In this study, by analyzing COL4A3–5 mRNAs from urine or skin fibroblasts, nine deep intronic pathogenic variants were identified in five unrelated Alport syndrome patients with negative NGS results. These findings indicate that our developed approach may be applied to help provide personalized evaluation and care of patients and their families. In addition, this is the first report on compound heterozygous deep intronic splicing mutations in COL4A4 gene in an Alport syndrome patient.
Numerous studies have shown that NGS is effective in finding single nucleotide variations and small indels in exons and the flanking intronic regions16. However, some genetic events such as deep intronic variants, copy number variants, and somatic cell mosaicism may be missed by NGS17. Therefore, for a patient with clinically diagnosed or suspected AS and no pathogenic variants detected by NGS, it is necessary to further analyze COL4A3–5 genes by mRNA sequencing, chromosome microarray analysis, droplet digital PCR or other approaches to improve genetic diagnosis18,19.
According to the literature and public databases (Human Gene Mutation Database and Leiden Open source DNA Variation Database), pathogenic splicing variants account for 14.9–24.5% in the COL4A5 gene20,21. Approximately 70.4% (112/159) occurred at consensus splice sites, and only seven splicing variants occurred in introns at more than 100 base pairs up/downstream from exon-intron junctions. Approximately 70% (23/32) of the pathogenic COL4A3 splicing variants occurred at consensus splice sites and only two variants were located in introns at more than 100 base pairs upstream from the exons. No deep intronic COL4A4 splicing variants have been reported to date. These findings indicate that deep intronic COL4A3–5 mutations are rare. The nine novel deep intronic pathogenic variants obtained in the present study extend the mutational spectrum of AS. These findings also highlight COL4A3–5 mRNA analysis as an effective supplementary approach for NGS in molecular diagnosis of this disease.
Previous studies have reported that GBM collagen α3α4α5(IV) is synthesized solely by podocytes22, and the urine podocyte detachment rate (assessed by podocin mRNA in urine pellets) is increased in AS patients23,24. Therefore, extraction of RNA directly from patient-originated urine may be a valuable approach to the analysis of all three Alport gene variants, which was demonstrated by the findings of the present study. In addition, compared with the method that extracted RNA from urine-derived podocyte-lineage cells, our developed approach for isolation of RNA directly from urine is simpler and more practical. A weak point of our approach is the requirement for patient cooperation to obtain enough fresh urine, which means that young patients who cannot rapidly drink 1000–1500 ml water are not suitable for urine mRNA analysis.
Given that the deep intronic variants identified in the present study could be detected using whole genome sequencing, and in silico splicing prediction tools are usually used to select variants that are predicted to have an effect on splicing in a molecular diagnostic setting25, we assessed the reliability of HSF, NNSPLICE, and NetGene2 in discriminating between neutral and pathogenic variants. Assuming that the splice outcomes obtained from one tool were consistent with transcript analysis results, eight out of the nine variants detected in this study were correctly predicted, which indicated that these tools are useful to select deep intronic variants that are likely to be worth RNA analysis. However, extensive in silico analysis should be compared with transcript analysis results to determine their benefit in the context of molecular diagnosis.
In summary, three novel pathogenic COL4A4 variants and six novel pathogenic COL4A5 splicing variants were detected in five unrelated AS patients with negative NGS test results. All identified variants were deep intronic variants. As obtaining urine is feasible and non-invasive, we suggest analyzing COL4A3–5 mRNA from urine as the preferred method for evaluation of patients with clinically diagnosed or suspected AS with negative NGS results.