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

Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene # # 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 2 3 first-author 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:dongmy@zju.edu.cn †E-mail:dongmy@zju.edu.cn dongmy@zju.edu.cn Department of Reproductive Genetics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Department of Reproductive Genetics, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006, China Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, Hangzhou 310006, China Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of Education, Hangzhou 310006, China Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Hangzhou 310006, China Key Laboratory of Women’s Reproductive Health of Zhejiang Province, Hangzhou 310006, China 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. 目的 2 针对100例无亲缘关系的假肥大型肌营养不良症(DMD/BMD)患者,联合应用多种检测技术进行遗传学诊断,并且建立个体化产前诊断及胚胎植入前遗传学诊断方案,以最大限度地降低DMD/BMD患儿的出生. 创新点 2 通过minigene剪接实验分析 DMD :c.1149+1G > A和c.1150−2A > G突变是否导致剪接异常,并确定剪接方式. 方法 2 收集100例无亲缘关系DMD/BMD患者的临床资料,应用多重连接依赖式探针扩增技术(MLPA)、第二代测序(NGS)、minigene剪接实验(HMSA)进行遗传学诊断,并通过单体型分析及性别鉴定进行胚胎植入前遗传学诊断. 结论 2 联合应用多种检测技术可以尽早地对患者进行遗传学诊断,为临床遗传咨询和产前诊断及胚胎植入前遗传学诊断提供了科学依据. Dystrophin gene Variation Genetic diagnosis Splice site mutation Hybrid minigene splicing assay DMD 基因 突变 遗传学诊断 剪接突变 Minigene剪接实验 Introduction 1 Dystrophinopathies are common neuromuscular disorders in childhood. There are two main phenotypes: Duchenne muscular dystrophy (DMD, OMIM: 310200) and Becker muscular dystrophy (BMD, OMIM: 300376). DMD, with an approximate incidence of 1/3500 in live male births, is characterized by early-onset, rapidly progressive muscle weakness and being wheel-chair-dependent before age 13, while BMD presents milder clinical features, later-onset and slower progression (Jefferies et al., 2005 2005 ). Both DMD and BMD are X-linked recessive neuromuscular diseases due to DMD gene (OMIM: 300377) mutations. This is the largest gene in the human genome (Muntoni et al., 2003 2003 ). The DMD gene spans more than 2.5 million base pairs (bp) of genomic DNA on chromosome X p21.2-21.1 and consists of 79 exons. It encodes a 14 kilobases (kb) mRNA transcript and a 427 kDa full-length dystrophin protein comprising four domains (amino terminal domain, central rod domain, cysteine-rich domain, and carboxy terminal domain) (Muntoni et al., 2003 2003 ). The dystrophin protein interacts with integral membrane proteins and they assemble into a dystrophin–glycoprotein complex, which plays an important role in stabilizing the sarcolemma and protecting muscle fibers from damage and necrosis (Rando, 2001 2001 ). Mutations of DMD include intragenic large deletions/duplications and point mutations. Intragenic deletions and duplications refer to one or more exons (Fairclough et al., 2013 2013 ), whereas point mutations include nonsense mutations, small frame-shift deletions or insertions, missense, and splice site mutations (Roberts et al., 1994 1994 ). Multiplex polymerase chain reaction (PCR) is used to be a standard diagnostic method. It could identify 98% of deletions in male patients, but neither duplication and point mutation nor female carriers are detectable (Beggs et al., 1990 1990 ). Multiplex ligation-dependent probe amplification (MLPA) is currently the most widely used to detect exonic deletions and duplications of DMD both in male patients and female carriers (Schouten et al., 2002 2002 ; Zimowski et al., 2014 2014 ). Recently, with the dramatically reduced cost of sequencing, next-generation sequencing (NGS) has become clinically available for the detection of large deletions/duplications and point mutations (nonsense mutation, missense mutation, small insertions/deletions (indels) spanning several base pairs, and splice site mutation) throughout all the exons and flanking introns of DMD (Haas et al., 2011 2011 ). Splicing regulation has been attracting much more attention since exon-skipping therapy began to appear (Echevarria et al., 2018 2018 ). So far, a splicing pattern is essential for therapy and diagnosis of disease. Hybrid minigene splicing assay (HMSA) has allowed a relatively fast functional test to build connections between splicing and disease. Though the detection technologies develop rapidly, 1% to 