Integrated detection and population-genetic analysis of SNPs and copy number variation

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.     

Science policy and science policy research

This assessment of the value of science policy research in the making of policies is based on a lecture given at a meeting of the Sociology of Science Study Group of the British Sociological Association last March. The thoughts it contains are of course those of Dr Blume himself, and not of the Department of Education and Science or the Council for Scientific Policy.     

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.     

Nova regulates brain-specific splicing to shape the synapse

Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2-/- mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.