Genetic features of Tibetans have been broadly investigated, but the properties of copy number variation (CNV) have not been well examined. To get a preliminary view of CNV in Tibetans, we scanned 29 Tibetan genomes with the Illumina Human-1 M high-resolution genotyping microarray and identified 139 putative copy number variable regions (CNVRs), consisting of 70 deletions, 61 duplications, and 8 multi-allelic loci. Thirty-four of the 139 CNVRs showed differential allele frequencies versus other East-Asian populations, with P values <0.0001. These results indicated a distinct pattern of CNVR allele frequency distribution in Tibetans. The Tibetan CNVRs are enriched for genes in the disease class of human reproduction (such as genes from the DAZ, BPY2, CDY, and HLA-DQ and -DR gene clusters) and biological process categories of “response to DNA damage stimulus” and “DNA repair” (such as RAD51, RAD52, and MRE11A). These genes are related to the adaptive traits of high infant birth weight and darker skin tone of Tibetans, and may be attributed to recent local adaptation. Our results provide a different view of genetic diversity in Tibetans and new insights into their high-altitude adaptation.
Citation: Zhang Y-B, Li X, Zhang F, Wang D-M, Yu J (2012) A Preliminary Study of Copy Number Variation in Tibetans. PLoS ONE 7(7): e41768. doi:10.1371/journal.pone.0041768
Editor: Jeong-Sun Seo, Seoul National University College of Medicine, Republic of Korea
Received: January 20, 2012; Accepted: June 27, 2012; Published: July 23, 2012
Copyright: © 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Environmental hypoxia, high ultraviolet (UV) radiation, low temperatures, and low precipitation are the harsh nature of the Tibetan Plateau. Modern Tibetans have adapted to live in these rigorous conditions, as evidenced by their efficient oxygen utilization, absence of chronic mountain sickness, and high infant survival rate . Although it is known that Tibetans are a branch of East-Asian (EA) populations , , , long-term endurance in the harsh environment has facilitated the divergence of Tibetans from other East-Asian populations with a distinct set of genetic characteristics .
Studies on Tibetans have mostly focused on disease susceptibility , , migration history , , , and high-altitude adaptation , . Great success was achieved with the use of genetic markers from SNP, STR, and haplotypes of mitochondria, as well as Y chromosomes. However, very few CNV studies were reported of Tibetans so far. DNA copy number variation is an important type of genomic variation that can influence phenotype through gene dosage effect and/or alteration of the genomic architecture , and is a good candidate for natural selection . Copy number variable region (CNVR) usually contains higher nucleotide count per genome than that of SNP , and may contain more than one gene and/or their regulatory regions . CNVs have been shown to account for nearly 18% of variation in gene expression . In humans, breakpoints of a CNV preferentially possess sequence motifs related to chromosomal rearrangement or genome instability . These suggest that the impacts of CNVs on genomes may easily turn into phenotypic alterations.
Recently, more CNVs have been implicated in pathogenesis, from rare genetic disorders to a wide-range of common diseases . Most CNV-related diseases are systemic autoimmune diseases, mood and psychotic disorders, and AIDS , , , . In addition to the pathogenic CNVs, extensive amounts of common CNVs have been identified from healthy individuals. Discovery studies of CNVs with diverse populations found significant differences in the frequencies of CNVs among distinct ethnic groups , . With genotyping microarrays, the number of CNVs identified per individual genome ranged from 3.5 in African Americans  to 40.3 in Koreans . Despite advances in discovery and functional studies, the exhaustion of the non-pathogenic CNV pool with larger sample sizes and more ethnic groups will portray a better genomic landscape in humans.
To characterize the allele frequency distribution of CNVs in Tibetans and to explore the role of CNVs in high-altitude adaptation, we scanned 29 Tibetan genomes, using high-resolution genotyping microarrays. This preliminary study will further improve our understanding of the genetic diversity of Tibetans and will benefit research on high-altitude adaptation and disease susceptibility of Tibetans.