2% of DMD cases remain undiagnosed (Bovolenta et al., 2008 2008 ). Identification of pathogenic DMD variants can confirm the diagnosis of DMD/BMD patients and determine the carrier status of their family members. Once the familial mutation is determined, prenatal diagnosis or preimplantation genetic diagnosis (PGD) could be adopted for the family members to prevent the recurrence of the disease. Here, we combined MLPA, NGS, and Sanger sequencing to identify causative mutations in 100 unrelated DMD patients. Eleven novel mutations were detected and two splice site mutations were verified by HMSA. Prenatal diagnosis or PGD was performed on 50 families with known mutations. Materials and methods 1 Patients 2 A total of 100 unrelated DMD/BMD families referred to the Department of Reproductive Genetics, Women’s Hospital, School of Medicine, Zhejiang University (Hangzhou, China) were recruited. All subjects were Han Chinese. The diagnosis of DMD/BMD was based on serum creatine kinase (CK) measurements, muscle biopsy, electromyography, electrocardiogram, progression of the disease, and family history. There were 100 DMD/BMD probands and 100 mothers of probands in our study. This study was approved by the Ethical Committee of the Women’s Hospital, School of Medicine, Zhejiang University. Informed consents were obtained from all participants. Genomic DNA extraction 2 DNA was isolated from peripheral blood lymphocytes and amniocytes according to the manufacturer’s standard protocol (QIAamp DNA Blood Mini Kit, Hilden, Germany). Amniotic fluids were obtained by an obstetrician through amniocentesis and then amniocytes were cultured for eleven days before DNA extraction. MLPA 2 MLPA was used to screen all exons of DMD for all patients. The assay was performed with two independent MLPA kits SALSA P034 and P035 (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer’s instruction and the PCR products were separated by ABI PRISM ® 3100 Genetic Analyzer (Applied Biosystems, CA, USA). Then, the statistical and quantitative analyses were calculated by Coffalyser Net software ( http://www.coffalyser.net http://www.coffalyser.net ). NGS and Sanger sequencing 2 NGS was performed as previously described (Wei et al., 2013 2013 ; Li et al., 2017 2017 ). All primers were designed by Primer 5.0 (Premier Biosoft International, CA, USA) (Table S1 S1 ). The sequencing data and the standard DMD reference sequence (GenBank accession number NM_004006.2) were aligned by Mutation Surveyor software (Soft Genetics, State College, PA, USA) to determine nucleotide variations. All mutations identified in this study were checked on the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk http://www.hgmd.cf.ac.uk www.hgmd.cf.ac.uk www.hgmd.cf.ac.uk ), the Leiden Open Variation Database (LOVD, http://www.dmd.nl http://www.dmd.nl ), and the Universal Mutation Database (UMD, http://www.umd.be http://www.umd.be ). Novel mutations were evaluated according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015 2015 ). Missense mutations were evaluated by Sorts Intolerant from Tolerant (SIFT, http://sift.jcvi.org http://sift.jcvi.org ) and PolyPhen-2 ( http://genetics.bwh.harvard.edu/pph2 http://genetics.bwh.harvard.edu/pph2 ). Splicing impact was scored using the Human Splicing Finder (HSF, http://www.umd.be/HSF3 http://www.umd.be/HSF3 ). HMSA 2 To investigate the effects of the splice site mutations, HMSA including minigene construct, transfection, and reverse transcription PCR (RT-PCR) was performed as previously described (Xia et al., 2018 2018 ). Human genomic DNA fragments covering from exon 10 to exon 11 were amplified using forward primer DMD-1149/50-F (5'-GATCCTCGAG ctccctattgtctgtatctgc-3') and reverse primer DMD-1149/50-R (5'-GATCGGATCCtacacaatctcctgtgcatgc-3'). The vector primers were as follows: SD6 (5'-TTGTCTACCCATGGACCCAGA-3') and SA2 (5'-CCCCCTGAACCTGAAACATAAAAT-3'). The PCR products were analyzed on 2% (0.02 g/mL) agarose gel and sequencing was performed. PGD 2 In vitro fertilization (IVF) procedure was performed as previously described (Ye et al., 2014 2014 ). Two or five trophocytes were aspirated from each blastocyst on Day 5 and transferred to PCR tube for whole genome amplification (WGA). One or two non-affected embryos were selected to be transferred. Clinical pregnancy