, a 5' point mutation causing cardiomyop-athy with minimal skeletal myopathy has been found inthe mdx mouse.54-56Other familial DCMs with X-linked inheritance have

been reported, but clinical and biochemical differencesexist when compared with XLCM. The initial report ofthis type appeared in 1979 when Neustein et al57reported a pedigree with X-linked recessive inheritance,DCM, and abnormal cardiac mitochondria. Later,Barth and coworkers58 reported a family with X-linkedinheritance, DCM, neutropenia, and abnormalities ofcardiac and skeletal mitochondria; recently, the map-ping of this disorder was localized to Xq28 by cosegre-gation analysis.59 Because Xq28 is the region in whichEmery-Dreifuss muscular dystrophy (EDMD) is found,we would propose that this cardioskeletal myopathypossibly is another phenotype within an EDMD spectralarray similar to that which we have shown for XLCMand DMD-BMD.One third of patients with DMD or BMD are appar-

ent "sporadic" cases due to new mutations in thedystrophin gene.60 It is likely that some sporadic cases ofXLCM also represent new mutations in the 5' region ofthe dystrophin gene. Immunoblotting with dystrophinantibody against total protein extracts from cardiac



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tissue obtained from endomyocardial biopsy or ex-planted hearts could be used to identify males whoseDCM is due to a mutation in the dystrophin gene. Atpresent, detection of such a mutation in females wouldrequire identifying it in the affected female's DNA. Theaddition of MM-CK measurements in both males andfemales with DCM and no skeletal myopathy could alsobe used as presumptive evidence for XLCM, leading tofurther workup of this disorder. In the pedigrees re-ported herein, concurrence between the clinical presen-tation and the diagnosis of genetically affected males,manifesting carrier females, and presently unaffectedcarrier females is seen. With these results, prediction of

overt manifestation of cardiac disease in thus-far-unaf-fected young males and carriers can be considered.Furthermore, prenatal diagnosis for XLCM is now

possible in these families. In addition to potentiallyleading to the diagnosis of a dystrophin abnormality insome sporadic cases of DCM, it is possible that thesefindings will lead ultimately to a specific therapy for thistype of DCM.61 62

AppendixX-Chromosome Probes Used for Linkage Analysis ofX-Linked Cardiomyopathy

A. Xp Genomic Probes

Physical Insert Allele AlleleLocus Probe location size (kb) Enz size (kb) frequencyDXS41 99-6 Xp22.1 1.7 PstI 22,13 0.56,0.44DXS269 P20 Xp21.2 2.65 EcoRV 7.5,7.0 0.40,0.60

MspI 6.0,3.5 0.60,0.40DXS270 JBir Xp21.2 1.1 BamHI 21,5 0.21,0.79DXS164 pERT 87-30 Xp21.2 1.8 BglII 8,30 0.37,0.63

87-15 1.5 BamHI 9.4,7.1/2.3 0.38,0.6287-8 1.3 TaqI 2.7/1.1,3.8 0.74,0.2687-1 1.35 BstNI 3.1,2.5/0.6 0.63,0.37

XmnI 8.7,7.5 0.66,0.34DXS206 XJ2.3 Xp21.2 1.1 TaqI 6.4,7.8 0.70,0.30

XJ1.1 1.0 TaqI 3.8,3.1 0.28,0.72XJ1.2 0.6 BclI 2.0,1.7 0.70,0.30

DXS142 pERT 84-10 Xp21.1 2.5 TaqI 4.5,2.8 0.83,0.17DXS84 754 Xp21.1 2.2 PstI 12,9 0.62,0.38OTC HU731 Xp21.1 1.2 BamHI 18,5.2 0.75,0.25

MspI 6.6,6.2 0.61,0.39pOTC Xp21.1 BamHI 5.1,4.4 0.73,0.27

16,14 0.75,0.25B. DMD cDNA Probes

Physical Insert Allele AllelecDNA Probe Location size (kb) enz size (kb) frequency1-2a 9-7 Xp21.3-21 1.5 HindIII 8.3,7.5 0.10,0.90

BglII 23,8.2 0.71,0.29TaqI 3.4,3.2 0.26,0.74

C. PCR Polymorphisms

Physical Insert AllelePCR Probe location size (kb) Enz Allele size (kb) frequencyMaeIII (Pm) Xp21.1 MaeIII 0.128,0.108(841Q+) 0.24

0.236(841Q-) 0.76Intron 44 (CA)n Xp21.2 0=0.204 0.158

- 1=0.202 0.035-3=0.198 0.018-7=0.190 0.193-10=0.182 0.053

Intron 45 (CA)n Xp21.2 0=0.184 0.018-2=0.180 0.035-3=0.176 0.035-7=0.168 0.035-8=0.166 0.085-9=0.164 0.058

DMD, Duchenne muscular dystrophy; PCR, polymerase chain reaction.



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AcknowledgmentsThe authors wish to thank Bob Grier, PhD, for providing the

tissue specimens and biochemical analyses for XLCM-2; PeterRay, Ronald Worton, and Dennis Bulman for dystrophinantibodies and DXS206 probes; and David Barker for RX andQST probes. Joel Perlman provided assistance with dystrophinanalysis and helpful discussions. Excellent secretarial supportwas provided by Derrellyn Yates and Melba Koegele.

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J A Towbin, J F Hejtmancik, P Brink, B Gelb, X M Zhu, J S Chamberlain, E R McCabe and M Swiftmuscular dystrophy (dystrophin) gene at the Xp21 locus.

X-linked dilated cardiomyopathy. Molecular genetic evidence of linkage to the Duchenne

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