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May 20, 1992, Vol 267, No. 19 >
ARTICLE | May 20, 1992

Diagnosis of Duchenne and Becker Muscular Dystrophies by Polymerase Chain Reaction: Title and subTitle BreakA Multicenter Study FREE

[+] Author Affiliations

A complete list of the group's participants and their affiliations appears at the end of the article.

Reprint requests to The Institute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (Dr Caskey).


JAMA. 1992;267(19):2609-2615. doi:10.1001/jama.1992.03480190051030
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References

Objective.  —To assess the efficiency, reliability, and ease of use of DNA diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) using the polymerase chain reaction (PCR).

Design.  —DNA from the patients was screened for deletion mutations using multiplex PCR, and the results were compared with those obtained by Southern blot analysis. The PCR multiplex reaction detects nine specific "hot-spot" exons in the dystrophin gene while the Southern analysis detects 66 specific dystrophin gene restriction fragments. The multiplex reaction requires 50-fold less DNA than Southern analysis and thus is considerably more sensitive.

Setting.  —Fourteen university-affiliated and private genetic disease diagnostic laboratories.

Patients.  —Male patients with clinical signs of DMD/BMD. Cases were selected for analysis randomly, without knowledge of whether a deletion was present within the dystrophin gene.

Main Outcome Measures.  —The percentage of cases that were detectable by multiplex PCR in comparison with Southern analysis, the frequency, extent, and location of the detected deletion mutations. In some cases, duplication mutations were monitored.

Results.  —The accuracy of a single PCR multiplex amplification (nine exons) was compared with Southern analysis with 10 cDNA probes that cover the full length of the gene. The multiplex PCR analytic method detected 82% of those deletions detected by Southern analysis methods. In one of 745 analyses, the multiplex method suggested a single exon deletion, which was not confirmed by Southern analysis, representing a false-positive rate of 0.013%.

Conclusions.  —Multiplex PCR represents a sensitive and accurate method for deletion detection of 46% of all cases of DMD/BMD. The method requires 1 day for analysis, is easy to perform, and does not use radioactive tracers. As such, multiplex PCR represents an efficient and rapid method for prenatal or postnatal diagnosis of DMD/BMD.(JAMA. 1992;267:2609-2615)