was confirmed by serum β-human chorionic gonadotrophin (β-hCG) and sonography. Amniocentesis was performed at 20 weeks of gestation. Multiple displacement amplification (MDA) was performed according to the manufacturer’s instructions (REPLI-g Single Cell Kit for WGA, QIAGEN, Germany). The MDA products were used for PCR immediately or stored at −20 °C. Preimplantation genetic haplotyping (PGH) was performed by NGS-based single nucleotide polymorphism (SNP) haplotyping. SNP markers located 1 Mb upstream and downstream of the DMD gene in the genomes from the 1000 Genomes Project and known mutation in DMD gene were used for NGS-based SNP haplotyping. The detailed procedure of NGS was performed according to the protocol as previously described (Chen et al., 2016 2016 ). The data from NGS was further confirmed by standard PCR and Sanger sequencing. Sex determining region Y ( SRY ) gene was amplified by primers (SRY-F: AGTAAAGGCAACGTCCAGGAT; SRY-R: TTCCGACGAGGTCGATACTTA) and then the amplified products were analyzed by 1.5% (0.015 g/mL) agarose gel and Sanger sequencing. Results 1 Deletion and duplication detection 2 In total, 69 deletions and 9 duplications were identified in this study (Table S2 S2 and Table S3 S3 ). Notably, two deletion hotspots (exons 45–55 and exons 2–19) were observed (Fig. 1 1 ). The number of deleted or duplicated exons ranged widely from 1 to 47. Two or more continuous exon deletions could be easily and reliably determined by MLPA. For the cases with only one exon deletion, the results should be further confirmed by standard PCR and Sanger sequencing. Three-exon deletion/duplication was the most frequent (21.79%, 17/78), followed by single-exon (15.38%, 12/78), five-exon (10.26%, 8/78), and two-exon (8.97%, 7/78). As for the 12 single-exon deletion/duplication mutations, exon 51 was the commonest affected exon (41.67%, 5/12), followed by exon 50 (16.67%, 2/12). Among the 78 affected patients due to deletions/duplications, 47 of them (60.25%, 47/78) inherited the mutation from their mothers, while 31 (39.74%, 31/78) were de novo mutations. Deletions and duplications are located above the schematic representation of exon and dystrophin structural domains of DMD . Black and red lines represent deletion and duplication, respectively. The length of line corresponds to the coverage of deleted and duplicated exons. Point mutations are all located below the schematic representation of exon and dystrophin structural domains of DMD . The types of mutations are distinguished by different symbols (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) Point mutation detection 2 Because of the negative MLPA results of the rest probands, NGS and Sanger sequencing were provided for these subjects. Mutations were found in all 22 probands (Table S4 S4 ), including 13 nonsense (59.10%, 13/22), 6 indels (27.27%, 6/22), 2 splice site (9.10%, 2/22), and 1 missense (4.55%, 1/22) mutations. Of note, unlike two hotspots observed in deletions, point mutations were scattered throughout the whole DMD gene (Fig. 1 1 ). Among them, only three were de novo (13.64%, 3/22). Importantly, two splice site mutations were identified in this study: a novel mutation c.1149+1G > A and a previously reported mutation c.1150−2A > G, which were both inherited from their mothers (Fig. 2 2 ). The variation c.1149+1G > A might lead to 5' motif change from GAGgtaaac to GAGataaac, with the wild-type score being 83.90 in comparison with the mutant score of 57.07 as predicted by splicing prediction software HSF. As for c.1150−2A > G, HSF showed the wild-type score was 90.99, while the mutant score was 62.05. Meanwhile, the variation was predicted to break the exonic splicing enhancer (ESE) element and exonic splicing silencer (ESS) element. Therefore, it was suspected that these splice site mutations could break the original splice site. A reported missense mutation c.497G > T was found in a patient. This mutation was predicted to be deleterious by SIFT (affecting protein function with a score of 0.00) and PolyPhen-2 (probably damaging with a score of 1.00). Black boxes indicate affected males; dotted circles indicate female carriers; clear boxes indicate unaffected males. Partial sequencing chromatograms of genomic DNA from patients are shown. The arrows denote the positions