The value of avian genomics to the conservation of wildlife.

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 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m 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.

Sex chromosomes and sex determination in reptiles.

Reptiles occupy a crucial position with respect to vertebrate phylogeny, having roamed the earth for more than 300 million years and given rise to both birds and mammals. To date, this group has been largely ignored by contemporary genomics technologies, although the green anole lizard was recently recommended for whole genome sequencing. Future experiments using flow-sorted chromosome libraries and high-throughout genomic sequencing will help to discover important findings regarding sex chromosome evolution, early events in sex determination, and dosage compensation. This information should contribute extensively toward a general understanding of the genetic control of development in amniotes.

Haplotype analysis of the HSD17B1 gene and risk of breast cancer: a comprehensive approach to multicenter analyses of prospective cohort studies.

The 17beta-hydroxysteroid dehydrogenase 1 gene (HSD17B1) encodes 17HSD1, which catalyzes the final step of estradiol biosynthesis. Despite the important role of HSD17B1 in hormone metabolism, few epidemiologic studies of HSD17B1 and breast cancer have been conducted. This study includes 5,370 breast cancer cases and 7,480 matched controls from five large cohorts in the Breast and Prostate Cancer Cohort Consortium. We characterized variation in HSD17B1 by resequencing and dense genotyping a multiethnic sample and identified haplotype-tagging single nucleotide polymorphisms (htSNP) that capture common variation within a 33.3-kb region around HSD17B1. Four htSNPs, including the previously studied SNP rs605059 (S312G), were genotyped to tag five common haplotypes in all cases and controls. Conditional logistic regression was used to estimate odds ratios (OR) for disease. We found no evidence of association between common HSD17B1 haplotypes or htSNPs and overall risk of breast cancer. The OR for each haplotype relative to the most common haplotype ranged from 0.98 to 1.07 (omnibus test for association: X2 = 3.77, P = 0.58, 5 degrees of freedom). When cases were subdivided by estrogen receptor (ER) status, two common haplotypes were associated with ER-negative tumors (test for trend, Ps = 0.0009 and 0.0076; n = 353 cases). HSD17B1 variants that are common in Caucasians are not associated with overall risk of breast cancer; however, there was an association among the subset of ER-negative tumors. Although the probability that these ER-negative findings are false-positive results is high, these findings were consistent across each cohort examined and warrant further study.

Analysis of genes critical for growth regulation identifies Insulin-like Growth Factor 2 Receptor variations with possible functional significance as risk factors for osteosarcoma.

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.

MCP-1-MCP-3-Eotaxin gene cluster influences HIV-1 transmission.

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.

Genetic variation in the CCL18-CCL3-CCL4 chemokine gene cluster influences HIV Type 1 transmission and AIDS disease progression.

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.

CCL3L1 and CCL4L1 chemokine genes are located in a segmental duplication at chromosome 17q12.

Sixteen CC chemokine genes localize to a 2.06-Mb interval at 17q11.2-q12 on genomic contig NT_010799.13. Four of these genes comprise two closely related paralogous pairs: CCL3-CCL3L1 and CCL4-CCL4L1. Members within each pair share 95% sequence identity at both the genomic and the amino acid levels. One BAC clone (AC131056.5) on the contig with substantial internal sequence duplication contains two complete copies of CCL3L1 and CCL4L1 and one truncated copy of CCL3L1, while a partially overlapping clone (AC003976.1) contains one copy each of CCL3 and CCL4. Dot-matrix comparison of the regions of AC131056.5 with those of AC003976.1 containing the four genes reveals 90% sequence similarity over 37 kb. These observations support the idea that the multiple copies of CCL3L1 and CCL4L1 present in a single diploid genome are the result of segmental duplication.

Concerted evolution and higher-order repeat structure of the 1.709 (satellite IV) family in bovids.

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.

Chromosomal localization of six repeated DNA sequences among species of Microtus (Rodentia).