REFERENCES

1 +
Chamberlain JS, Caskey CT.  Duchenne muscular dystrophy.  In: Appel SH, ed. Current Neurology, X . Chicago, Ill: Year Book Medical Publishers Inc; 1990;:65-103.
2 +
Witkowski J.  Dystrophin-related muscular dystrophies. J Child Neurol . 1990;;4:251-271.
3 +
Moser H.  Review of studies on the proportion and origin of new mutants in Duchenne muscular dystrophy , In: Ten Kate LP, Pearson PL, Stadhouders AM, eds. Current Clinical Practices, Series 20a . Amsterdam, the Netherlands: Excerpta Medica; 1984;:41-52.
4 +
Emery AEH.  Duchenne muscular dystrophy.  In: Oxford Monography on Medical Genetics, XV . New York, NY: Oxford University Press; 1987;.
5 +
Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM.  Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature . 1986;;323:646-650.
6 +
Burghes AHM, Logan C, Hu X, Belfall B, Worton RG, Ray PN.  A cDNA clone from the Duchenne/ Becker muscular dystrophy gene. Nature . 1987;; 328:434-437.
7 +
Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM.  Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell . 1987;;50: 509-517.
8 +
Hoffman EP, Fischbeck KH, Brown RH, et al.  Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med . 1988;;318: 1363-1368.
9 +
Ward PA, Hejtmancik JF, Witkowski JA, et al.  Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis. Am J Hum Genet . 1989;; 44:270-281.
10 +
Hoffman EP, Kunkel LM, Angelini C, Clark A, Johnson M, Harris JB.  Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology . 1989;;39:1011-1017.
11 +
Bulman DE, Murphy EG, Zubrzycka-Gaarn EE, Worton RG, Ray PN.  Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin. Am J Hum Genet. 1991;;48:295-304.
12 +
Hu X, Ray PN, Murphy EG, Thompson MW, Worton RG.  Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotype/genotype correlation. Am J Hum Genet . 1990;;46:682-695.
13 +
Bakker EB, Bonten EJ, Veena H, et al.  Prenatal diagnosis of Duchenne muscular dystrophy: a three year experience in a rapidly evolving field. J Inherited Metab Dis . 1989;;12:174-190.
14 +
Mullis KB, Faloona FA.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol . 1987;;155:335-350.
15 +
Chamberlain JS,Gibbs RA,Ranier JE,Nguyen PN, Caskey CT.  Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res . 1988;;16:11141-11156.
16 +
Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT.  Multiplex PCR for the diagnosis of Duchenne muscular dystrophy.  In: Innis M, Geltand D, Sninsky J, White T, eds. PCR Protocols: A Guide to Methods and Applications . Orlando, Fla: Academic Press; 1989;:272-281.
17 +
Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT.  Detection of gene deletions using multiplex polymerase chain reactions.  In: Mathew C, ed. Methods in Molecular Biology, IX: Protocols in Human Molecular Genetics . Clifton, NJ: The Humana Press; 1991;:299-312.
18 +
Darras BT, Koenig M, Kunkel LM, Francke U.  Direct method for prenatal diagnosis and carrier detection in Duchenne/Becker muscular dystrophy using the entire dystrophin cDNA. Am J Med Genet . 1988;;29:713-726.
19 +
Koenig M, Beggs AH, Moyer H, et al.  The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet . 1989;;45:498-506.
20 +
Baumbach LL, Chamberlain JS, Ward PA, Farwell NJ, Caskey CT.  Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology . 1989;;39:465-474.
21 +
Den Dunnen JT, Grootscholten PM, Bakker E, et al.  Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet . 1989;;45:835-847.
22 +
Fenwick RG, Ward PA, Alvarez FV.  Mutation and carrier detection for Duchenne muscular dystrophy by quantitative Southern analysis and multiplex PCR analysis. Am J Hum Genet . 1990;;47 ( (suppl) ):A216.
23 +
Abbs S, Yau SC, Clark S, Mathew CG, Bobrow M.  A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridization shows mistyping with both methods. J Med Genet . 1991;; 28:304-311.
24 +
Fujimura FK, Northrup H, Beaudet AL, O'Brien WE.  Genotyping errors with the polymerase chain reaction. N Engl J Med . 1990;;322:61.
25 +
Beggs AH, Koenig M, Boyce FM, Kunkel LM.  Detection of 98% of DMD/BMD deletions by PCR. Hum Genet . 1990;;86:45-48.
26 +
Roberts RG, Cole CG, Hart KA, Bobrow M, Bentley DR.  Rapid carrier and prenatal diagnosis of Duchenne and Becker muscular dystrophy. Nucleic Acids Res . 1989;;17:5961-5971.
27 +
Oudet C, Heilig R, Mandel JL.  An informative polymorphism detectable by polymerase chain reaction at the 3' end of the dystrophin gene. Hum Genet . 1990;;84:283-285.
28 +
Beggs AH, Kunkel LM.  A polymorphic CACA repeat in the 3' untranslated region of dystrophin. Nucleic Acids Res . 1990;;18:1931.
29 +
Clemens PR, Fenwick RG, Chamberlain JS, et al.  Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Genet . 1991;;49:951-960.
30 +
Roberts RG, Barby TFM, Manners E, Bobrow M, Bentley DR.  Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Hum Genet . 1991;;49:298-310.

