Abstract: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions (69%), with two hotspots located in the 5' end (exons 2–19) and the central of the DMD gene (exons 45–55), while point mutations were observed in 22% patients. Further, c.1149+1G>A and c.1150−2A>G were confirmed by hybrid minigene splicing assay (HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis (PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.

假肥大型肌营养不良症(DMD/BMD)遗传学诊断及剪接突变的致病性分析

[1]Bakker E, Veenema H, den Dunnen JT, et al., 1989. Germinal mosaicism increases the recurrence risk for ‘new’ Duchenne muscular dystrophy mutations. J Med Genet, 26(9):553-559.

[2]Baralle D, Baralle M, 2005. Splicing in action: assessing disease causing sequence changes. J Med Genet, 42(10):737-748.

[3]Beggs AH, Koenig M, Boyce FM, et al., 1990. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet, 86(1):45-48.

[4]Bovolenta M, Neri M, Fini S, et al., 2008. A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies. BMC Genomics, 9:572.

[5]Buratti E, Baralle FE, 2004. Influence of RNA secondary structure on the pre-mRNA splicing process. Mol Cell Biol, 24(24):10505-10514.

[6]Buratti E, Baralle M, Baralle FE, 2006. Defective splicing, disease and therapy: searching for master checkpoints in exon definition. Nucleic Acids Res, 34(12):3494-3510.

[7]Burset M, Seledtsov IA, Solovyev VV, 2001. SpliceDB: database of canonical and non-canonical mammalian splice sites. Nucleic Acids Res, 29(1):255-259.

[8]Chen LJ, Diao ZY, Xu ZP, et al., 2016. The clinical application of preimplantation genetic diagnosis for the patient affected by congenital contractural arachnodactyly and spinal and bulbar muscular atrophy. J Assist Reprod Genet, 33(11):1459-1466.

[9]Echevarria L, Aupy P, Goyenvalle A, 2018. Exon-skipping advances for Duchenne muscular dystrophy. Hum Mol Genet, 27(R2):R163-R172.

[10]Fairclough RJ, Wood MJ, Davies KE, 2013. Therapy for Duchenne muscular dystrophy: renewed optimism from genetic approaches. Nat Rev Genet, 14(6):373-378.

[11]Flanigan KM, von Niederhausern A, Dunn DM, et al., 2003. Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet, 72(4):931-939.

[12]Girardet A, Hamamah S, Dechaud H, et al., 2003. Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy. Mol Hum Reprod, 9(7):421-427.

[13]Haas J, Katus HA, Meder B, 2011. Next-generation sequencing entering the clinical arena. Mol Cell Probes, 25(5-6):206-211.

[14]Habara Y, Takeshima Y, Awano H, et al., 2009. In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G→A mutations in introns of the dystrophin gene. J Med Genet, 46(8):542-547.

[15]Hertel KJ, 2008. Combinatorial control of exon recognition. J Biol Chem, 283(3):1211-1215.

[16]Jefferies JL, Eidem BW, Belmont JW, et al., 2005. Genetic predictors and remodeling of dilated cardiomyopathy in muscular dystrophy. Circulation, 112(18):2799-2804.

[17]Juan-Mateu J, González-Quereda L, Rodríguez MJ, et al., 2013. Interplay between DMD point mutations and splicing signals in dystrophinopathy phenotypes. PLoS ONE, 8(3):e59916.

[18]Kralovicova J, Hwang G, Asplund AC, et al., 2011. Compensatory signals associated with the activation of human GC 5' splice sites. Nucleic Acids Res, 39(16):7077-7091.

[19]Krawczak M, Thomas NST, Hundrieser B, et al., 2007. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat, 28(2):150-158.

[20]Lalic T, Vossen RHAM, Coffa J, et al., 2005. Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet, 13(11):1231-1234.

[21]Lee SH, Kwak IP, Cha KE, et al., 1998. Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis. Mol Hum Reprod, 4(4):345-349.

[22]Li Y, Pan Q, Gu YS, 2017. Phenotype-genotype correlation with Sanger sequencing identified retinol dehydrogenase 12 (RDH12) compound heterozygous variants in a Chinese family with Leber congenital amaurosis. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 18(5):421-429.

[23]Ma PP, Zhang S, Zhang H, et al., 2018. Comprehensive genetic characteristics of dystrophinopathies in China. Orphanet J Rare Dis, 13:109.

[24]Muntoni F, Torelli S, Ferlini A, 2003. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol, 2(12):731-740.

[25]Parada GE, Munita R, Cerda CA, et al., 2014. A comprehensive survey of non-canonical splice sites in the human transcriptome. Nucleic Acids Res, 42(16):10564-10578.

[26]Pico AR, Bader GD, Demchak B, et al., 2014. The Cytoscape app article collection. F1000Res, 3:138.

[27]Rando TA, 2001. The dystrophin–glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve, 24(12):1575-1594.

[28]Roberts RG, Gardner RJ, Bobrow M, 1994. Searching for the 1 in 2,400,000: a review of dystrophin gene point mutations. Hum Mutat, 4(1):1-11.

[29]Roca X, Sachidanandam R, Krainer AR, 2003. Intrinsic differences between authentic and cryptic 5' splice sites. Nucleic Acids Res, 31(21):6321-6333.

[30]Richards S, Aziz N, Bale S, et al., 2015. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 17(5):405-424.

[31]Sakthivel Murugan SM, Arthi C, Thilothammal N, et al., 2013. Carrier detection in Duchenne muscular dystrophy using molecular methods. Indian J Med Res, 137(6):1102-1110.

[32]Schouten JP, McElgunn CJ, Waaijer R, et al., 2002. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res, 30(12):e57.

[33]Stockley TL, Akber S, Bulgin N, et al., 2006. Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies. Genet Test, 10(4):229-243.

[34]Takeshima Y, Yagi M, Okizuka Y, et al., 2010. Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J Hum Genet, 55(6):379-388.

[35]Tuffery-Giraud S, Béroud C, Leturcq F, et al., 2009. Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD–DMD database: a model of nationwide knowledgebase. Hum Mutat, 30(6):934-945.

[36]van Essen AJ, Abbs S, Baiget M, et al., 1992. Parental origin and germline mosaicism of deletions and duplications of the dystrophin gene: a European study. Hum Genet, 88(3):249-257.

[37]Wei XM, Dai Y, Yu P, et al., 2013. Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study. Eur J Hum Genet, 22(1):110-118.

[38]Xia ZJ, Lin J, Lu LP, et al., 2018. An intronic mutation c.6430-3C>G in the F8 gene causes splicing efficiency and premature termination in hemophilia A. Blood Coagul Fibrinoly, 29(4):381-386.

[39]Ye Y, Yu P, Yong J, et al., 2014. Preimplantational genetic diagnosis and mutation detection in a family with duplication mutation of DMD gene. Gynecol Obstet Invest, 78(4):272-278.

[40]Zhang XHF, Leslie CS, Chasin LA, 2005. Dichotomous splicing signals in exon flanks. Genome Res, 15(6):768-779.

[41]Zimowski JG, Massalska D, Holding M, et al., 2014. MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy. Neurol Neurochir Pol, 48(6):416-422.

[42]List of electronic supplementary materials

[43]Table S1 Oligonucleotide primers for detection of mutations in DMD gene

[44]Table S2 Deletions in DMD gene detected by MLPA

[45]Table S3 Duplications in DMD gene detected by MLPA

[46]Table S4 Point mutations in DMD gene detected by NGS