The General Characteristics of CNV and CNVR
With high-resolution genotyping microarrays, we identified 562 candidate CNVs from 29 Tibetans, with an average of 19.38 CNVs per genome and a corresponding average length of 6.07 Mb per genome. The mean size of our CNVs was 313 kb, ranging from 75.5 kb to 4.2 Mb. These 562 CNVs corresponded to 139 CNVRs (70 deletions, 61 duplications, and 8 multi-allelic loci), covering a total of 1.67 percent of the human genome (Figure 1, Table S1). Of the 139 CNVRs, 123 (88%) were found to be overlapped with CNVRs from the Database of Genomic Variants (DGV, latest updated: Nov 02, 2010) and were thus considered known CNVRs. About half of our known CNVRs (62 out of the 123 CNVRs) were common, with allele frequency greater than 5%. The remaining 16 CNVRs (12%; 12 deletions and 4 duplications) were novel, all with allele frequency less than 5% and 14 of them were validated by real-time quantitative PCR (Table S2). The 139 CNVRs encoded 344 genes as annotated by RefSeq (release 50), of which 184 genes had an Entrez gene summary. About 70% of our CNVRs overlapped with RefSeq genes, with no strong bias of harboring RefSeq genes between the deletion and duplication regions (78% and 57%, respectively).
Figure 1. Structural variation map of Tibetan samples.
The schematic summarizes the distribution of duplications, deletions and multi-allelic loci on each human chromosome.doi:10.1371/journal.pone.0041768.g001
Compare with East-Asian Populations
To better understand the properties of CNVs found in Tibetans, we compared some relevant statistics of our results with those of East-Asian (EA) populations and HapMap Phase II Samples (Table 1) , , , , . The analyzed EA populations were composed of 300 Han Taiwanese , 692 Han Chinese , 800 Han Taiwanese , and 3578 Koreans . With the Human-1 M chip, we found that in Tibetans, the average sizes of CNVs (313 kb) and CNVRs (370.6 kb), as well as the average CNV coverage per genome (6.07 Mb), are larger than those in other EA populations (Table 1). Our results may be influenced by our detection platform, which is sensitive to the larger-sized CNVs . Comparing the shared CNVRs between and among populations, we noticed that Tibetans share fewer CNVRs with Han Chinese than with other populations. In total, 74 shared CNVRs were found between Tibetan and other EA populations. Of the shared CNVRs, 25 showed significant differences in allele frequency (P values <0.0001; Table S1), and an additional 9 were exclusively present in Tibetans, all with high allele frequency (>10%). Combining these two observations, we extracted 34 CNVRs as a putative distinct set, of which allele frequency distributions were significantly deviated from other EA populations. This set of CNVRs may offer information for our subsequent analyses on the high-altitude adaptation of Tibetans.
Table 1. Characteristic summary of CNVRs found in Tibetan and other populations.doi:10.1371/journal.pone.0041768.t001
To understand the Tibetan-specific biological effects from our CNVs, we focused on the aforementioned 34 CNVRs, including 18 duplications, 13 deletions, and 3 multi-allelic loci. Among the 34 CNVRs, 14 CNVRs overlapped with Entrez genes and are listed in Table 2. Each of the 14 CNVRs was larger than 100 kb, and over half of them (57%) were in the form of deletion. In addition, autosomal chromosomes had mostly deletion type of CNVRs, whereas sex chromosomes had mostly duplication. The allele frequency discrepancy of variants among populations can be affected by factors, such as selection, genetic drift, and gene flow. Still, local adaptation is a major selection force to shape the discrepancy among geographically close populations , . Therefore, these 14 CNVRs that showed significant differences in allele frequency between Tibetan and other EA populations were potentially due to recent adaptation. Furthermore, we found 4 CNVRs encoded genes that can affect human reproductive ability (such as genes from the gene clusters of DAZ, CDY, and XKRY) and may play an important role in high-altitude adaptation of Tibetans .
Table 2. CNVRs with Entrez gene and significant difference in allele frequency.doi:10.1371/journal.pone.0041768.t002
Gene Ontology and Genetic Pathway Analyses
To further understand the functional implications of our CNVRs, in total, 184 Entrez genes from the 139 CNVRs were subjected to functional annotation and classification analysis with DAVID v6.7 . We found 28 disease-related genes, classified into three classes: reproduction, cancer, and infection (Table 3). Cancer and infection related genes are frequently reported in other CNV studies , , , whereas human reproductive genes are rarely mentioned. Within these reproductive genes, HLA-DRB1, HLA-DQA1, and HLA-DQB1 have been implicated in high infant birth weight and pregnancy loss , , and several genes present in the gene clusters of DAZ, BPY2, CDY, and XKRY have been reported to affect male germ cell development and fertilization , , . The corresponding CNVRs of these reproductive genes exhibited lower frequencies in Tibetan than that in other EA populations (P values <0.009), as well as CEU (P values <0.0009) and YRI (P values <0.005).