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1 +
Chamberlain JS, Caskey CT.  Duchenne muscular dystrophy.  In: Appel SH, ed. Current Neurology, X . Chicago, Ill: Year Book Medical Publishers Inc; 1990;:65-103.
2 +
Witkowski J.  Dystrophin-related muscular dystrophies. J Child Neurol . 1990;;4:251-271.
3 +
Moser H.  Review of studies on the proportion and origin of new mutants in Duchenne muscular dystrophy , In: Ten Kate LP, Pearson PL, Stadhouders AM, eds. Current Clinical Practices, Series 20a . Amsterdam, the Netherlands: Excerpta Medica; 1984;:41-52.
4 +
Emery AEH.  Duchenne muscular dystrophy.  In: Oxford Monography on Medical Genetics, XV . New York, NY: Oxford University Press; 1987;.
5 +
Monaco AP, Neve RL, Colletti-Feener C, Bertelson CJ, Kurnit DM, Kunkel LM.  Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature . 1986;;323:646-650.
6 +
Burghes AHM, Logan C, Hu X, Belfall B, Worton RG, Ray PN.  A cDNA clone from the Duchenne/ Becker muscular dystrophy gene. Nature . 1987;; 328:434-437.
7 +
Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM.  Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell . 1987;;50: 509-517.
8 +
Hoffman EP, Fischbeck KH, Brown RH, et al.  Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med . 1988;;318: 1363-1368.
9 +
Ward PA, Hejtmancik JF, Witkowski JA, et al.  Prenatal diagnosis of Duchenne muscular dystrophy: prospective linkage analysis and retrospective dystrophin cDNA analysis. Am J Hum Genet . 1989;; 44:270-281.
10 +
Hoffman EP, Kunkel LM, Angelini C, Clark A, Johnson M, Harris JB.  Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology . 1989;;39:1011-1017.
11 +
Bulman DE, Murphy EG, Zubrzycka-Gaarn EE, Worton RG, Ray PN.  Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin. Am J Hum Genet. 1991;;48:295-304.
12 +
Hu X, Ray PN, Murphy EG, Thompson MW, Worton RG.  Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotype/genotype correlation. Am J Hum Genet . 1990;;46:682-695.
13 +
Bakker EB, Bonten EJ, Veena H, et al.  Prenatal diagnosis of Duchenne muscular dystrophy: a three year experience in a rapidly evolving field. J Inherited Metab Dis . 1989;;12:174-190.
14 +
Mullis KB, Faloona FA.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol . 1987;;155:335-350.
15 +
Chamberlain JS,Gibbs RA,Ranier JE,Nguyen PN, Caskey CT.  Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res . 1988;;16:11141-11156.
16 +
Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT.  Multiplex PCR for the diagnosis of Duchenne muscular dystrophy.  In: Innis M, Geltand D, Sninsky J, White T, eds. PCR Protocols: A Guide to Methods and Applications . Orlando, Fla: Academic Press; 1989;:272-281.
17 +
Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT.  Detection of gene deletions using multiplex polymerase chain reactions.  In: Mathew C, ed. Methods in Molecular Biology, IX: Protocols in Human Molecular Genetics . Clifton, NJ: The Humana Press; 1991;:299-312.
18 +
Darras BT, Koenig M, Kunkel LM, Francke U.  Direct method for prenatal diagnosis and carrier detection in Duchenne/Becker muscular dystrophy using the entire dystrophin cDNA. Am J Med Genet . 1988;;29:713-726.
19 +
Koenig M, Beggs AH, Moyer H, et al.  The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet . 1989;;45:498-506.
20 +
Baumbach LL, Chamberlain JS, Ward PA, Farwell NJ, Caskey CT.  Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology . 1989;;39:465-474.
21 +
Den Dunnen JT, Grootscholten PM, Bakker E, et al.  Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet . 1989;;45:835-847.
22 +
Fenwick RG, Ward PA, Alvarez FV.  Mutation and carrier detection for Duchenne muscular dystrophy by quantitative Southern analysis and multiplex PCR analysis. Am J Hum Genet . 1990;;47 ( (suppl) ):A216.
23 +
Abbs S, Yau SC, Clark S, Mathew CG, Bobrow M.  A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridization shows mistyping with both methods. J Med Genet . 1991;; 28:304-311.
24 +
Fujimura FK, Northrup H, Beaudet AL, O'Brien WE.  Genotyping errors with the polymerase chain reaction. N Engl J Med . 1990;;322:61.
25 +
Beggs AH, Koenig M, Boyce FM, Kunkel LM.  Detection of 98% of DMD/BMD deletions by PCR. Hum Genet . 1990;;86:45-48.
26 +
Roberts RG, Cole CG, Hart KA, Bobrow M, Bentley DR.  Rapid carrier and prenatal diagnosis of Duchenne and Becker muscular dystrophy. Nucleic Acids Res . 1989;;17:5961-5971.
27 +
Oudet C, Heilig R, Mandel JL.  An informative polymorphism detectable by polymerase chain reaction at the 3' end of the dystrophin gene. Hum Genet . 1990;;84:283-285.
28 +
Beggs AH, Kunkel LM.  A polymorphic CACA repeat in the 3' untranslated region of dystrophin. Nucleic Acids Res . 1990;;18:1931.
29 +
Clemens PR, Fenwick RG, Chamberlain JS, et al.  Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am J Hum Genet . 1991;;49:951-960.
30 +
Roberts RG, Barby TFM, Manners E, Bobrow M, Bentley DR.  Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Hum Genet . 1991;;49:298-310.

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