of identified mutations Splicing study by HMSA 2 HMSA was performed to investigate the effect of the two splice site mutations. The wide-type minigene construct was 1507 bp, across from partial intron 9 (152 bp), exon 10 (189 bp), intron 10 (650 bp), exon 11 (182 bp) to partial intron 11 (334 bp). A full length of 371 bp RT-PCR product (exclusive SD6 and SA2) was detected, which included exon 10 and exon 11 as expected. There was no product of normal size obtained for the mutant c.1149+1G > A minigene, but instead two abnormal larger fragments of 686 bp and 914 bp were detected resulting from two activations of cryptic donor splice sites in intron 10. Sequencing revealed that the two products contained 50 bp and 278 bp of intron 10, respectively (Fig. 3 3 ). They resulted from the activation of a cryptic splice site at different positions in the beginning of intron 10. Theoretically, both mutant isoforms would induce a premature termination codon (PTC) at amino acid 386. The motifs of two cryptic 5' splice sites (5'ss) activated were ATGgcaagt and ATGgtattg, with the score 67.46 and 71.24 calculated by HSF, respectively. (a) Electrophoretogram of RT-PCR products. The mutant plasmid showed two types of splicing fragments of 686 bp ( *** ) and 914 bp ( ** ), while the wild-type plasmid showed a normal splicing fragment of 636 bp ( * ). The empty plasmid including SD6 and SA2 showed 265 bp. (b) Splicing models schematic representation. (c) Sequencing result of the mutated (aberrantly spliced) cDNA showing 278 bp retention of intron 10. (d) Sequencing result of the mutated (aberrantly spliced) cDNA showing 50 bp retention of intron 10 Another minigene construct containing the c.1150−2A > G mutation yielded two shortened transcripts: a primary 454 bp transcript and a 614 bp transcript (Fig. 4 4 ). The 454 bp transcript was caused by complete skipping of exon 11 that led to the deletion of 182 bp. Thus, it included exon 10 (189 bp) and 265 bp fragment of exogenous pSPL3 vector by SD6 and SA2 primers. The skipping of exon 11 would cause frameshift that introduced stop codon 9 nucleotides downstream of the junction of exons 10 and 12. The 614 bp transcript resulted from the lack of a part of exon 11 (22 bp), which would cause frameshift that consequently introduced stop codon 46 nucleotides downstream of exon 11. The motif of cryptic 3' splice site (3'ss) activated was ggatttgacagCC, and the score of this cryptic 3'ss was 79.18 calculated by HSF. (a) Electrophoretogram of RT-PCR products. The mutant plasmid showed two types of splicing fragments of 454 bp ( *** ) and 614 bp ( ** ), while the wild-type plasmid showed a normal splicing fragment of 636 bp ( * ). The empty plasmid including SD6 and SA2 showed 265 bp. (b) Splicing models schematic representation. (c) Sequencing result of the mutated (aberrantly spliced) cDNA showing 22 bp lack of exon. (d) Sequencing result of the mutated (aberrantly spliced) cDNA showing skipping of exon 11 Prenatal diagnosis and PGD 2 Prenatal diagnosis was performed on 50 mothers of probands (34 carriers and 16 non-carriers). Thirty normal females, 12 normal males, 5 female carriers, and 8 affected males were diagnosed (Table 1 1 ). The female carrier of exons 48–50 deletion (P30 family) and female carrier of c.1886C > G (P80 family) underwent PGD by haplotyping and gender determination. For the P30 family, biopsies were undertaken for four embryos: 1 and 4 were male, while embryos 2 and 3 were female. PGH identified 110 SNPs and 8 informative SNPs were selected. PGH and specific detection of deletions showed that all 4 embryos were normal (Fig. 5 5 ). Pregnancy was achieved after three cycles of embryo transfer. Amniocyte diagnosis confirmed the PGD results and then a healthy boy was delivered. In the P80 family, embryo biopsies were performed on 5 embryos. PGH identified 91 SNPs and 7 informative SNPs were selected (Fig. 5 5 ). Embryos 1, 2, and 4 demonstrated an affected haplotype inherited from the mother, whereas embryos 3 and 5 demonstrated an unaffected haplotype. PGH results were consistent with mutation and sex identification. Results indicated that embryos 1, 2, and 4 were female carriers, while embryos 3 and 5 were normal female and male, respectively. Finally, one embryo was transferred, resulting in a biochemical