A point mutation in the 5' splice site of the dystrophin gene first intron responsible for X-linked dilated cardiomyopathy
A point mutation in the 5 ' splice site of the dystrophin gene first intron responsible for X-linked dilated cardiomyopathyJelena Milasin1, Francesco Muntoni2, Giovanni Maria Severini1, Lucia Bartoloni3, Matteo Vatta1, Maja Krajinovic1, Anna Mateddu6, Corrado Angelini3, Fulvio Camerini4, Arturo Falaschi1, Luisa Mestroni1,4 and Mauro Giacca1,* and the Heart Muscle Disease Study Group+,4,5
1International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy, 2Department of Pediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0NN, UK, 3Department of Biology and Neurology, University of Padova, 35121 Padova, Italy, 4Departments of Cardiology and 5Morbid Anatomy, Ospedale Maggiore and University of Trieste, 34100 Trieste, Italy and 6Department of Neuroscience, University of Cagliari, 09100 Cagliari, Italy
Received July 24, 1995; Revised and Accepted September 29, 1995
X-linked dilated cardiomyopathy (XLDC) is a familial heart disease presenting in young males as a rapidly progressive congestive heart failure, without clinical signs of skeletal myopathy. This condition has recently been linked to the dystrophin gene in some families and deletions encompassing the genomic region coding for the first muscle exon have been detected. In order to identify the defect responsible for this disease at the molecular level and to understand the reasons for the selective heart involvement, a family with a severe form of XLDC was studied. In the affected members, no deletions of the dystrophin gene were observed. Analysis of the muscle promoter, first exon and intron regions revealed the presence of a single point mutation at the first exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the first intron. This mutation introduced a new restriction site for MseI, which cosegregates with the disease in the analyzed family. Expression of the major dystrophin mRNA isoforms (from the muscle-, brain- and Purkinje cell-promoters) was completely abolished in the myocardium, while the brain- and Purkinje cell- (but not the muscle-) isoforms were detectable in the skeletal muscle. Immunocytochemical studies with anti-dystrophin antibodies showed that the protein was reduced in quantity but normally distributed in the skeletal muscle, while it was undetectable in the cardiac muscle. These findings indicate that expression of the muscle dystrophin isoform is critical for myocardial function and suggest that selective heart involvement in dystrophin-linked dilated cardiomyopathy is related to the absence, in the heart, of a compensatory expression of dystrophin from alternative promoters.
The dystrophin gene spans a ~3 megabase region of chromosome Xp21 and consists of at least 79 exons. The gene is characterized by a very complex developmental- and tissue-dependent regulation (1 -4 ). It encodes for a high molecular weight cytoskeletal protein expressed primarily in skeletal and cardiac muscle and, to a lesser extent, in smooth muscle and brain tissue (5 ). Different isoforms are produced by alternative splicing events and by transcription initiating from at least seven distinct promoters. Three of these promoters, the brain-, muscle- and Purkinje cell-promoters, generate full length transcripts of 14 kb (reviewed in ref. 6 ).
Mutations of the dystrophin gene cause a severe degenerative disease, Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD). Deletions are the most common type of mutations responsible for both DMD and BMD (~65% of the cases). Almost invariably, the deletions found in DMD patients predict an out-of-frame and grossly altered product, while those found in BMD patients maintain the correct reading frame and lead to the production of a partially functional protein, often also reduced in quantity (7 ). In addition to skeletal muscle involvement, a myocardial involvement is often present in both conditions.
Recently, it has been suggested that specific dystrophin defects could be responsible for a familial form of dilated cardiomyopathy, X-linked dilated cardiomyopathy (XLDC). This is a progressive heart disease, presenting generally with heart failure in young males in the absence of overt signs of skeletal myopathy. From the clinical standpoint, the disease in these patients does not differ from that found in other forms of familial dilated cardiomyopathy with different patterns of inheritance (8 ,9 ). In two kindred with XLDC, Towbin et al. (10 ) established linkage between the centromeric portion of the dystrophin gene and XLDC, but failed to find any specific defect in the gene. Evidence supporting the involvement of this gene was brought up by Muntoni et al. (11 ), who reported about a large deletion in the 5' portion of the muscle isoform region, removing the muscle promoter and the first muscle exon. Similar findings were observed also by Yoshida et al. (12 ), who described a deletion in the first muscle exon and the 5' portion of the first intron in two unrelated cases of XLDC.
