Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer.

Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2ß and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2ß-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.

A resolved discrepancy between multiplex PCR and multiplex ligation-dependent probe amplification by targeted next-generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene.

BACKGROUND: The genetic diagnosis of Duchenne muscular dystrophy (DMD) has been complicated by the large size of the gene and its heterogeneous mutational spectrum. Multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) are two well-established mutation screening methods. Here, we applied targeted next-generation sequencing (NGS) to clarify discrepant results between multiplex PCR and MLPA in a Chinese patient with DMD. METHODS: MLPA was performed to confirm multiplex PCR results obtained previously. Targeted NGS was then used to analyze the full-length DMD gene including introns. RESULTS: Multiplex PCR had previously identified an apparent deletion of exon 43 in the patient with DMD, but current MLPA indicated that exon 43 was present. Targeted NGS to clarify the genetic diagnosis identified a novel mutation, c.6241_c.6290 + 1109del1159insAC, which caused partial deletion of exon 43. This mutation removed the annealing sequence of the exon 43 reverse primer in multiplex PCR but had no influence on the hybridization site of the MLPA probe. Therefore, the discrepancy between the two methods was caused by partial exonic deletion that escaped MLPA detection. CONCLUSION: Targeted NGS disclosed a novel partial exonic deletion in the DMD gene as the cause of discrepancy between multiplex PCR and MLPA. Targeted NGS could be used to provide a more accurate genetic diagnosis of DMD, particularly in cases of partial exonic deletions, which will be of benefit in patient management and the identification of disease carriers.

[Survey of the prevalence of chronic prostatitis in men with premature ejaculation].

OBJECTIVE: To investigate the prevalence of chronic prostatitis in men with premature ejaculation. METHODS: The segmented urine specimens before and after prostatic massage and the expressed prostatic secretion specimens from 106 patients with premature ejaculation and 38 controls were evaluated by microscopic and/or bacteriological studies. The prevalence of premature ejaculation was also investigated in 120 patients with chronic prostatitis. RESULTS: Prostatic inflammation was found in 46.2% and chronic bacterial prostatitis in 34.7% of the subjects with premature ejaculation, respectively. Compared with the controls, the findings were statistically significant (P < 0.05). The prevalence of premature ejaculation in the patients with chronic prostatitis was 47.5% (57/120). CONCLUSIONS: Chronic prostatic inflammation may play a role in the pathogenesis of some cases of premature ejaculation and it is important to give a careful examination of the prostate before initiating any therapy for premature ejaculation.