Stargardt disease is caused by variants in the gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.

Purpose Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation‐scanning methods. We aimed to develop a cost‐effective sequencing method for ABCA4 exons and regions carrying known causal deep‐intronic variants. Methods Fifty exons and 12 regions containing 14 deep‐intronic variants of ABCA4 were sequenced using double‐tiled single molecule Molecular Inversion Probe (smMIP)‐based next‐generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. Results Thirty‐four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep‐intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep‐intronic variant (c.4539+2065C>G) resulted in a 170‐nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). Conclusions smMIPs‐based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost‐effective mutation detection in STGD1 cases in previously unsolved cases.

Background The cervicovaginal microbiome (CVM) plays a significant role in women’s cervical health and disease. Microbial alterations at the species level and characteristic community state types (CST) have been associated with acquisition and persistence of high-risk human papillomavirus (hrHPV) infections that may result in progression of cervical lesions to malignancy. Current sequencing methods, especially most commonly used multiplex 16S rRNA gene sequencing, struggle to fully clarify these changes because they generally fail to provide sufficient taxonomic resolution to adequately perform species-level associative studies. To improve CVM species designation, we designed a novel sequencing tool targeting microbes at the species taxonomic rank and examined its potential for profiling the CVM. Results We introduce an accessible and practical circular probe-based RNA sequencing (CiRNAseq) technology with the potential to profile and quantify the CVM. In vitro and in silico validations demonstrate that CiRNAseq can distinctively detect species in a mock mixed microbial environment, with the output data reflecting its ability to estimate microbes’ abundance. Moreover, compared to 16S rRNA gene sequencing, CiRNAseq provides equivalent results but with improved sequencing sensitivity. Analyses of a cohort of cervical smears from hrHPV-negative women versus hrHPV-positive women with high-grade cervical intraepithelial neoplasia confirmed known differences in CST occurring in the CVM of women with hrHPV-induced lesions. The technique also revealed variations in microbial diversity and abundance in the CVM of hrHPV-positive women when compared to hrHPV-negative women. Conclusions CiRNAseq is a promising tool for studying the interplay between the CVM and hrHPV in cervical carcinogenesis. This technology could provide a better understanding of cervicovaginal CST and microbial species during health and disease, prompting the discovery of biomarkers, additional to hrHPV, that can help detect high-grade cervical lesions.

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