pregnancy. At 20 weeks of gestation, amniocyte diagnosis was performed and confirmed the PGD results. (a) Haplotype analysis of P30 family. Diagram shows that all embryos have normal haplotype. (b) Direct mutation detection by amplification of exons 47, 50, and 51 of DMD for P30 family. All embryos had no deletion of exon 50. (c) Haplotype analysis of P80 family. E1, E2, and E4 demonstrated an affected haplotype pattern (red-line allele) inherited from mother, whereas E3 and E5 demonstrated an unaffected haplotype pattern (black-line allele). (d) Direct detection c.1886C/C in exon 16 of DMD for P80 family. E3 had wild homozygote c.1886C/C, whereas E5 had hemizygote. However, E1, E2, and E4 had heterozygous c.1886C > G (p.Ser629X). P: proband; M: proband’s mother; E1−E5: embryos 1–5; −: negative control. Black boxes indicate affected males; dotted circles indicate female carriers; clear boxes and circles indicate unaffected males and females, respectively (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) 1/2 : number of pregnance; NF: normal female fetuses; NM: normal male fetuses; d: double fetuses Family No. Carrier status of mother Prenatal diagnosis  P4 Yes NF  P91 No dNF  P27 No NF  P35 No NF  P36 No NF  P48 No NM  P51 No NF  P52 No NF  P74 No NM  P56 No NF  P57 No NF  P60 No NF  P62 Yes NF  P63 Yes NM  P65 Yes Carrier  P67 Yes Carrier  P68 Yes NF  P11 Yes NM  P7 No NF  P40 No NF  P84 Yes Patient  P46 No NF 1 /NM 2  P69 No NF  P16 Yes Patient  P20 Yes NF  P86 Yes Patient  P92 Yes Patient  P94 Yes NF  P95 Yes NM  P96 Yes NF  P97 Yes Patient 1 /Carrier 2  P31 Yes NM  P39 Yes dNF  P73 Yes NF  P43 Yes Patient  P49 Yes Carrier  P50 Yes Patient  P58 Yes NF  P77 Yes NM  P30 Yes NM  P80 Yes NF  P18 Yes NF  P34 Yes NF  P8 Yes NM  P15 No NM  P33 Yes Patient 1 /NM 2  P12 Yes NF  P32 Yes NF  P76 Yes NF  P82 Yes Carrier Discussion 1 In this study, all pathogenic variants in DMD were successfully identified in all 100 cases, and 11 variants were novel (Tables 2 2 and 3 3 ). Nonsense, indels and splice site mutations at ±1/±2 were pathogenic, as evaluated according to ACMG guidelines (Richards, et al., 2015 2015 ). Our findings expanded the spectrum of the Human Mutation Database. MLPA was used as the first-line screening method and our results showed that the percentage of deletion and duplication detection by MLPA is 78%, which was similar to other studies (70%) (Lalic et al., 2005 2005 ; Takeshima et al., 2010 2010 ). As for the cases with negative MLPA results, we further used NGS to detect possible point mutations (Flanigan et al., 2003 2003 ). In total, 22 point mutations out of 100 cases (22%) were identified. Point mutations were dominated by nonsense mutation, which was consistent with previous studies (Tuffery-Giraud et al., 2009 2009 ; Takeshima et al., 2010 2010 ). Mutation type Pathogenic Known Novel De novo Hereditary  Deletion 69 67 2 29 40  Duplication 9 8 1 2 7  Nonsense 13 9 4 3 10  Splicing 2 1 1 0 2  Missense 1 1 0 0 1  Indels 6 3 3 0 6 6  Total 100 89 11 34 66 Patient No. Variant Inheritance  P4 EX5–6 deletion Maternal  P43 EX12–33 deletion Maternal  P77 EX56–70 duplication Maternal  P79 c.3487C>T, p.(Gln1163Ter) De novo  P82 c.8086_8087insC, p.(Leu2696ProfsX14) Maternal  P85 c.9689delA, p.(Asp3230AlafsX53) Maternal  P88 c.9669C>T, p.(Arg3223Ter) Maternal  P89 c.2664 C>T, p.(Gln888Ter) De novo  P92 c.7899G>A, p.(Trp2633Ter) Maternal  P94 c.4131delA, p.(Lys1377AsnfsX5) Maternal  P99 c.1149+1G>A Maternal We identified a potential pathogenic missense mutation c.497G > T (p.Gly166Val) which was reported previously (Juan-Mateu et al., 2013 2013 ). This mutation was located at highly conserved residues and was predicted to be pathogenic. In pedigree 81, there were three affected males who had hemizygote c.497G > T. The first affected male had severe muscle weakness and died from respiratory failure at 20 years of age. The second one was early-onset and lost ambulation as disease progressed at 15 years of age, while the third one was 7-year-old now and suffered from progressive muscle weakness from 4 years of age. Juan-Mateu et al. ( 2013 2013 ) had reported that mutation c.497G > T was found in an intermediate muscular dystrophy patient (loss of ambulation between 13 and 16 years of age), which was almost consistent with the patient’s