Supplementary Tables 1S-7S, Figure 1S from Haplotype Analysis of the HSD17B1 Gene and Risk of Breast Cancer: A Comprehensive Approach to Multicenter Analyses of Prospective Cohort Studies

Most behavioral traits are complex and are the product of interactions between multiple genes and/or environments. Therefore, ideal models in which to examine the relative roles of genetic effects should have, 1) variation in behavioral phenotypes, 2) the potential to identify the genetic bases of these behavioral traits, 3) an obvious association between phenotype and genotype, 4) the potential to identify relevant environmental conditions contributing to the establishment of behavioral phenotypes so that partitioning of gene-by-environment effects is possible, and 5) a strong understanding of the evolutionary forces influencing the system. Given these desires, an unexpected new animal model emerges for the study of behavioral genomics – morphs of the white-throated sparrow (Zonotrichia albicollis) exhibit alternative strategies of monogamy/high parental effort vs. promiscuity/low parental effort. These behaviors are absolutely correlated with the presence or absence of a large chromosomal rearrangement. We have amassed 22+ years of detailed behavioral, physiological, ecological, and evolutionary data on this species making it possible to identify the genetic, epigenetic, and environmental bases of behavior. Here we further outline the utility of the species, as well as present current cytogenetic and molecular data showing that rearrangements and linkage in multiple chromosomes are key to the evolution of alternative phenotypes. In addition, comparative analyses among the Zonotrichia suggest an interesting and slightly counterintuitive evolutionary pathway in this group. Genomic studies in the white-throated sparrow will identify the gene(s) associated with complex behaviors, as well as provide us with information on how environment interacts with genetic architecture to affect aggressive, social, sexual, and parental phenotypes. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate, yet linked evolutionary trajectories. Such results can transform our understanding of the evolution of genomes.

Background Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. Results Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2 m and 3 a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2 m existed in the common ancestor of the four North American species of Zonotrichia , but not in the one South American species, and that the 2 m form, originally thought to be the derived condition, might actually be the ancestral one. Conclusion Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease. In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology.

The Interleukln-2 (IL-2) receptor y chain (IL-2RY) Is a component of high and intermediate affinity IL-2 receptors that Is required to achieve full llgand binding affinity and internalization. We have localized the IL-2Ry gene to human chromosome Xq13. Genetic linkage analysis Indicates that the IL-2Rγ gene and the locus for X-linked severe combined immunodeficiency (XSCID) appear to be at the same position. Moreover, we demonstrate that each of three unrelated patients with XSCID has a different mutation In his IL-2Rγ gene resulting in a different premature stop codon and predicted C-terminal truncation. These data establish that XSCID Is associated with mutations of the IL-2Rγ gene product. Since XSCID Is characterized by absent or markedly reduced numbers of T cells, our findings Imply that IL-2Ry plays a vital role in thymic maturation of T cells. These results also have Important Implications for prenatal and postnatal diagnosis, carrier female detection, and gene therapy for XSCID.

In all studied neognathous birds, the Z–W pair of sex chromosomes shows strictly localized recombination in a very short pseudoautosomal region. There are five published W-linked genes in this region that have homologs on the Z, reflecting their common origin from an ancestral homologous chromosome pairs: CHD1, HINT, SPIN, UBAP2, and ATP5A1. Using BAC-based FISH, we have investigated in detail the genes located on the chicken Z and W chromosomes, especially those in the pseudoautosomal region. For one of them, UBAP2Z, we identified its precise cytogenetic location on the Z chromosome and mapped its W-linked counterpart, UBAP2W. The fine FISH mapping of two more homologs, ATPA5A1Z and ATPA5A1W, has been completed. By screening two gridded genomic jungle fowl BAC libraries, TAMU 031-JF256-BI and CHORI-261, with labeled amplified gene fragments or overgos, we detected four and five specific clones, respectively. BAC clones TAM31-100C09, TAM31-099N01, TAM31-027P16 and TAM31-095L18 were assigned to GGAZ at Zp23-p22 site (Flpter, 0.12 ± 0.034 to 0.15 ± 0.033). Clones CH261-046G16, CH261-033F10 and CH261-064F22 were detected on GGAZ (Flpter, 0.12 ± 0.033 to 0.14 ± 0.033). They also co-localized on GGAW, along with the CH261-114G22 BAC clone containing the GGAW-specific UBAP2W DNA sequence. We cytogenetically mapped in the chicken genome the sixth, previously unknown pair of Z- and W-linked homologs, UBE2R2Z and UBE2R2W. Using a chicken UBE2R2W-specific overgo, a positive clone was identified in the California condor BAC library and is being used for FISH in this endangered species and other birds.