In order to define the molecular basis of the disease, and to test the hypothesis of a 5' end dystrophin gene defect in the pathogenesis of selective heart involvement, a clinical, immunohistochemical and molecular genetic study was carried out in a family with a severe form of XLDC.
In 1987, a 24 year old male was admitted to the Cardiology Department in Trieste, Italy for a severe heart failure developed within the preceding 6 months. The diagnosis was idiopathic dilated cardiomyopathy. He was completely free of any clinical and laboratory sign of skeletal muscle disease, including increased serum creatine kinase (CK) level. Additionally, he had been a competitive basketball player for several years. He died 2 years later from post-surgical complications after heart transplantation. After several years, his brother was examined because of atypical chest pain and found to have a severely dilated and hypokinetic left ventricle. The levels of the MM isoform of creatine kinase (MM-CK) were moderately increased, without clinical evidence for muscle disease and with normal electromyography, despite some reported episodes of urine pigmentation after physical exercise and sporadic myalgias. These observations prompted us for a careful examination of the other family members. The pedigree of this family is reported in Figure 1 A (the proband was individual II-1 and his brother II-2) and some of the relevant clinical and immunohistological findings of the affected and unaffected family members are reported in Table 1 . The proband's mother (I-1) and the second brother (II-3) were found to be normal at physical examination, electrocardiography and echocardiography. Individual II-4 died at birth for unknown causes and individual I-2 died for a non-cardiac disease. The reported history of first- and second-degree relatives was completely negative.
Analysis of dystrophin expression was performed in left ventricular endomyocardial and skeletal muscle biopsies obtained from individual II-2.
Histological examination of the skeletal muscle biopsy showed a mild variability in fiber size with the presence of mildly scattered atrophic fibers, rare splittings and no increase in interstitial connective tissue. The number of internal nuclei was slightly augmented. At immunocytochemistry with antibodies directed toward the N-terminus, mid-rod and C-terminus of dystrophin, all fibers appeared continuously labelled, but the intensity of fluorescent labelling was paler than in control muscle (Fig. 3 A-C).
The present study reports the identification of a splice-site point mutation at the 5' end of the dystrophin gene, responsible for dilated cardiomyopathy in a family. Our findings, in agreement with other studies (10 -12 ), indicate a specific involvement of the 5' end of the dystrophin gene in determining isolated heart `dystrophy'. It should be further stressed that the phenotype examination of the two affected family members did not reveal signs of skeletal muscle disease and, unlike all previously reported cases (10 -12 ), the proband (II-1) had constantly normal creatine kinase values. On the other hand, clinical investigations were indicative of a severe myocardial dilatation and dysfunction.
Standard multiplex PCR method (16 ,17 ) failed to detect any typical deletion of the dystrophin gene in these patients. However, the selective involvement of the dystrophin gene 5' end was strongly suggested by the finding of a critical recombination in the unaffected subject II-3, detected by haplotype analysis. Sequence analysis of the DNA fragment corresponding to the first muscle exon-intron junction demonstrated a single base substitution. This mutation replaces the invariant G at the position +1 of the splice donor sequence G100 T100 A/G95 A70 G80 T45 [where the numbers indicate the percentage of nucleotide conservation (18 )], with a T. Since the G at position +1 of the 5' splice site consensus sequence is completely conserved in eukaryotes, this mutation is undoubtedly affecting mRNA maturation. The abolition of the 5' splice site of the very large first intron [>200 kb (21 )] predicts the production of an unstable mRNA, with consequent absence of the muscle isoform expression. The utilization of an alternative cryptic splice site located at a downstream position of the first intron could not be detected by RT-PCR amplification, neither in heart nor in muscle samples, using a pair of primers located in the first and second exons. Although this does not exclude that an aberrant mRNA is produced longer than the maximum PCR amplification limit, this would be expected to lead to the production of a grossly altered protein.