C-banding and fluorescence in situ hybridization (FISH) document the distribution of constitutive heterochromatin and six highly repeated DNA families (MSAT2570, MSAT21, MSAT160, MS2, MS4 and STR47) in the chromosomes of nine species of Microtus (M. chrotorrhinus, M. rossiaemeridionalis, M. arvalis, M. ilaeus, M. transcaspicus, M. cabrerae, M. pennsylvanicus, M. miurus and M. ochrogaster). Autosomal heterochromatin is largely centromeric and contains different repeated families in different species. Similarly, large C-band positive blocks on the sex chromosomes of four species contain different repeated DNAs. This interspecific variation in the chromosomal distribution and copy number of the repeats suggests that a common ancestor to modern species contained most of the repetitive families, and that descendant species selectively amplified or deleted different repeats on different chromosomes.

MORPHOLOGICAL VARIABILITY AND ASYMMETRY IN THE CHEETAH (ACINONYX JUBATUS), A GENETICALLY UNIFORM SPECIES.

The African cheetah (Acinonyx jubatus) is an unusual species because of its extremely low amount of biochemical genetic variation. A comparative analysis of morphological variation of 16 cranial characters from four species of Felidae (ocelot, Leopardus pardalus; margay, L. wiedii; leopard, Panthera pardus; and cheetah) was undertaken to evaluate the consequence of biochemical monomorphism on morphological variation. The species were selected because the cheetah has been shown previously to possess extremely low amounts of biochemical genetic variation as opposed to the other three species which retain comparatively high levels of allozyme heterozygosity. The cheetah sample showed dramatically greater fluctuating asymmetry but was not outstanding in morphological variability. Elevated levels of fluctuating asymmetry have been interpreted as a reflection of developmental instability, which is a common consequence of inbreeding. The inverse correlation of genetic variation and developmental stability (homeostasis) observed here fulfills prior expectations and further emphasizes the genetic invariability of the cheetah species.

Functional redundancy of the human CCL4 and CCL4L1 chemokine genes.

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.

Haplotype diversity in the interleukin-4 gene is not associated with HIV-1 transmission and AIDS progression.

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.

HLA-DR, HLA-DQ, and TAP genes in familial Hodgkin disease.

The HLA region has long been implicated in sporadic and familial Hodgkin disease (HD), with recent case-control studies suggesting that HLA class II loci predispose to sporadic nodular sclerosis HD (NSHD). To determine whether this predisposition extends to familial HD, HLA class II loci (DRB1, DQA1, DQB1, DRB3, DRB4, and DRB5) and transporter associated with antigen processing (TAP) loci (TAP1, TAP2) were investigated in 100 members of 16 families with at least 2 confirmed cases of HD. With the use of the transmission disequilibrium test, evidence for linkage disequilibrium with familial HD and, in particular, familial NSHD was obtained for the DRB1*1501-DQA1*0102-DQB1*0602 haplotype, the TAP1 allele encoding Ile at residue 333, and the DRB5-0101 allele. These 3 markers were in linkage disequilibrium and may not represent independent susceptibility regions. Use of a family-based approach excludes population stratification as an explanation for these findings.

Modulating influence on HIV/AIDS by interacting RANTES gene variants.

RANTES (regulated on activation normal T cell expressed and secreted), a ligand for the CC chemokine receptor 5, potently inhibits HIV-1 replication in vitro. We tested the influence of four RANTES single nucleotide polymorphism (SNP) variants and their haplotypes on HIV-1 infection and AIDS progression in five AIDS cohorts. Three SNPs in the RANTES gene region on chromosome 17 (403A in the promoter, In1.1C in the first intron, and 3'222C in the 3' untranslated region) are associated with increased frequency of HIV-1 infection. The common In1.1C SNP allele is nested within an intronic regulatory sequence element that exhibits differential allele binding to nuclear proteins and a down-regulation of gene transcription. The In1.1C allele or haplotypes that include In1.1C display a strong dominant association with rapid progression to AIDS among HIV-1-infected individuals in African-American, European-American, and combined cohorts. The principal RANTES SNP genetic influence on AIDS progression derives from the down-regulating RANTES In1.1C allele, although linkage disequilibrium with adjoining RANTES SNPs including a weaker up-regulating RANTES promoter allele (-28G), can modify the observed epidemiological patterns. The In1.1C-bearing genotypes account for 37% of the attributable risk for rapid progression among African Americans and may also be an important influence on AIDS progression in Africa. The diminished transcription of RANTES afforded by the In1.1C regulatory allele is consistent with increased HIV-1 spread in vivo, leading to accelerated progression to AIDS.