Of note, 19% of the variants affect the essential GT-AG splice sites, 71% affect the extended donor (5ʹ) splice site or acceptor (3ʹ) splice-site regions, 27% were exonic variants ( Figure 1D), and 2 were structural/copy number variants. RT-PCR and Sanger sequencing were performed for 129 individuals (2 quads, 18 trios, 13 duos, and 41 singletons) encompassing 79 splicing variants ( Figure 1D and E).
STRANDED DEEP TRAINER .12 SKIN
RNA was derived from blood (65/74), skin fibroblasts (13/74), urothelial cells (7/74), and/or an available biopsy specimen (3/74) ( Figure 1C). A total of 74 families with diverse Mendelian conditions were recruited with 6% with prenatal, 25% with severe-congenital, 47% with early childhood, and 22% with teenage-adult onset ( Figure 1B Supplemental Table 4). Our devised inclusion criteria to ascertain variants with high clinical suspicion of causality were (1) a high likelihood of a monogenic Mendelian disorder, (2) variant allele frequency consistent with disease incidence, (3) putative splicing variant in a clinician-defined, phenotypically concordant gene, and (4) preferably the variant segregates with disease (some cases studied in parallel with segregation because of clinical urgency). CSS, cryptic splice site EBV, Epstein-Barr virus LCLs, lymphoblastoid cell lines PGD, preimplantation genetic diagnosis PGS, preimplantation genetic screening RT-PCR, reverse transcription polymerase chain reaction TPM, transcripts per million TSS, transcription start site. Schematic design of a junctional PCR primer bridging 2 exon junctions. Schematic illustrating the range of theoretical mis-splicing outcomes that may arise from a splicing variant. Summary of clinical impact metrics returned from referring clinicians by survey.
Summary of mis-splicing events detected and their effect on protein reading frame. Variant classification of putative splicing variants (E) before and (F) after splicing studies. Black: variants maintaining normal splicing. Red: variants shown to induce mis-splicing. Position of putative splicing variants analyzed in this study relative to the donor splice site or acceptor splice site.
Venn diagram summarizing the biospecimens used as a source of RNA for this study. Pie chart depicting age of disease onset for affected individuals from 74 families subject to RNA diagnostics. Informatics analyses of RNA sequencing data from whole blood, EBV-LCLs, skin fibroblasts, or urothelial cells shows that 81.3% of OMIM genes linked to clinically relevant Mendelian disorders are expressed at TPM >0.5 levels feasible for analysis by RT-PCR. Figure 1 Overview of the cohort of 74 families triaged in real time from clinical genomics into RNA diagnostics.
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