Table 3. Genes in Tibetan CNVRs that are associated with disease risk.doi:10.1371/journal.pone.0041768.t003
By investigating enriched Gene Ontology categories, we found an overrepresentation of genes in categories of “sensory perception” (P values <0.0001) and “defense response to pathogens” (P values <0.008), which were also enriched in other CNV studies with East-Asian populations , . Considering Tibetan-specific traits, we paid more attention to the categories of “female pregnancy,” “response to DNA damage stimulus,” and “DNA repair” (P values <0.004) (Table 4). The category of female pregnancy was enriched for the overrepresentation of the PSG gene family members. The allele frequencies of their corresponding CNVRs were similar between Tibetan and other EA populations. The last two categories contained 11 genes (RAD51, RAD52, ANKRD17, MICA, EYA2, MRE11A, UBR5, RRM2B, ESCO2, GTF2H2, and ATRX) corresponding to 10 CNVRs. It is worth noting that 7 of the 10 CNVRs were only observed in Tibetans.
Table 4. GO term enrichment of genes within Tibetan CNVRs.doi:10.1371/journal.pone.0041768.t004
To further investigate the roles of CNVRs in high-altitude adaptation, we interrogated our data with annotations from the KEGG Pathway Database and Pathway Commons Database. Within all enriched pathways, we found 6 genes (RAD51, RAD52, RRM2B, POLR2J, GTF2H2, and MRE11A) involved in pathways related to DNA repair, such as “double-strand break repair,” “homologous recombination repair,” and “ATM-mediated response to DNA double-strand break”. Both results from genetic pathway and gene ontology analyses suggest that the DNA repair genes identified from our CNVRs may facilitate the adaptive trait of Tibetans.
Studies of Tibetan populations often provide knowledge of human migration and evolution for endurance of harsh natural conditions at high altitude. We generated a CNV profile with Tibetan genomes and added more understanding to the genetic diversity of Tibetans under the influence of high-altitude adaptation.
In this study, 139 putative CNVRs were found with a majority of them reported in DGV. Only 14 of them were newly identified CNVRs. We didn’t observe any Tibetan-specific common CNVRs (≥5%), which may provide clues to the migration and adaptation of Tibetan population. The detectable rate of a common CNVR was dependent on sample size. With only 29 Tibetan genomes, the Tibetan-specific common CNVRs may be under-ascertained here. Thus far, very few population-specific common CNVRs have been identified, even with large sample sizes . A study using 3,578 Koreans reported that 15 out of 4,003 identified CNVRs were Korean-specific common CNVRs, with all frequencies less than 10% . The lack of population-specific common CNVRs may be due to most de novo CNVRs being deleterious and having been eliminated by subsequent selection , .
For all the 139 CNVRs, the percentage of deletions overlapping with RefSeq genes is slightly higher than that of duplications (78% vs. 57%; P>0.05). Furthermore, a significant preference for overlapping of RefSeq genes in deletions over duplications (68% vs. 32%; P<0.02) was observed in a subset of the 139 CNVRs, which contained the 14 novel CNVRs and the 34 CNVRs showing allele frequency discrepancy with other EA populations. This preference is contrary to previous findings that deletions were found to contain less RefSeq genes than that of duplications , . Some factors, such as under-ascertainment of small-size CNVs (<100 kb) with current microarrays, CNV reproducibility among different platforms and analytic tools , and different detection rates between duplication and deletion , , may influence our observations. However, our findings may be due to the need to improve fitness in high-altitude harsh environments. In this case, functional genes containing deletions may serve as substrates for natural selection in Tibetans , .
In the past two years, various beneficial genes, such as EPAS1, EGLN1, ANGPT1, and PPARA, were reported in studies of high-altitude adaptation , . None of these genes were found to overlap with our CNVRs. Using all the identified Entrez genes (184) in this study, we conducted a functional annotation and classification analysis, and identified several enriched categories related to infant survival, such as birth weight, male infertility, and female pregnancy. One of the adaptive traits of Tibetans is high infant survival rate, which tightly correlates with heavier birthweight . Genetic factors associated with heavier birthweight contribute to high-altitude adaptation. Our findings are consistent with previous results that the genes related to infant survival were under positive selection in Tibetans . The enriched categories contain genes from the DAZ, BYP, and HLA-DR and -DQ gene clusters. Their corresponding CNVRs exhibited significant discrepancy in allele frequency between Tibetan and EA populations, which suggested important roles of these genes in recent adaptation of Tibetans.
