Novel PHEX Variants and Splicing Mutations in Patients with X-Linked Hypophosphatemia

Background: X-linked hypophosphatemia rickets (XLH) is a genetic disorder of phosphate wasting that causes the majority of inherited hypophosphatemic rickets. The disease is caused by mutations in the phosphate-regulating endopeptidase gene (PHEX). All types of mutations have been detected in the PHEX gene. There is no clear preference for the position and the type of mutation.Methods: In this study, we performed DNA sequencing and ex vivo splicing analysis to study the PHEX gene in ten sporadic patients and six core families with XLH. Results: A total of 25 patients were studied. Fifteen different mutations were detected in these patients, including five missense mutations, five splicing mutations, two nonsense mutations, two small deletions, and one small insertion. Five mutations were not previously reported. We also characterized the splicing mutations identified in our study, which consisted of exon skipping and activation of new splice sites in an exon or intron. Most of the observed changes resulted in a frameshift of the PHEX open reading frame.Conclusions: The genetic etiology in patients with XLH were successfully identified. This study expands the mutation database of the PHEX gene. We also explored the pathogenesis of XLH caused by splice site mutations. These results will contribute to the diagnosis of XLH and the pathogenicity analysis of mutations in patients.

APC Splicing Mutations Leading to In-Frame Exon 12 or Exon 13 Skipping Are Rare Events in FAP Pathogenesis and Define the Clinical Outcome

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.

Comprehensive characterisation of intronic mis-splicing mutations in human cancers

Previous studies studying mis-splicing mutations were based on exome data and thus our current knowledge is largely limited to exons and the canonical splice sites. To comprehensively characterise intronic mis-splicing mutations, we analysed 1134 pan-cancer whole genomes and transcriptomes together with 3022 normal control samples. The ratio-based splicing analysis resulted in 678 somatic intronic mutations, with 46% residing in deep introns. Among the 309 deep intronic single nucleotide variants, 245 altered core splicing codes, with 38% activating cryptic splice sites, 12% activating cryptic polypyrimidine tracts, and 36% and 12% disrupting authentic polypyrimidine tracts and branchpoints, respectively. All the intronic cryptic splice sites were created at pre-existing GT/AG dinucleotides or by GC-to-GT conversion. Notably, 85 deep intronic mutations indicated gain of splicing enhancers or loss of splicing silencers. We found that 64 tumour suppressors were affected by intronic mutations and blood cancers showed higher proportion of deep intronic mutations. In particular, a telomere maintenance gene, POT1, was recurrently mis-spliced by deep intronic mutations in blood cancers. We validated a pseudoexon activation involving a splicing silencer in POT1 by CRISPR/Cas9. Our results shed light on previously unappreciated mechanisms by which noncoding mutations acting on splicing codes in deep introns contribute to tumourigenesis.

APC Splicing Mutations Leading to In-Frame Exon Skipping are Rare Events in FAP Pathogenesis and Define the Clinical Outcome

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature me-ta-analysis of APC splicing mutations. We found that 123 unique APC splice site mutations, in-cluding the one described here, have been reported in FAP patients, 69 of which have been char-acterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with 4 mutations causing skipping of exon 12 and/or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and 5 mutations leading to skipping of exon 5, 7, 8, and (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations considered in this study cluster with the AFAP phenotype and delineate a novel molecular mechanism of pathogenesis in FAP disease.