The discrepant clinical phenotype of the patients (apparent normal skeletal muscle and selective heart involvement) is consistent with the normal distribution of dystrophin in skeletal muscle biopsy, where only its quantitative reduction could be suggestive of a dystrophinopathy, in contrast to the absolute absence of the protein in the heart. Production of the protein is likely to be sustained in the muscle by translation of the brain- and the Purkinje cell-isoform mRNAs. Thus, it is conceivable that, in the skeletal muscle, the absence of the muscle dystrophin isoform can drive a compensatory overexpression of the other full-length isoforms, able to prevent the myopathy in the affected males. Surprisingly, transcription of these isoforms is absent in the heart, where even the normal expression of the brain mRNA isoform is suppressed. This finding is consistent with the selective cardiac disease in these patients. These observations point to the existence of selective mechanisms of regulation of transcription initiation at the different dystrophin promoters in these two tissues. It is still unclear by which mechanisms the detected single point mutation, that is expected to act primarily at the post-transcriptional level, is able to exert such effects on the dystrophin promoters. Similar findings have been recently reported for another family with XLDC, characterized by a large deletion in the 5' end of the gene (19 ). In this case, it was hypothesized that deletions involving the muscle promoter, the first muscle exon and part of the intron 1, could remove a distinct cardiac enhancer responsible for transcription also from the brain and Purkinje cell promoter (12 ,19 ). However, the present report does not support this idea. In fact, it would be extremely unlikely that a putative cardiac specific enhancer is situated at the intron-exon splice site and that a point mutation at this level could completely knock out its function. Thus, the molecular reasons why the compensatory transcription mechanism from the brain and Purkinje cell promoters is occurring in the skeletal muscle but not in the myocardium still remain to be elucidated. Additionally, it is also intriguing to observe that, in other families, the deletion of the first muscle exon of dystrophin is associated with a clear muscular dystrophy phenotype of different severity (20 -23 ). A better understanding of the genetic differences between these cases will await the definition of the exact boundaries of these deletions, of the transcriptional patterns of dystrophin in these patients and the identification of the cis-elements involved in transcriptional control of the different gene isoforms.
In the heart of the patient analyzed in this study, as well as in patients of another family with XLDC (24 ), the only detectable dystrophin mRNA was the one coding for Dp71, indicating that there is no transcriptional interference between the 5' promoters and the Dp71 promoter. Recent published work on transgenic animals indicates that the expression of Dp71 fails to compensate for the absence of the dystrophin in the skeletal muscle (36 ,37 ). These data are in accordance with the presence of a severe cardiac impairment in our XLDC patients despite Dp71 expression in the heart. However, since the mdx mouse does not necessarily represent the best model to study the involvement of dystrophin in heart muscle disease, it is still possible that the presence of Dp71 could play a role in XLDC, partially compensating for the absence of the major isoforms, thus delaying the onset and mitigating the progression of the disease.
In conclusion, the data presented in this work define for the first time a precise point mutation at the 5' end of the dystrophin gene as responsible for a selective dilated cardiomyopathy. Since the most apparent phenotype for this mutation is the absence of the muscle dystrophin isoform mRNA, it appears that expression of this isoform is essential for the heart function. Therefore, it is conceivable that, in addition to splice junction defects, also different types of mutations resulting in altered levels of this mRNA isoform could lead to selective heart myopathy, since its absence can not be compensated in the heart by transcriptional initiation at alternative promoters.
A detailed history of the family was taken and all the family members included in the study were evaluated with physical examination, resting electrocardiogram, M-mode and cross sectional echocardiography, as previously described (8 ). Normal values for echocardiographic measurements were determined according to standard protocols (25 ). Relatives showing signs of cardiomyopathy (II-1 and II-2, Fig. 1 ) underwent a comprehensive clinical evaluation including ventriculography, coronary angiography and endomyocardial biopsy. Patient II-1 underwent also neurological evaluations, including electromyography and skeletal muscle biopsy.
A needle biopsy of skeletal muscle and a left ventricular endomyocardial biopsy were obtained from individual II-2, after informed consent. The samples were studied according to standard techniques (26 ), including immunocytochemistry using a biotin-streptavidin Texas-red method. Six micron unfixed cryostat sections were immunostained using a panel of antibodies to dystrophin (Novocastra Laboratories, Newcastle-upon Tyne, UK), as described (11 ).
Total RNA was isolated from frozen samples according to a published procedure (27 ) and cDNA synthesis was performed using random hexanucleotide primers. PCR was carried out in a reaction volume of 25 [mu]l containing the cDNA template (2 [mu]g) and oligonucleotide primers designed to amplify the muscle isoform of dystrophin (forward oligonucleotide in the first muscle exon and reverse oligonucleotide located in exons 2 or 4) and the Dp71 transcript (19 ,24 ). PCR reactions (25 [mu]l) utilized 0.5 U Taq DNA polymerase,0.25 [mu]M each primer, 200 [mu]M each dNTP, in 10 mM Tris-HCl, pH 8.3, 50 mM KCl and 1.5 mM MgCl2. After 10 min denaturation at 94oC, PCR amplification was carried out with the following cycle profile: denaturation at 94oC for 30 s; annealing at 58oC for 60 s; extension at 72oC for 120 s; 26 cycles. Nine microlitres of the reaction were resolved on 2.5% agarose gels containing 0.2 g/ml of ethidium bromide.
