[Mutation screening of 433 families with Duchenne/Becker muscular dystrophy]

Mutation analysis of unrelated families with Duchenne/Becker muscular dystrophy (DMD/BMD) was performed to investigate the characteristic of DMD gene mutation, especially the distribution pattern of point mutation of DMD gene in Chinese population.

A total of 433 unrelated DMD/BMD families were collected at the Center of Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University from March 2010 to December 2014. The deletions or duplications in 79 exons of DMD gene were screened using multiplex ligation-dependent probe amplification (MLPA). Any single-exon deletion detected by MLPA was further validated by PCR amplification. In the 117 unrelated Chinese families in which large-scale deletions and duplications had been excluded by MLPA, the point mutation in 79 exons of DMD gene were tested in the propositus using next-generation sequencing (NGS), and further verified the point mutation using Sanger sequencing.

In the 433 unrelated DMD/BMD families, 316 families with DMD deletions/duplications were identified by MLPA. Out of 57 single-exon deletions detected by MLPA, 3 were found as point mutations by PCR and Sanger sequencing, including 2 nonsense mutation (c.1729G>T [p.Glu577X], c. 3346A>T [p.Lys1116X]) and 1 frame-shift mutation (c.8605_8606delGT [p.Val2869ThrfsX25]). Direct sequencing with Ion PGM and Sanger sequencing in 117 families with negative results in MLPA detected 92 different point mutations in 96 families, including 46 novel mutations, 42 previously reported ones, and 4 possible polymorphisms (rs189143447, rs202008454, rs200213555, rs187617705). The 46 novel mutations consisted of 16 nonsense mutations (c.100A>T [p.Lys34X], c. 1201C>T [p.Gln401X], c. 1707C>A [p.Cys569X], c. 1831G>T [p.Glu611X], c. 1912C>T [p.Gln638X], c. 2213C>G [p.Ser738X], c. 3673_3673delA [p.Ile1225X], c. 3774C>A [p.Cys1258X], c. 4858G>T [p.Glu1620X], c. 5764A>T [p.Lys1922X], c. 6035T>G [p.Leu2012X], c. 6408G>A [p.Trp2136X], c. 7717C>T [p.Gln2573X], c. 7864G>T [p.Glu2622X], c. 8184_8185insT [p.Lys2729X], c. 8215C>T [p.Gln2739X]), 5 missense mutations (c.139G>A [p.Gly47Arg], c. 238G>C [p.Ala80Pro], c. 335G>T [p.Trp112Leu], c. 804A>C [p.Leu268Phe], c. 1149G>T [p.Glu383Asp]), 6 splice-site mutations (c.2293-3C>A, c. 2380+ 1G>T, c. 3277-1G>C, c.4519-7A>G, c. 5740-15G>T, c. 7661-1G>C), 16 small deletions (c.688_688delA [p.Met230CysfsX14], c.1760_1791del32 [p.Thr587IlefsX37], c. 2271_2271delA [p.Asp774ThrfsX22], c. 2281_2285delGAAAA [p.Glu761SerfsX10], c. 2527_2527delG [p.Glu843SerfsX3], c. 3405_3405delC [p.Asn1135LysfsX18], c. 4450_4450delC [p.His1484ThrfsX14], c. 4770_4770delA [p.Thr1590ThrfsX5], c. 4937_4937delA [p.Glu1646GlyfsX11], c. 5253_5256delATTA [p.Lys1751LysfsX2], c. 5654_5654delA [p.Gln1885ArgfsX6], c. 7441_7441delG [p.Glu2481AsnfsX13], c. 7860_7860delC [p.Ile2620IlefsX18], c. 8668-8668delG /c.8668+ 1-8668+ 1delG, c. 9009_9009delC [p.Thr3003ThrfsX18], c. 9021_9021delT [p.Ile3007IlefsX14]), and 3 small insertions (c.305_306insG [p.Gly102GlyfsX4], c. 3116_3117insA [p.His1039GlufsX11], c. 9197_9198insATCTC [p.Ser3066SerfsX25]). And 87.4% (83/95) of the pathologic point mutations disrupted the translational reading frame (46 nonsense mutations, 24 frame-shift mutations, and 13 splice-site mutations).

Inexpensive and efficient genetic/prenatal diagnosis of DMD/BMD may be plausible by MLPA analysis, NGS, and Sanger sequencing. Most of the mutations identified in this study led to a predictable premature stop codon or splicing defects, resulting in defective function of dystrophin.