U2AF1 mutations alter splice site recognition in hematological malignancies.

Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 alter its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in patients, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1's zinc finger domains.

Efficient identification of rare variants in large populations: deep re-sequencing the CRP locus in the CARDIA study.

Effect sizes of many common single nucleotide polymorphisms identified in genome-wide association studies generally explain only a modest fraction of the total estimated heritability in a variety of traits. One hypothesis is that rare variants with larger effects might account for the missing heritability. Despite advances in sequencing technology, discovering rare variants in a large population is still economically challenging. Sequencing pooled samples can reduce the cost, but detecting rare variants and identifying individual carriers is difficult and requires additional experiments. To address these issues, we have developed a rare variant-detection algorithm V-Sieve to screen for rare alleles in pooled DNA samples which, in combination with a unique pooling strategy, is able to efficiently screen a candidate gene for idiosyncratic variants in thousands of samples. We applied this method to 2283 individuals, and identified >100 polymorphisms in the C-reactive protein locus at an allele frequency as low as 0.02%, with a positive predictive rate of 93%. We believe this algorithm will be useful in both screening for rare variants in genomic regions known to associate with particular phenotypes and in replicating rare variant associations identified in large-scale studies, such as exome re-sequencing projects.

SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition.

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.