The California condor is among the endangered avian species under captive management for which genetic and genomic investigation technologies are being developed. A genetic form of chondrodystrophy in condors was previously identified that appears to be inherited as an autosomal recessive allele. The ongoing research project is aimed at (i) initiating development of a genetic map of the California condor, (ii) evaluating the candidate genes responsible for heritable chondrodystrophy, and (iii) producing a carrier test for individuals possessing the gene. To address genome research and genetic management of California condors, we have begun to develop genetic, cytogenetic and comparative maps as a prerequisite for identification of candidate loci for the chondrodystrophy mutation. We produced a highly redundant California condor genomic BAC library that was used to construct a first-generation chicken-condor comparative physical map and to identify specific condor BACs carrying candidate genes for chondrodystrophy. Sequencing and characterization of candidate loci are underway that will enable us to identify carriers of the chondrodystrophy allele and provide tools for improved genetic management of this disease.

Background: Osteosarcoma, the most common malignant primary bone tumor, typically occurs during the adolescent growth spurt. Germ-line genetic variation in genes critical in growth regulation could confer altered risk of osteosarcoma.Methods: Fifty-two common single nucleotide polymorphisms (SNP) in 13 genes were genotyped in a prospective case-control study of osteosarcoma (104 osteosarcoma cases and 74 orthopedic controls). Genotype data analyzed with contingency tables suggested the strongest association with insulin-like growth factor 2 receptor (IGF2R) SNPs. Additional SNPs were genotyped to capture IGF2R common haplotypes and resequencing was done across the IGF2R block associated with osteosarcoma risk. Percentage methylation was determined by pyrosequencing of the IGF2R variant allele located in a CpG island.Results: IGF2R Ex16+88G > A (rs998075) and IVS16+15C > T (rs998074) SNPs were associated with increased risk for osteosarcoma compared with orthopedic controls (haplotype odds ratio, 2.04; 95% confidence interval, 1.29-3.24). Follow-up genotyping showed that IGF2R IVS15+213C > T was also associated with increased osteosarcoma risk. Resequence analysis identified two additional SNPs linked to the risk-associated SNPs; linkage disequilibrium was strongest in a 1-kb pair region around them. The Ex16+88G > A SNP is located within a CpG island and alters methylation at that site.Conclusion: This pilot study of germ-line genetic variation in growth pathway genes and osteosarcoma identified a haplotype block in IGF2R associated with increased risk of osteosarcoma. The presence of a SNP in this block results in loss of methylation at a CpG island, providing corroborative evidence of a possible functional variant. Our analysis of the IGF2R haplotype structure will be applicable to future studies of IGF2R and disease risk.

CCL3 (MIP-1 alpha), CCL4 (MIP-1 beta), and CCL18 (DC-CK1/PARC/AMAC-1) are potent chemoattractants produced by macrophages, natural killer cells, fibroblasts, mast cells, CD4(+) T cells, and CD8(+) T cells. CCL3 and CCL4 are natural ligands for the primary human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 and are also known to activate and enhance the cytotoxicity of natural killer cells. Genomic DNAs from >3,000 participants enrolled in five United States-based natural-history cohorts with acquired immunodeficiency syndrome (AIDS) were genotyped for 21 single-nucleotide polymorphisms (SNPs) in a 47-kb interval on chromosome 17q12 containing the genes CCL3, CCL4, and CCL18. All 21 SNPs were polymorphic in African Americans (AAs), whereas 7 of the 21 had minor-allele frequencies <0.01 in European Americans (EAs). Substantial linkage disequilibrium was observed in a 37-kb interval containing 17 SNPs where many pairwise D' values exceeded 0.70 in both racial groups, but particularly in EAs. Four and three haplotype blocks were observed in AAs and EAs, respectively. Blocks were strongly correlated with each other, and common haplotype diversity within blocks was limited. Two significant associations are reported that replicate an earlier study. First, among AA members of the AIDS Link to the Intravenous Experience cohort of injection drug users, frequencies of three correlated SNPs covering 2,231 bp in CCL3 were significantly elevated among highly exposed, persistently HIV-1-uninfected individuals compared with HIV-1-infected seroconvertors (P = .02-.03). Second, seven highly correlated SNPs spanning 36 kb and containing all three genes were significantly associated with more-rapid disease progression among EAs enrolled in the Multicenter AIDS Cohort Study cohort (P = .01-.02). These results reiterate the importance of chemokine gene variation in HIV-1/AIDS pathogenesis and emphasize that localized linkage disequilibrium makes the identification of causal mutations difficult.

