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We describe the variation in copy number of a ~ 10 kb region overlapping the long intergenic noncoding RNA (lincRNA) gene, TTTY22, within the IR3 inverted repeat on the short arm of the human Y chromosome, leading to individuals with 0-3 copies of this region in the general population. Variation of this CNV is common, with 266 individuals having 0 copies, 943 (including the reference sequence) having 1, 23 having 2 copies, and two having 3 copies, and was validated by breakpoint PCR, fibre-FISH, and 10× Genomics Chromium linked-read sequencing in subsets of 1234 individuals from the 1000 Genomes Project. Mapping the changes in copy number to the phylogeny of these Y chromosomes previously established by the Project identified at least 20 mutational events, and investigation of flanking paralogous sequence variants showed that the mutations involved flanking sequences in 18 of these, and could extend over > 30 kb of DNA. While either gene conversion or double crossover between misaligned sister chromatids could formally explain the 0-2 copy events, gene conversion is the more likely mechanism, and these events include the longest non-allelic gene conversion reported thus far. Chromosomes with three copies of this CNV have arisen just once in our data set via another mechanism: duplication of 420 kb that places the third copy 230 kb proximal to the existing proximal copy. Our results establish gene conversion as a previously under-appreciated mechanism of generating copy number changes in humans and reveal the exceptionally large size of the conversion events that can occur.

High-altitude adaptation in Tibetans is influenced by introgression of a 32.7-kb haplotype from the Denisovans, an extinct branch of archaic humans, lying within the endothelial PAS domain protein 1 (EPAS1), and has also been reported in Sherpa. We genotyped 19 variants in this genomic region in 1507 Eurasian individuals, including 1188 from Bhutan and Nepal residing at altitudes between 86 and 4550 m above sea level. Derived alleles for five SNPs characterizing the core Denisovan haplotype (AGGAA) were present at high frequency not only in Tibetans and Sherpa, but also among many populations from the Himalayas, showing a significant correlation with altitude (Spearman's correlation coefficient = 0.75, p value 3.9 × 10(-11)). Seven East- and South-Asian 1000 Genomes Project individuals shared the Denisovan haplotype extending beyond the 32-kb region, enabling us to refine the haplotype structure and identify a candidate regulatory variant (rs370299814) that might be interacting in an additive manner with the derived G allele of rs150877473, the variant previously associated with high-altitude adaptation in Tibetans. Denisovan-derived alleles were also observed at frequencies of 3-14% in the 1000 Genomes Project African samples. The closest African haplotype is, however, separated from the Asian high-altitude haplotype by 22 mutations whereas only three mutations, including rs150877473, separate the Asians from the Denisovan, consistent with distant shared ancestry for African and Asian haplotypes and Denisovan adaptive introgression.

We have assessed copy number variation (CNV) in the male-specific part of the human Y chromosome discovered by array comparative genomic hybridization (array-CGH) in 411 apparently healthy UK males, and validated the findings using SNP genotype intensity data available for 149 of them. After manual curation taking account of the complex duplicated structure of Y-chromosomal sequences, we discovered 22 curated CNV events considered validated or likely, mean 0.93 (range 0-4) per individual. 16 of these were novel. Curated CNV events ranged in size from <1 kb to >3 Mb, and in frequency from 1/411 to 107/411. Of the 24 protein-coding genes or gene families tested, nine showed CNV. These included a large duplication encompassing the AMELY and TBL1Y genes that probably has no phenotypic effect, partial deletions of the TSPY cluster and AZFc region that may influence spermatogenesis, and other variants with unknown functional implications, including abundant variation in the number of RBMY genes and/or pseudogenes, and a novel complex duplication of two segments overlapping the AZFa region and including the 3' end of the UTY gene.

We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ~300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.

We have surveyed 15 high-altitude adaptation candidate genes for signals of positive selection in North Caucasian highlanders using targeted re-sequencing. A total of 49 unrelated Daghestani from three ethnic groups (Avars, Kubachians, and Laks) living in ancient villages located at around 2,000 m above sea level were chosen as the study population. Caucasian (Adygei living at sea level, N = 20) and CEU (CEPH Utah residents with ancestry from northern and western Europe; N = 20) were used as controls. Candidate genes were compared with 20 putatively neutral control regions resequenced in the same individuals. The regions of interest were amplified by long-PCR, pooled according to individual, indexed by adding an eight-nucleotide tag, and sequenced using the Illumina GAII platform. 1,066 SNPs were called using false discovery and false negative thresholds of ~6%. The neutral regions provided an empirical null distribution to compare with the candidate genes for signals of selection. Two genes stood out. In Laks, a non-synonymous variant within HIF1A already known to be associated with improvement in oxygen metabolism was rediscovered, and in Kubachians a cluster of 13 SNPs located in a conserved intronic region within EGLN1 showing high population differentiation was found. These variants illustrate both the common pathways of adaptation to high altitude in different populations and features specific to the Daghestani populations, showing how even a mildly hypoxic environment can lead to genetic adaptation.

Three populations from northern Pakistan, the Burusho, Kalash, and Pathan, claim descent from soldiers left behind by Alexander the Great after his invasion of the Indo-Pak subcontinent. In order to investigate their genetic relationships, we analyzed nine Alu insertion polymorphisms and 113 autosomal microsatellites in the extant Pakistani and Greek populations. Principal component, phylogenetic, and structure analyses show that the Kalash are genetically distinct, and that the Burusho and Pathan populations are genetically close to each other and the Greek population. Admixture estimates suggest a small Greek contribution to the genetic pool of the Burusho and Pathan and demonstrate that these two northern Pakistani populations share a common Indo-European gene pool that probably predates Alexander's invasion. The genetically isolated Kalash population may represent the genetic pool of ancestral Eurasian populations of Central Asia or early Indo-European nomadic pastoral tribes that became sequestered in the valleys of the Hindu Kush Mountains.