clinical phenotype in this study. In DMD patients, prediction of splicing outcomes is essential for determining the phenotype. As for novel mutation c.1149+1G > A, the HMSA results revealed that two abnormal transcripts of 50 bp and 278 bp retention partial intron 10 resulted from the activation of cryptic donor splice sites in intron 10 and both of them were out of frame. This was consistent with the patient’s DMD phenotype (early-onset and wheel-chair-dependent at 8 years of age). The mutation c.1150−2A > G was previously reported once in a DMD case (Ma et al., 2018 2018 ). However, no experimental evidence was available for this mutation. In our study, we not only verified that the mutation was maternal but the splicing outcome that arose from the mutation was conducted by HMSA. It revealed that two abnormal transcripts of complete skipping of exon 11 and lack of 22 bp of exon 11. These two abnormal splicing outcomes were out of frame, which was consistent with the patient’s DMD phenotype (early-onset and wheel-chair-dependent at 9 years of age). Mutations involving GT and AG dinucleotide at the 5' (donor) and 3' (acceptor) splice sites would lead to disease (Pico et al., 2014 2014 ). The major consequences of mutation at splice sites were exon skipping and cryptic splice site activation (Baralle and Baralle, 2005 2005 ). The decision between two patterns of splicing outcomes was rather a matter of exon and intron length, exon flanking sequence, RNA secondary structure, open reading frame conservation, and DNA sequence environment (Roca et al., 2003 2003 ; Buratti and Baralle, 2004 2004 ; Zhang et al., 2005 2005 ; Buratti et al., 2006 2006 ; Hertel, 2008 2008 ; Pico et al., 2014 2014 ). Exon skipping was more common than cryptic donor splice site activation in the dystrophin gene mutations in cases of intron +1G > A, while cryptic splice site activation was likely only seen in strong exons ( > 170 bp) (Habara et al., 2009 2009 ). As for the c.1149+1G > A case, it was suspected that cryptic splice site activation was influenced by the long length of exon 10 (189 bp). Minigene assay identified two cryptic 5'ss, and the scores calculated by HSF were lower than that of wild-type 5'ss, but higher than that of the mutant one. Actually, several potential 5'ss predicted by HSF were between the mutated authentic splice site and cryptic 5'ss. These potential 5'ss were completely silent and likely to be pseudo 5'ss. Roca et al. ( 2003 2003 ) observed that the cryptic 5'ss activated were usually located close to the authentic 5'ss, whereas a few of cryptic 5'ss located further away were also activated. We suggested that the distance to the mutated splice site played an important role, but not the decisive factor for cryptic 5'ss activation. However, we observed the difference in splicing efficiency between these two cryptic 5'ss, which might be associated with proximity to the mutated splice site. It was interesting to note that one of cryptic splice sites activated at 50 bp downstream of exon 10 was activated, though the following nucleotides are “GC.” The GT/AG splice sites were present in the majority of eukaryotic introns. In a database (SpliceDB, http://www.softberry.com/spldb/SpliceDB.html http://www.softberry.com/spldb/SpliceDB.html ) of known mammalian splice sites, Burset et al. ( 2001 2001 ) observed that non-canonical GC sequence at 5'ss accounted for 0.56% and GC 5'ss possess a strong consensus sequence. Parada et al. ( 2014 2014 ) uncovered that the GC/AG splice sites comprise about 0.889% of the total splice sites in humans. Although the percentage of non-canonical GC 5'ss was low, the GC 5'ss existed in various species, and were not aberrant splice sites. Also, a GC/AG splice sites pair existed at intron 30 of DMD . The factors promoting such weak site efficient selection are still poorly understood. Kralovicova et al. ( 2011 2011 ) showed high density of GAA/CAA with splicing enhancers in exon making a contribution for efficient selection of GC 5'ss. In the absence of an alternative splice site, exon skipping was the preferred outcome at both donor and acceptor splice sites (Krawczak et al., 2007 2007 ). As for c.1150−2A > G, exon 11 skipping was the major splicing outcome, and in contrast to the cryptic splice site was activated