Decoding splicing complexity: integrated genomic analysis reveals two novel pathogenic variants in Chinese dystrophinopathy pedigrees.

Background: Dystrophinopathy is severe X-linked recessive muscle disease caused by mutations in DMD gene. There is an increasing number of deep intronic variants in DMD gene, and understanding the pathogenic mechanisms of intronic variants can help the diagnosis and treatment of patients with DMD.

Objective: To identify two novel splice site variants in two families affected with Dystrophinopathy.

Methods: Two children with Duchenne muscular dystrophy (DMD) caused by DMD gene variants diagnosed at Women and Children's Hospital of Ningbo University in April 2024 and August 2024 were selected as the subject. Genomic DNA was extracted from peripheral blood samples of the children and subjected to whole exome sequencing. Suspected splicing variant was verified by Sanger sequencing of family members. The splicing effects of the variant on DMD was assessed in a minigene assay, and X chromosome inactivation (XCL) analysis was performed for female carriers in 2 families.

Results: Two rare intronic variants were identified in DMD patients. One variant, NM_004006.3: c.9807 + 2dup of DMD identified in patient 1, leading to the retention of 21 intronic nucleotides, introducing a truncated protein (p.Ala3270_Met3685delinsValLysLeuPheThrPhe). The other variant, NM_004006.3: c.5739 + 326T > G of DMD identified in patient 2, resulted in the retention of 43 or 19 intronic nucleotides, introducing a truncated protein(p.Glu1914Metfs*3). X-chromosome inactivation (XCI) patterns identified the mother of patient 2 were moderately skewed with 83.1% (normal allele)/16.9% (mutant allele) when compared with the sister of patient 2 with 45.1%/54.9%.

Conclusion: Our study discovered two novel splicing mutation of DMD in two DMD patients, which expand the variant spectrum of DMD and provide precise genetic diagnosis of DMD for timely therapy. XCI may explained the asymptomatic of female carriers of DMD.