DNA samples from individuals I-1, II-2 and II-3 (Fig. 1 ) were extracted according to the salting out procedure (28 ), either from fresh blood samples or from continuous B-lymphocyte cell lines immortalized with the Epstein-Barr virus (29 ). In the case of the deceased patient II-1, DNA was extracted from a paraffin-embedded myocardial tissue sample (30 ).
The screening for deletions of the dystrophin gene was performed using multiplex PCR, according to methods described elsewhere (16 ,17 ). Amplifications of the first muscle exon, muscle promoter, brain promoter and second exon were carried out as reported (2 ,13 ,21 ,31 ).
For the haplotype analysis, the polymorphic microsatellite sequences (CA repeats) DYS-I, DYS-II, DYS-III, DYS MSB, DYS MSA, DXS 997, DXS 992 were PCR amplified using described conditions (32 -35 ). PCR products were resolved by a long run on a 10-12% polyacrylamide native gel and visualized by ethidium bromide staining.
For point mutations screening, the region including the muscle promoter, the first muscle exon and 250 bp of the first intron (13 ) were amplified by PCR using serial pairs of primers to obtain products of ~100-300 bp. PCR amplifications were carried out in 50 [mu]l of a solution containing Tris-HCl 10 mM (pH 8.0), KCl 50 mM, MgCl2 1.5 mM, gelatin 0.01%, each dNTP 200 mM, each primers 0.1 [mu]M, 200 ng of template DNA and 2.5 U of Taq DNA polymerase (Perkin Elmer, Roche Molecular Systems, Branchburg, NJ), with 40 repetitions of the following cycle: 30 s at 94oC, 30 s at the annealing temperature and 30 s at 72oC. Annealing temperatures varied from 56 to 62oC, according to the different primer pairs. The PCR product encompassing the 3' end of the muscle first exon and the beginning of intron 1 was obtained using the following primers: forward primer, 5'-TATCGCTGCCTTGATATACA-3'; reverse primer, 5'-ACTAAACGTTATGCCACAGT-3', with an annealing temperature of 60oC. All the oligonucleotides were synthesized by the ICGEB Oligonucleotide Synthesis Service on a Applied Biosystem 380B synthesizer.
PCR-SSCP samples were prepared by denaturing 5 [mu]l of PCR product with 7 [mu]l of formamide dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol) at 94oC for 8 min. After cooling in ice, the samples were quickly loaded on 10-20% non-denaturing polyacryamide gels with or without glycerol and run for 12-16 h at room temperature or at 4oC. DNA fragments on SSCP gels were visualized by silver staining using a commercial procedure (BioRad, Richmond, VI).
The PCR amplified fragments were eluted from polyacrylamide gels and directly cloned in a commercial vector (TA Cloning Kit, Invitrogen Corporation, San Diego, CA). Sequence analysis was performed on plasmid DNA extracted from at least three individual bacterial clones by the dideoxinucleotide chain termination method using a T7-based DNA sequencing kit (Pharmacia, Uppsala, Sweden). Sequence data were obtained for both strands of the insert by extension of the universal and reverse primers of the vector, as well as by using internal primers.
Since the mutation found at the 5' splice site of the first dystrophin intron predicts the generation of a new restriction site for MseI, the 110 bp fragment encompassing the mutation was digested with this enzyme to produce two fragments of 60 and 50 bp, that were visualized following electrophoresis on an ethidium bromide-stained 8% polyacrylamide gel.
We are grateful to Maurizio Porcu (Cardiology Div., Ospedale S. Michele, Cagliari, Italy) for examining the individual I-1 and to Pietro Gallo (Morbid Anatomy Institute, University La Sapienza, Rome, Italy) for providing samples from the explanted heart of individual II-1. We are particularly indebted to family members, without whose collaboration this study would not have been possible. This work was supported by grants from the Associazione Amici del Cuore of Trieste, the National Research Council, Italy (CNR 94, 00993.CT04), the Telethon, Italy (Grant no. E.11) and the Muscular Dystrophy Group of Great Britain and North Ireland (grant to F.M.). J.M. is on leave from the Institute of Biology and Human Genetics, School of Stomatology, University of Belgrade, Yugoslavia.