slightly. The sub-optimal, neighboring 3'ss activated was tggatttgacag/CC and the score calculated by HSF was lower for this cryptic 3'ss with respect to wild-type 3'ss. The variation c.1150−2A > G was predicted to break ESE and ESS, which regulate premature mRNA splicing. These regulatory elements may play important roles in the pre-mRNA splicing process and have an impact on transcriptional efficiency. It was supposed that skipping of the adjacent exon was the preferred outcome when cryptic splice site was of low efficiency or not sufficient. So far, it is still a challenge to establish a rule to predict splicing outcomes. Though HMSA is an effective validation technique in the molecular laboratory, the appropriate RNA analysis ideally comes from affected tissue because of the tissue-specific dystrophin functions (Muntoni et al., 2003 2003 ). So it is important to keep in mind that the splicing outcome would be different in artificial minigenes as compared to endogenous transcripts. Early diagnosis of DMD and genetic confirmation of the causative mutation in patients are important. Once a mutation is identified, carrier analysis of female relatives can be performed. In our study, DMD gene mutation was inherited from the mother in 66% (66/100) of cases, whereas the remaining 34% (34/100) were de novo, which was consistent with a previous report (30%) (Sakthivel Murugan et al., 2013 2013 ). However, it should be noted that a mother without any mutation in her peripheral blood lymphocytes is still at risk due to the possibility of germinal mosaicism (Bakker et al., 1989 1989 ; van Essen et al., 1992 1992 ). Prenatal diagnosis and PGD are valid tools to reduce the risk of having affected children. In 50 families with confirmed mutations, prenatal diagnoses were successfully performed by MLPA or Sanger sequencing. Eight fetuses were verified as affected boy, approximately theoretical value 25%, and five fetuses were carriers in 34 mother carriers. On the other hand, all fetuses were identified as unaffected in 16 mothers without mutation in their peripheral blood lymphocytes. In P30 family with deletion of exons 48–50, PGD was performed and a healthy boy was successfully delivered. Therefore, it is obvious that PGD is an alternative to prenatal diagnosis for a family at high risk of transmitting an inherited disease to offspring and avoiding the possible termination of pregnancy. Specific mutation detection (Girardet et al., 2003 2003 ) and haplotype analysis (Lee et al., 1998 1998 ) with gender determination have been universally used for PGD of DMD. In conclusion, it is a great challenge to perform comprehensive analyses of causative mutations in DMD because of its large size and the variety of mutation types (Stockley et al., 2006 2006 ). Our study provided a comprehensive combination of MLPA, NGS, and HMSA, which is an effective strategy to identify DMD pathogenic variants. Early and accurate genetic diagnosis and confirmation of variants are essential for therapy and prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families. References E Bakker , H Veenema , JT den Dunnen , 等 . 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Dichotomous splicing signals in exon flanks JG Zimowski , D Massalska , M Holding , 等 . MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy . Neurol Neurochir Pol , 2014 . 48 ( 6 ): 416 - 422 . 25482253 DOI: 10.1016/j.pjnns.2014.10.004 http://doi.org/10.1016/j.pjnns.2014.10.004 . MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy The authors thank the patients for their voluntary involvement in this study. We are grateful to Dr. Jiong GAO (BGI Genomics, Shenzhen, China) for analyzing the data and modifying the article. Compliance with ethics guidelines Yan-mei YANG, Kai YAN, Bei LIU, Min CHEN, Li-ya WANG, Ying-zhi HUANG, Ye-qing QIAN, Yi-xi SUN, Hong-ge LI, and Min-yue DONG declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. 10.1631/jzus.B1800541 Project supported by the National Key Research and Development Program of China (No. 2016YFC1000703), the Medicine and Health Science and Technology Plan Projects in Zhejiang Province (No. 2014KYA246), and the National Natural Science Foundation of China (Nos. 81801441 and 81300532) 2018-10-28 2019-03-25 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 20