4. Discussion
Duchenne muscular dystrophy is a rare inherited disease due to lack of dystrophin, which is caused by the mutation of DMD gene. To date, there are 4690 gene mutation sites in DMD gene recruited in The Human Gene Mutation Database (HGMD) (Stenson, et al., 2020). HGMD (http://www.hgmd.org) have been collating all known gene lesions associated with DMD that have been published in the peer-reviewed literature. Mutation catalogues in HGMD are divided into 11 categories by mutation type, including gross deletions/insertions/duplications mutation (> 20 bp) (account for 52.8%), frameshift mutation (17.1%), nonsense mutation (15.6%), Canonical-splice mutation (6.8%), missense mutation (4%), splice mutation (1.7%), noncoding mutation (1.2%), inframe mutation (0.7%), synonymous mutation (0.1%), initiation mutation (0.02%), and regulatory mutation (0.02%). The intron variant of DMD gene is rarely studied since it is assumed to be less lethal than exonic variants. This study reported a proband with clinically diagnosed DMD that carried a novel splicing intronic variant in the DMD gene. The in vitroMini-gene splicing assay demonstrated this new variant caused mRNA damage during gene expression, which resulting in pathological symptoms. This novel finding could be added to the DMD mutational repertoire.
Currently, there is no effective treatment for DMD (Li, et al., 2012; Ebrahimzadeh-Vesal, Teymoori, Azimi-Nezhad, &Hosseini, 2018). It is important to develop accurate molecular diagnosis for this fatal disease as preventing birth defect is the main approach to manage DMD. Therefore, the investigation of gene variant in DMD gene is necessary for providing the basic knowledge to design and invent the mutation-specific gene therapy (Takeshima, et al., 2010). For this disease, the genetic diagnosis usually starts with the proband. In our study, the proband is a five-year-old boy who has been diagnosed DMD, being characterized by slow walking, difficult squatting, weakness of both lower limbs and dependent on wheelchair. A novel heterozygous DMD splicing mutation, c.2293-1G>C on intron 18, was detected in the proband and his mother but not the father by WES and Sanger sequencing. The splicing mutation was predicted to be deleterious by multiple computational methods. Complying with the guideline of American College of Medical Genetics and Genomics (ACMG) (Richards, et al., 2015) and ClinGen Sequence Variant Interpretation (SVI) (Ahmad N Abou Tayoun, et al.,2018; Biesecker, &Harrison, 2018; Ghosh, Harrison, Rehm, Plon, &Biesecker,2018), it is suggested that the c.2293-1G > C mutation of gene DMD is a pathogenic mutation site and associated with X-linked recessive disease Duchenne muscular dystrophy (DMD).
Further, our study successfully constructed pcMINI- DMD- wt/mut and pcMINI-C-DMD-wt/mut vectors, and then transfected 293T and MCF-7 cells. In vitro experiment of mini-gene splicing assay verified that the mutation c.2293-1G > C on intron 18 alters the normal splicing of DMD mRNA. The variant in both pc-MINI-DMD and pc-MINI-C-DMD vectors resulted in the deletion of 7bp on the left side of Exon19, with a codon frameshift resulting in an abnormal dystrophin protein as Fig 3 and 4. Because of the deletion of 7bp on the left side of Exon19, the variation of cDNA is c.2292_ 2299del and the variation of protein is p. ala765ser FS * 26. We concluded it would produce a truncated body protein with a size of 789aa, or may take the NMD pathway (nonsense mediated mRNA degradation) on account of the deletion of 7bp on the left side of Exon19 by the software (http://www.mutationtaster.org/ ). Hence, we deduced that the novel splice site mutation on intron 18 was a pathogenic variant and influenced the normal splicing of mRNA leading to alternative on the DMD translation products.
Another valuable data included in the study is that we collected the amniotic fluid (10ml) of the fetus at the second pregnancy of the studies family. The splicing mutation c.2293-1G>C was reproduced in the prenatal investigation and the sex of this fetus was male as well, indicating that the new genetic variant could be closely linked with the incidence of DMD. Besides of knowing DMD as a severe X-linked recessive muscle-wasting disease, DMD is also characterized by pseudohypertrophy in the calf muscle and the rapidly progressive degeneration and necrosis of the proximal muscles (Birnkrant et al., 2018). DMD patients are commonly first diagnosed before age of 5 and many died of respiratory or cardiac failure around age of 20. Consistently, the fetus in our study was also diagnosed with this new DMD gene variant. After genetic counseling and an accurate prenatal diagnosis, this couple decided to terminate pregnancy at 18 weeks of gestation.
In conclusion, we found a novel DMD splicing mutation c.2293-1G>C on intron 18 and confirmed that the mutation was pathogenic in gene expression level. Our finding enriches the DMD mutation spectrum. and provide scientific tool to achieve an effective prenatal diagnosis for the studied family.