The yield of radiation on Tibetan plateau is about two times higher than that found at sea level at the same latitude . Darker skin tone is a distinctive trait of Tibetans and is mainly caused by high UV radiation. It is also known that UV radiation can lead to DNA damage  and impose a selection pressure on Tibetans. Within our CNVRs, at least 11 genes, such as MRE11A, RAD51, and RAD52, were involved in the gene ontology categories of “response to DNA damage stimulus” and “DNA repair” and genetic pathways of “double-strand break repair” and “homologous recombination repair”. All their corresponding CNVRs have low allele frequencies in Tibetans, but most of them are absent from other EA populations. In addition, three genes, ATP7A, KGFLP2, and KGFLP1, in epidermis morphogenesis, were also found in our CNVRs. These DNA repair and epidermis morphogenesis genes may be involved in UV radiation damage and play important roles in recent adaptation of Tibetans.
Our study presents a preliminary survey of copy number variation in Tibetans. We found that compared to other EA populations, Tibetans have a distinctive pattern of CNVR allele frequency distribution. The CNVRs are enriched for genes related to traits of infant survival rate and darker skin tone, thus, the suggestion of their involvement in local adaptation of Tibetans. Despite the need for more functional studies with larger sample size in the future, our results provide insights to high-altitude adaptation in Tibetans.
Materials and Methods
All donors signed informed consent forms for cell line establishment and subsequent biological investigations. This project was reviewed and approved by the Ethics Committee of the Beijing Institute of Genomics, Chinese Academy of Sciences.
Samples and CNV Discovery
The peripheral blood samples of 29 unrelated Tibetans (16 males and 13 females) from Lhasa (at an altitude of 3700 meters) were used to prepare immortalized cell lines in this study. All DNA samples were obtained with DNA-extraction kits (Tiangen Biotech, Beijing, China) and genotyped on the Human 1M-Duo v3 chip (Illumina, CA, USA), according to the manufacturer's specifications. The microarray chip contained nearly 1.2 million SNPs and an additional 60,000 probes designed for CNV detection.
Genotyping modules from Genomestudio (Illumina, CA, USA) were employed to call the raw data. CNV Partition v2.3.4 (Illumina, CA, USA) was used to detect the presence of a CNV and to estimate the copy number of a CNV, based on log of the signal intensity (log R ratio) and B allele frequency measurement at each probe. Only CNVs detected with more than 5 probes with confidence scores greater than 35 were considered. Copy number variable regions (CNVRs) were defined after consolidation of all the overlapping CNVs. The CNVs obtained from this study were compared to the CNVs in the Database of Genomic Variants (http://projects.tcag.ca/variation/) and the CNVs from recent publications, using genotyping microarrays with Han Taiwanese, Han Chinese, and Koreans , , , , . The P value of the allele frequency discrepancy was determined by a modified Chi-square test.
Validation of CNVs
To validate CNVs identified by our chip method, we conducted a genomic real-time quantitative PCR with the 7500 Real-Time PCR system (Applied Biosystems, CA, USA). All novel CNVs were included for validation. The primers of the PCR are listed in supplementary material Table S2. To a 25-ul PCR reaction, 12.5 ul of SYBR Green Master mix (Applied Biosystems, CA, USA), 50 ng of genomic DNA, and 10 pmol of each primer were included. We validated our 16 novel CNVRs according to the method from D’Haene et al . For each validation, the copy number of a CNVR was determined by the qPLUS software with the use of ZNF80 and GPR15 for normalization and a sample with copy number variation equal 2 as positive control and water as negative control. Each reaction was performed in replication. Of all 16 novel CNVRs, 14 of them were validated by quantitative PCR. We did not remove them from analysis for their neglectable effect on statistics.
Gene Set Enrichment Analysis
We submitted all 184 Entrez genes that overlapped with our CNVRs to DAVID v6.7 for functional annotation and classification analysis . The P value of gene set enrichment was calculated by a modified Fisher’s exact test. We kept GO terms and disease classes with P value less than 0.05. As for the pathway enrichment analysis, Web-based Gene Set Analysis Toolkit 2.0 was used with the pathway annotation from the KEGG Pathways Database and the Pathway Commons Database .