Characteristics of Different Splicing Factor Mutation Hotspots in Myelofibrosis

CONTEXT: Splicing factor mutations (SRSF2, U2AF1, SF3B1, and ZRSR2) are present in ~50% of myelofibrosis (MF) patients. SRSF2 and U2AF1 Q157 are considered to be high-risk mutations, while the prognostic significance of ZRSR2 and SF3B1 has not been well established. As a group, splicing mutations are associated with cytopenias, the management of which is an area of unmet clinical need in MF. OBJECTIVE: To describe the clinical characteristics, treatment approaches, and outcomes in MF patients with splicing mutations. DESIGN: This is a single-institution, retrospective analysis of 133 MF patients with splicing mutations who presented to our institution between 2006 and 2019. PMF, post-ET MF, and post-PV MF were defined according to the World Health Organization and International Working Group criteria, respectively. Baseline variables were compared between patients harboring different splicing factor mutations and different mutations within the same splicing gene. Median overall survival (OS) was measured from time of diagnosis to date of death or censored at last follow up or date of transplant. Kaplan-Meier plots were created to compare LFS and OS among treatment cohorts, and differences were assessed using Log-rank tests. RESULTS: Among 133 MF patients with a splicing mutation, SRSF2 mutations were most common (n = 48), followed by U2AF1 (n = 36), SF3B1 (n = 27) and ZRSR2 mutations (n = 24). Most SRSF2 mutations occurred at P95 (90%). Thirty (83%) U2AF1 mutations occurred at Q157, with 5 (14%) at S34. Fourteen (63%) SF3B1 mutations occurred K666, with 9 (33%) at K700. Thirteen (54%) ZRSR2 mutations were in-frame insertions/deletions, 4 (17%) frameshift mutations, 3 (13%) nonsense mutations and 4 (17%) missense. All frameshift/nonsense ZRSR2 mutations occurred in males. Spliceosome mutations were mutually exclusive but for 2 cases (one had U2AF1 and SRSF2 mutations and the other had SF3B1 and ZRSR2 mutations). Baseline characteristics were similar between splicing mutations. The presence of a U2AF1 mutation correlated with lower hemoglobin (p 0.018) and U2AF1 Q157 mutations were associated with thrombocytopenia p=0.051) and higher DIPSS-plus scores (p=0.006). Severe thrombocytopenia (platelets < 50 x 109/L) was present in 20 (17%) patients and enriched in those with U2AF1 mutations (n = 9). ASXL1 mutations rarely occurred in conjunction with SF3B1 mutations (p = 0.007). Among all patients with splicing mutations, median OS was 60.6 months. Median OS was decreased in patients with SRSF2 mutations (33 vs 106 months, p=0.001) compared to those with other splicing mutations. Median OS was increased in patients with SF3B1 mutations compared to patients with other splicing mutations (181 mo vs 42 mo, p = 0.002). Median OS for patients with U2AF1 and ZRSR2 mutations was 44 and 106 months, respectively. Among patients with U2AF1 mutations, the presence of severe thrombocytopenia was associated with inferior survival (13.9 mo vs not reached, p = 0.045). The presence of an SRSF2 mutation was associated with an increased risk of leukemic transformation (24% vs 3%, p = 0.002). Among patients with SRSF2 mutations, median OS in those with documented leukemic transformation was 32.9 mo compared to 48.7 mo in those without (p = 0.17). CONCLUSIONS: Splicing mutations in MF have unique phenotypic and prognostic correlations. While SRSF2 mutations appear detrimental, SF3B1 mutations correlate with favorable outcomes. While U2AF1 and SRSF2 mutations are considered high-risk in MF, the impact appears driven by cytopenias in the former and leukemic transformation in the latter. This may hold relevance when considering therapeutic approaches in these patients. Disclosures Talati: AbbVie: Honoraria; Jazz: Speakers Bureau; Astellas: Speakers Bureau; BMS: Honoraria; Pfizer: Honoraria. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Sweet:Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Stemline: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria. Padron:Incyte: Research Funding; Kura: Research Funding; BMS: Research Funding; Novartis: Honoraria. Lancet:Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Komrokji:Geron: Honoraria; Novartis: Honoraria; Acceleron: Honoraria; Incyte: Honoraria; Abbvie: Honoraria; Agios: Speakers Bureau; BMS: Honoraria, Speakers Bureau; Jazz: Honoraria, Speakers Bureau. Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding.

Author Correction: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background splicing and genetic disease

We report that low level background splicing by normal genes can be used to predict the likely effect of splicing mutations upon cryptic splice site activation and exon skipping, with emphasis on the DBASS databases, BRCA1, BRCA2 and DMD. In addition we show that background RNA splice sites are also involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer. We discuss how background splicing information might inform splicing therapy.