Rare and low-frequency coding variants in CXCR2 and other genes are associated with hematological traits.

Hematological traits are important clinical parameters. To test the effects of rare and low-frequency coding variants on hematological traits, we analyzed hemoglobin concentration, hematocrit levels, white blood cell (WBC) counts and platelet counts in 31,340 individuals genotyped on an exome array. We identified several missense variants in CXCR2 associated with reduced WBC count (gene-based P = 2.6 × 10(-13)). In a separate family-based resequencing study, we identified a CXCR2 frameshift mutation in a pedigree with congenital neutropenia that abolished ligand-induced CXCR2 signal transduction and chemotaxis. We also identified missense or splice-site variants in key hematopoiesis regulators (EPO, TFR2, HBB, TUBB1 and SH2B3) associated with blood cell traits. Finally, we were able to detect associations between a rare somatic JAK2 mutation (encoding p.Val617Phe) and platelet count (P = 3.9 × 10(-22)) as well as hemoglobin concentration (P = 0.002), hematocrit levels (P = 9.5 × 10(-7)) and WBC count (P = 3.1 × 10(-5)). In conclusion, exome arrays complement genome-wide association studies in identifying new variants that contribute to complex human traits.

Differential methylation of genes and repeats in land plants.

The hypomethylated fraction of plant genomes is usually enriched in genes and can be selectively cloned using methylation filtration (MF). Therefore, MF has been used as a gene enrichment technology in sorghum and maize, where gene enrichment was proportional to genome size. Here we apply MF to a broad variety of plant species spanning a wide range of genome sizes. Differential methylation of genic and non-genic sequences was observed in all species tested, from non-vascular to vascular plants, but in some cases, such as wheat and pine, a lower than expected level of enrichment was observed. Remarkably, hexaploid wheat and pine show a dramatically large number of gene-like sequences relative to other plants. In hexaploid wheat, this apparent excess of genes may reflect an abundance of methylated pseudogenes, which may thus be more prevalent in recent polyploids.

Sequencing the maize genome.

Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

Maize genome sequencing by methylation filtration.

Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.