Details of CNVRs and CNVs observed in Tibetans.
Genomic qPCR validation of the 16 novel CNVRs identified in Tibetans.
Conceived and designed the experiments: FZ YBZ. Performed the experiments: XL YBZ. Analyzed the data: YBZ XL. Contributed reagents/materials/analysis tools: FZ. Wrote the paper: YBZ XL DMW JY.
- 1. Beall CM (2007) Detecting natural selection in high-altitude human populations. Respir Physiol Neurobiol 158: 161–171.
- 2. Zhao M, Kong QP, Wang HW, Peng MS, Xie XD, et al. (2009) Mitochondrial genome evidence reveals successful Late Paleolithic settlement on the Tibetan Plateau. Proc Natl Acad Sci U S A 106: 21230–21235.
- 3. Zhang Y, Zhang F, Lin H, Shi L, Wang P, et al. (2010) Nucleotide polymorphism of the TNF gene cluster in six Chinese populations. J Hum Genet.
- 4. Kang L, Li S, Gupta S, Zhang Y, Liu K, et al. (2010) Genetic structures of the Tibetans and the Deng people in the Himalayas viewed from autosomal STRs. J Hum Genet 55: 270–277.
- 5. Wang B, Zhang YB, Zhang F, Lin H, Wang X, et al. (2011) On the origin of Tibetans and their genetic basis in adapting high-altitude environments. PLoS One 6: e17002.
- 6. Chen W, Liu Q, Wang H, Johnson RJ, Dong X, et al. (2011) Prevalence and risk factors of chronic kidney disease: a population study in the Tibetan population. Nephrol Dial Transplant 26: 1592–1599.
- 7. Buroker NE, Ning XH, Zhou ZN, Li K, Cen WJ, et al. (2010) Genetic associations with mountain sickness in Han and Tibetan residents at the Qinghai-Tibetan Plateau. Clin Chim Acta 411: 1466–1473.
- 8. Gayden T, Mirabal S, Cadenas AM, Lacau H, Simms TM, et al. (2009) Genetic insights into the origins of Tibeto-Burman populations in the Himalayas. J Hum Genet 54: 216–223.
- 9. Simonson TS, Yang Y, Huff CD, Yun H, Qin G, et al. (2010) Genetic evidence for high-altitude adaptation in Tibet. Science 329: 72–75.
- 10. Feuk L, Carson AR, Scherer SW (2006) Structural variation in the human genome. Nat Rev Genet 7: 85–97.
- 11. Itsara A, Wu H, Smith JD, Nickerson DA, Romieu I, et al. (2010) De novo rates and selection of large copy number variation. Genome Res 20: 1469–1481.
- 12. Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, et al. (2006) Global variation in copy number in the human genome. Nature 444: 444–454.
- 13. Stranger BE, Forrest MS, Dunning M, Ingle CE, Beazley C, et al. (2007) Relative impact of nucleotide and copy number variation on gene expression phenotypes. Science 315: 848–853.
- 14. Conrad DF, Pinto D, Redon R, Feuk L, Gokcumen O, et al. (2010) Origins and functional impact of copy number variation in the human genome. Nature 464: 704–712.
- 15. Fanciulli M, Petretto E, Aitman TJ (2010) Gene copy number variation and common human disease. Clin Genet 77: 201–213.
- 16. Chen X, Li X, Wang P, Liu Y, Zhang Z, et al. (2010) Novel association strategy with copy number variation for identifying new risk Loci of human diseases. PLoS One 5: e12185.
- 17. McCarroll SA, Altshuler DM (2007) Copy-number variation and association studies of human disease. Nat Genet 39: S37–42.
- 18. Roll P, Sanlaville D, Cillario J, Labalme A, Bruneau N, et al. (2010) Infantile convulsions with paroxysmal dyskinesia (ICCA syndrome) and copy number variation at human chromosome 16p11. PLoS One 5: e13750.
- 19. Wain LV, Armour JA, Tobin MD (2009) Genomic copy number variation, human health, and disease. Lancet 374: 340–350.
- 20. Li J, Yang T, Wang L, Yan H, Zhang Y, et al. (2009) Whole genome distribution and ethnic differentiation of copy number variation in Caucasian and Asian populations. PLoS One 4: e7958.
- 21. McElroy JP, Nelson MR, Caillier SJ, Oksenberg JR (2009) Copy number variation in African Americans. BMC Genet 10: 15.