1 Koenig, M., Hoffman, E.P., Bertelson, C.J., Monaco, A.P., Feener, C. and Kunkel, L.M. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell, 50, 509-517.MEDLINE Abstract
2 Koenig, M., Monaco, A.P. and Kunkel, L.M. (1988) The complete sequence of the dystrophin predicts a rod-shaped skeletal protein. Cell, 53, 219-228.MEDLINE Abstract
3 Roberts, R.G., Coffey, A.J., Bobrow, M. and Bentley, D.R. (1992) Determination of exon structure of the distal portion of the dystrophin gene by vectorette PCR. Genomics, 13, 942-950.MEDLINE Abstract
4 Bies, R.D., Phelps, S.F., Cortez, M.D., Roberts, R., Caskey, C.T. and Chamberlain, J.S. (1992) Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart and brain development. Nucleic Acids Res., 20, 1725-1731.MEDLINE Abstract
5 Chelly, J., Kaplan, J.C., Maire, P., Gautron, S. and Kahn, A. (1988) Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature, 333, 858-860.MEDLINE Abstract
6 Ahn, A.H. and Kunkel, L.M. (1993) The structural and functional diversity of dystrophin. Nature Genet., 3, 283-291.MEDLINE Abstract
7 Koenig, M., Beggs, A.H., Moyer, M., et al. (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am. J. Hum. Genet., 45, 498-506.MEDLINE Abstract
8 Mestroni, L., Krajinovic, M., Severini, G.M., Pinamonti, B., Di Lenarda, A., Giacca, M., Falaschi, A. and Camerini, F. (1994) Familial dilated cardiomyopathy. Br. Heart J., 72, 35-41.
9 Mestroni, L., Krajinovic, M., Severini, G.M., Falaschi, A., Giacca, M. and Camerini, F. (1994) Molecular genetics of dilated cardiomyopathy. Herz, 19, 97-104.MEDLINE Abstract
10 Towbin, J.A., Hejtmancik, F., Brink, P., Gelb, B.D., Zhu, X.M., Chamberlain, J.S., McCabe, E.R.B. and Swift, M. (1993) X-linked cardiomyopathy (XLCM): molecular genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus. Circulation, 87, 1854-1865.MEDLINE Abstract
11 Muntoni, F., Cau, M., Ganau, A., Congiu, R., Arvedi, G., Mateddu, A., Marrosu, M.G., Cianchetti, C., Realdi, G., Cao, A. and Melis, M.A. (1993) Deletion of the dystrophin muscle-promoter region associated with X-linked dilated cardiomyopathy. N. Engl. J. Med., 329, 921-925.MEDLINE Abstract
12 Yoshida, K., Ikeda, S.I., Nakamura, A., Kagoshima, M., Takeda, S., Shoji, S. and Yanagisawa, N. (1993) Molecular analysis of the Duchenne muscular dystrophy gene in patients with Becker muscular dystrophy presenting with dilated cardiomyopathy. Muscle Nerve, 16, 1161-1166.MEDLINE Abstract
13 Klamut, H.J., Gangopadhyay, S.B., Worton, R.G. and Ray, P.N. (1990) Molecular and functional analysis of the muscle specific promoter region of the Duchenne muscular dystrophy gene. Mol. Cell. Biol., 10, 193-205.MEDLINE Abstract
14 Hugnot, J.P., Gilgenkrantz, H., Vincent, N., Chafey, P., Morris, G.E., Monaco, A.P., Berwlad-Netter, Y., Koulakoff, A., Kaplan, J.C. and Kahn, A. (1992) Distal transcription of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues. Proc. Natl. Acad. Sci. USA, 89, 7506-7510.MEDLINE Abstract
15 Lederfein, D., Yaffe, D. and Nudel, U. (1993) A housekeeping type promoter, located in the 3' region of the Duchenne muscular dystrophy gene, controls the expression of Dp71, a major product of the gene. Hum. Mol. Genet., 2, 1883-1888.MEDLINE Abstract
16 Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., Nguyen, P.N. and Caskey, C.T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res., 23, 11 141-11 156.