CCL4 and CCL4L1 are two CC chemokine genes located at chromosome 17q21 whose mature proteins differ at only a single amino acid. Abundant functional information exists for CCL4, however, CCL4L1 has only recently been recognized as a distinct gene, thus information describing it is wanting. The CCL4L1 protein was synthesized in Escherichia coli and compared with the CCL4 protein. Competitive binding studies using HEK-293/CCR5 cells produced comparable EC50 values for the two proteins. Similarly, chemotaxis assays with cells expressing CCR1, CCR3, or CCR5 revealed no substantial differences. CCL4L1 was somewhat more effective at inhibiting HIV-1 replication in PBMCs than was CCL4, however the difference was not statistically significant. These data combined with the observation of individual variation in CCL4L1 gene copy number [Eur. J. Immunol. 32 (2002) 3016, Genomics 83 (2004) 735] support the contention that the CCL4 and CCL4L1 proteins have redundant functions.

The 1.709 or satellite IV repeated DNA family originally isolated from the domestic cow was analyzed using Southern blotting, pulsed field gel electrophoresis, fluorescence in situ hybridization, and DNA sequencing in species belonging to the genera Bos, Bison, Bubalus, Syncerus, Boselaphus, and Tragelaphus. Hybridization indicates that the family has been amplified in Bos, Bison, Bubalus, and Syncerus but not in Boselaphus or Tragelaphus. Pericentromeric, higher-order repeat substructure exists in all species, with multimeric arrays ranging in size from 10 to 1500 kb. Sequence analysis of a 492-bp PCR product revealed comparable levels (0.2–4.5%) of intra- and interspecific divergence when species of Bos and Bison were compared, supporting the idea that species of these two genera should be recognized under the genus Bos. Alternatively, all Syncerus sequences cluster as a monophyletic group on an evolutionary tree and differ from those of Bos/Bison by about 13%. Comparing these findings with the fossil record indicates that concerted evolution has occurred since Bos/Bison and Syncerus last shared a common ancestor (5.0 MYA) but before the radiation of the genus Bos (2.5 MYA): GenBank accession numbers AY517856-AY517904.

Background: MCP-1 (CCL2), MCP-3 (CCL7), and eotaxin (CCL11) are genes for CC chemokines clustered on the long arm of chromosome 17. Previous studies have implicated these chemokines in monocyte recruitment, viral replication, and anti-HIV cytotoxic T cell responses. An epidemiological analysis identified genetic variants influencing HIV-1 transmission and disease progression. Methods: Genomic DNA from over 3000 participants enrolled in five natural history cohorts in the United States were analyzed. Nine single nucleotide polymorphisms (SNP) covering 33 kb containing these three genes were genotyped using the polymerase chain reaction. Distortions in allele, genotype, and haplotype frequencies were assessed with respect to HIV-1 transmission and rates of disease progression using categorical and survival analyses. Results: Extensive linkage disequilibrium was observed. Three SNP (−2136T located in the MCP-1 promoter region, 767G in intron 1 of MCP-1, and −1385A in the Eotaxin promoter) were nearly always found together on a 31 kb haplotype (H7) containing the three genes. Frequencies of the three variants and the H7 haplotype were significantly elevated (odds ratio, 0.6; P = 0.005–0.01) in uninfected European-Americans repeatedly exposed to HIV-1 through high-risk sexual behavior or contaminated blood products. Conclusions: Although the extensive linkage disequilibrium precludes positive identification of the causal variant, the results suggest that genetic variation in the H7 region influences susceptibility to HIV-1 infection. Since these chemokines do not bind the primary HIV-1 coreceptors CCR5 or CXCR4, the observed influence on transmission may result from activation of the immune system in response to infection rather than receptor blockage.

Interleukin-4 (IL-4) is a pleiotropic cytokine produced primarily by activated CD4+ T lymphocytes, mast cells, and basophils. It modulates the functions of a variety of cell types involved with the immune response. This cytokine differentially regulates two major HIV-1 coreceptors and activates viral expression, and is thus a reasonable candidate gene for analyses in HIV-1/AIDS cohort studies. Population genetic variation in five single nucleotide polymorphisms (SNPs) in the 5′ region of the IL-4 gene was assessed in five racial groups. Neutrality tests reveal that the populations are evolving in accord with the infinite-sites model. However, coalescent simulations suggest greater haplotype diversity among African Americans than expected. This increased variation is presumably attributable to recombination or gene conversion. Genetic epidemiological analyses were conducted among European American and African American participants enrolled in five USA-based HIV-1/AIDS cohorts. One SNP, −589T, known to influence IL-4 transcription was previously shown to be associated with HIV-1/AIDS in both Japanese and French populations. Present analyses failed to identify any significant associations with HIV-1 infection or progression to AIDS.