Splicing Outcomes of 5' Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays.

Combining data derived from a meta-analysis of human disease-associated 5' splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15-18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

TSC1 intragenic deletion transmitted from a mosaic father to two siblings with cardiac rhabdomyomas: Identification of two aberrant transcripts.

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder characterized by non-cancerous tumors in multiple organs including the brain, kidney, lung, heart, and skin. We encountered a Japanese family consisting of two siblings (a four-year-old boy and a one-year-old girl) with multiple cardiac rhabdomyomas conveying a high risk of TSC and apparently unaffected sibling (a two-year-old girl) and parents. Whole exome sequencing and application of Integrative Genomic Viewer revealed an identical intragenic TSC1 deletion with the breakpoints on intron 15 and exon 19 in the affected siblings, but not in the apparently unaffected sibling and parents. Subsequently, PCR-based analyses were performed using primers flanking the deletion, showing that the deletion was also present in the father and that the deletion occurred between chr9:135,777,038 (bp) and chr9:135,780,540 (bp) in association with a one bp overlap. Furthermore, RT-PCR analyses were carried out using lymphoblastoid cell lines, revealing a major in-frame insertion/deletion transcript produced by aberrant splicing using a cryptic ″ag″ splice acceptor motif at intron 15 (r.1998_2438delinsTTCATTAGGTGG) and a minor frameshift transcript generated by aberrant splicing between exon 15 and exon 20 (r.1998_2502del, p.Lys666Asnfs*15) in the affected siblings. These findings imply that the intragenic deletion producing two aberrant transcripts was generated as a somatic pathogenic variant involving the germline in the father and was transmitted to the affected siblings.

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

Exome sequencing identifies a novel intronic mutation in ENG that causes recurrence of pulmonary arteriovenous malformations.

Hereditary hemorrhagic telangiectasia (HHT) occasionally can be discovered in patients with cerebrovascular disease. Pulmonary arteriovenous malformation (PAVM) is one of the complications in HHT and occasionally is causative for life-threatening embolic stroke. Several genetic defects have been reported in patients with HHT. The broad spectrum of phenotype and intrafamilial phenotype variations, including age-at-onset of vascular events, often make an early diagnosis difficult. We present here a Japanese family with a novel intronic heterozygous mutation of ENG, which was identified using whole exome sequencing (WES). The intronic mutation, IVS3+4delAGTG, results in in-frame deletion of exon 3 and would produce a shorter ENG protein lacking the extracellular forty-seven amino acid sequences, which is located within the orphan domain. Our findings highlight the importance of the domain for the downstream signaling pathway of transforming growth factor-beta and bone morphogenesis protein superfamily receptors. Considering the phenotype variations and the available treatment for vascular complications, an early diagnosis using genetic testing, including WES, should be considered for individuals at risk of HHT.

Characterization of aberrant FHIT transcripts in gastric adenocarcinomas

Aberrant transcripts of FHIT (fragile histidine triad) have been reported in several types of primary tumors and cell lines, including gastric carcinoma. The role of these aberrant transcripts in tumorigenesis is not clear yet. Forty-eight aberrant-sized FHIT transcripts with various lengths and number in 35 cases of gastric adenocarcinomas were further characterized. Aberrant transcripts, with deletions and/or insertions, were frequently observed in 20 cases of tumors. Sequence analysis demonstrated that different types of aberrant transcripts used normal splice sites but skipped exons, contained the inserts with the part of intron 5 sequences, or used the FHIT cDNA sequence 179-180 as a cryptic splice acceptor site. Most of aberrant transcripts lacked exon 5 and were presumably non-functional as the translation initiation codon is located in exon 5. Additionally, other transcripts, indicative of additional splice processing, either deletions or insertions, were expressed in several tumors. Taken together, our data indicate that the FHIT gene expression is frequently altered in gastric adenocarcinomas by aberrant splicing, and suggest that different types of aberrant transcripts may result during the multi-step splice processing.