- 22. Yim SH, Kim TM, Hu HJ, Kim JH, Kim BJ, et al. (2010) Copy number variations in East-Asian population and their evolutionary and functional implications. Hum Mol Genet 19: 1001–1008.
- 23. Lin CH, Lin YC, Wu JY, Pan WH, Chen YT, et al. (2009) A genome-wide survey of copy number variations in Han Chinese residing in Taiwan. Genomics 94: 241–246.
- 24. McCarroll SA, Kuruvilla FG, Korn JM, Cawley S, Nemesh J, et al. (2008) Integrated detection and population-genetic analysis of SNPs and copy number variation. Nat Genet 40: 1166–1174.
- 25. Lin CH, Li LH, Ho SF, Chuang TP, Wu JY, et al. (2008) A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan. BMC Genet 9: 92.
- 26. Halper-Stromberg E, Frelin L, Ruczinski I, Scharpf R, Jie C, et al. (2011) Performance assessment of copy number microarray platforms using a spike-in experiment. Bioinformatics 27: 1052–1060.
- 27. Coop G, Pickrell JK, Novembre J, Kudaravalli S, Li J, et al. (2009) The role of geography in human adaptation. PLoS Genet 5: e1000500.
- 28. Barreiro LB, Laval G, Quach H, Patin E, Quintana-Murci L (2008) Natural selection has driven population differentiation in modern humans. Nat Genet 40: 340–345.
- 29. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57.
- 30. Thompson PA, Brewster AM, Kim-Anh D, Baladandayuthapani V, Broom BM, et al. (2011) Selective genomic copy number imbalances and probability of recurrence in early-stage breast cancer. PLoS One 6: e23543.
- 31. Park H, Kim JI, Ju YS, Gokcumen O, Mills RE, et al. (2010) Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing. Nat Genet 42: 400–405.
- 32. Aroviita P, Partanen J, Sistonen P, Teramo K, Kekomaki R (2004) High birth weight is associated with human leukocyte antigen (HLA) DRB1*13 in full-term infants. Eur J Immunogenet 31: 21–26.
- 33. Kruse C, Steffensen R, Varming K, Christiansen OB (2004) A study of HLA-DR and -DQ alleles in 588 patients and 562 controls confirms that HLA-DRB1*03 is associated with recurrent miscarriage. Hum Reprod 19: 1215–1221.
- 34. Tse JY, Wong EY, Cheung AN, O WS, Tam PC, et al. (2003) Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility. Biol Reprod 69: 746–751.
- 35. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 10: 383–393.
- 36. Machev N, Saut N, Longepied G, Terriou P, Navarro A, et al. (2004) Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility. J Med Genet 41: 814–825.
- 37. Itsara A, Cooper GM, Baker C, Girirajan S, Li J, et al. (2009) Population analysis of large copy number variants and hotspots of human genetic disease. Am J Hum Genet 84: 148–161.
- 38. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, et al. (2011) Copy number variation and selection during reprogramming to pluripotency. Nature 471: 58–62.
- 39. Conrad DF, Andrews TD, Carter NP, Hurles ME, Pritchard JK (2006) A high-resolution survey of deletion polymorphism in the human genome. Nat Genet 38: 75–81.
- 40. Pinto D, Darvishi K, Shi X, Rajan D, Rigler D, et al. (2011) Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants. Nat Biotechnol 29: 512–520.
- 41. Nguyen DQ, Webber C, Ponting CP (2006) Bias of selection on human copy-number variants. PLoS Genet 2: e20.
- 42. Cooper GM, Nickerson DA, Eichler EE (2007) Mutational and selective effects on copy-number variants in the human genome. Nat Genet 39: S22–29.
- 43. Nuosang Cuojia, Zhaduo, Ouzhu B, STAMNES J (1998) Discusses Lhasa urban district rat damage and the measure of science preventing and controlling shallowly. J Tibet Univ 24: 5–9.
- 44. Zasloff M (2005) Sunlight, vitamin D, and the innate immune defenses of the human skin. J Invest Dermatol 125: xvi-xvii.
- 45. D’Haene B, Vandesompele J, Hellemans J (2010) Accurate and objective copy number profiling using real-time quantitative PCR. Methods 50: 262–270.
- 46. Zhang B, Kirov S, Snoddy J (2005) WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res 33: W741–748.