17 Beggs, A.H., Koenig, M., Boyce, F.M. and Kunkel, L.M. (1990) Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet., 86, 45-48.MEDLINE Abstract
18 Horowitz, D.S. and Kreiner, A.R. (1994) Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet., 10, 100-106.MEDLINE Abstract
19 Muntoni, F., Melis, M.A., Ganau, A. and Dubowitz, V. (1995) Transcription of the dystrophin gene in normal tissues and in skeletal muscle of a family with X-linked dilated cardiomyopathy. Am. J. Hum. Genet., 56, 151-157.MEDLINE Abstract
20 Beggs, A.H., Hoffmann, E.P., Snyder, J.R., Arahata, K., Specht, L., Shapiro, F. and Angelini, C. (1991) Exploring the molecular basis for variability among patients with Becker muscular dystrophy: dystrophin gene and protein studies. Am. J. Hum. Genet., 49, 54-67.MEDLINE Abstract
21 Boyce, F.M., Beggs, A.H., Feener, C. and Kunkel, L.M. (1991) Dystrophin is transcribed in brain from a distant upstream promoter. Proc. Natl. Acad. Sci. USA, 88, 1276-1280.MEDLINE Abstract
22 Bulman, D.E., Murphy, E.G., Zubrzycka-Gaarn, E.E., Worton, R.G. and Ray, P.N. (1991) Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin. Am. J. Hum. Genet., 48, 295-304.MEDLINE Abstract
23 Malhotra, S.B., Hart, K.A., Klamut, H.J., Thomas, N.S.T., Bodrug, S.E., Burghes, A.H.M. and Bobrow, M. (1988) Frameshifts deletions in patients with Duchenne and Becker muscular dystrophy. Science, 242, 755-759.MEDLINE Abstract
24 Muntoni, F., Wilson, L., Marrosu, M.G., Cianchetti, C., Mestroni, L., Ganau, A., Dubowitz, V. and Sewry, C. (1995) A mutation in the dystrophin gene selectively affecting dystrophin expression in the heart. J. Clin. Invest., in press.
25 American Society of Echocardiography (1989) Recommendations for quantitation of the left ventricle by two dimentional echocardiography. J. Am. Soc. Echocard., 2, 358-367.
26 Dubowitz, V. (1985). Muscle Biopsy: A Practical Approach. 2nd edn, Bailliere Tindall, Eastbourne.
27 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156-159.MEDLINE Abstract
28 Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res., 16, 1215.MEDLINE Abstract
29 Neitzel, H. (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum. Genet., 73, 320-326.MEDLINE Abstract
30 Jackson, D.P., Hayden, J.D. and Quirke, P. (1991) Extraction of nucleic acids from fresh and archival material. In Mc Pherson, M.J., Quicke, P. and Taylor, G.R. (eds), PCR. A Practical Approach. IRL Press, Oxford, pp. 29-50.
31 Muntoni, F. and Strong, P.N. (1989) Transcription of the dystrophin gene in Duchenne muscular dystrophy muscle. FEBS Lett., 252, 95-98.MEDLINE Abstract
32 Feener, C.A., Boyce, F.M. and Kunkel, L.M. (1991) Rapid detection of CA polymorphisms in cloned DNA: application to the 5' region of the dystrophin gene. Am. J. Hum. Genet., 48, 621-627.MEDLINE Abstract
33 Oudet, C., Heilig, R., Hanauer, A. and Mandel, J.L. (1991) Nonradioactive assay for new microsatellite polymorphisms at the 5' end of the dystrophin gene and estimation of intragenic recombination. Am. J. Hum. Genet., 49, 311-319.MEDLINE Abstract
34 Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc, S., Bernardi, G., Lathrop, M. and Weissenbach, J. (1994) The 1993-94 Genethon human genetic linkage map. Nature Genet., 7, 246-339.MEDLINE Abstract
35 Saad, F.A., Busque, C., Vitiello, L. and Danieli, G. (1993) DXS997 localized to intron 48 of dystrophin. Hum. Mol. Genet., 2, 2199.MEDLINE Abstract
36 Cox, G.A., Sunada, Y., Campbell, K.P. and Chamberlain, J.S. (1994) Dp71 can restore the dystrophin-associated glicoprotein complex in muscle but fails to prevent dystrophy. Nature Genet., 8, 333-338.MEDLINE Abstract
37 Greenberg, D.S., Sunada, Y., Campbell, K.P., Yaffe, D. and Nudel, U. (1994) Exogenous Dp71 restores the level of dystrophin associated proteins but does not alleviate muscle damage in mdx mice. Nature Genet., 8, 340-344.MEDLINE Abstract
*To whom correspondence should be addressed+Heart Muscle Disease Study Group: Bruno Pinamonti, Gianfranco Sinagra, Andrea Di Lenarda, Furio Silvestri, Rossana Bussani and Milla Davanzo
This page is maintained by OUP admin. Last updated Thu Oct 31 15:08:29 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
