PTHR1 polymorphisms influence BMD variation through effects on the growing skeleton

We investigated whether polymorphisms in PTHR1 are associated with bone mineral density (BMD), to determine whether the association of this gene with BMD was due to effects on attainment of peak bone mass or effects on subsequent bone loss. The PTHR1 gene, including its 14 exons, their exon-intron boundaries, and 1,500 bp of its promoter region, was screened for polymorphisms by denaturing high-performance liquid chromatography (dHPLC) and sequencing in 36 osteoporotic cases. Eleven single-nucleotide polymorphisms (SNPs), one tetranucleotide repeat, and one tetranucleotide deletion were identified. A cohort of 634 families, including 1,236 men (39%) and 1,926 women (61%) ascertained with probands with low BMD (Z< -2.0) and the Children in Focus subset of the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (785 unrelated individuals, mean age 118 months), were genotyped for the five most informative SNPs (minor allele frequency >5%) and the tetranucleotide repeat. In our osteoporosis families, association was noted between lumbar spine BMD and alleles of a known functional tetranucleotide repeat (U4) in the PTHR1 promoter region (P = 0.042) and between two and three marker haplotypes of PTHR1 polymorphisms with lumbar spine, femoral neck, and total hip BMD (P = 0.021-0.047). This association was restricted to the youngest tertile of the population (age 16-39 years, P = 0.013-0.048). A similar association was found for the ALSPAC cohort: two marker haplotypes of SNPs A48609T and C52813T were associated with height (P = 0.006) and total body less head BMD (P = 0.02), corrected for age and gender, confirming the family findings. These findings suggest a role for PTHR1 variation in determining peak BMD.

Evidence of genetic association between TNFRSF1A encoding the p55 tumour necrosis factor receptor, and ankylosing spondylitis in UK Caucasians

Objectives: To replicate the possible genetic association between ankylosing spondylitis (AS) and TNFRSF1A. Methods: TNFRSF1A was re-sequenced in 48 individuals with AS to identify novel polymorphisms. Nine single nucleotide polymorphisms (SNPs) in TNFRSF1A and 5 SNPs in the neighbouring gene SCNN1A were genotyped in 1604 UK Caucasian individuals with AS and 1019 matched controls. An extended study was implemented using additional genotype data on 8 of these SNPs from 1400 historical controls from the 1958 British Birth Cohort. A meta-analysis of previously published results was also undertaken. Results: One novel variant in intron 6 was identified but no new coding variants. No definite associations were seen in the initial study but in the extended study there were weak associations with rs4149576 (p=0.04) and rs4149577 (p=0.007). In the metaanalysis consistent, somewhat stronger associations were seen with rs4149577 (p=0.002) and rs4149578 (p=0.006). Conclusions: These studies confirm the weak genetic associations between AS and TNFRSF1A. In view of the previously reported associations of TNFRSF1A with AS, in Caucasians and Chinese, and the biological plausibility of this candidate gene, replication of this finding in well powered studies is clearly indicated.

The koala immunological toolkit: Sequence identification and comparison of key markers of the koala (Phascolarctos cinereus) immune response

The koala (Phascolarctos cinereus) is an Australian marsupial that continues to experience significant population declines. Infectious diseases caused by pathogens such as Chlamydia are proposed to have a major role. Very few species-specific immunological reagents are available, severely hindering our ability to respond to the threat of infectious diseases in the koala. In this study, we utilise data from the sequencing of the koala transcriptome to identify key immunological markers of the koala adaptive immune response and cytokines known to be important in the host response to chlamydial infection in other species. This report describes the identification and preliminary sequence analysis of (1) T lymphocyte glycoprotein markers (CD4, CD8); (2) IL-4, a marker for the Th2 response; (3) cytokines such as IL-6, IL-12 and IL-1β, that have been shown to have a role in chlamydial clearance and pathology in other hosts; and (4) the sequences for the koala immunoglobulins, IgA, IgG, IgE and IgM. These sequences will enable the development of a range of immunological reagents for understanding the koala’s innate and adaptive immune responses, while also providing a resource that will enable continued investigations into the origin and evolution of the marsupial immune system.

Evaluation of the relationship between Chlamydia pecorum sequence types and disease using a species-specific multi-locus sequence typing scheme (MLST)

Chlamydia pecorum is globally associated with several ovine diseases including keratoconjunctivitis and polyarthritis. The exact relationship between the variety of C. pecorum strains reported and the diseases described in sheep remains unclear, challenging efforts to accurately diagnose and manage infected flocks. In the present study, we applied C. pecorum multi-locus sequence typing (MLST) to C. pecorum positive samples collected from sympatric flocks of Australian sheep presenting with conjunctivitis, conjunctivitis with polyarthritis, or polyarthritis only and with no clinical disease (NCD) in order to elucidate the exact relationships between the infecting strains and the range of diseases. Using Bayesian phylogenetic and cluster analyses on 62 C. pecorum positive ocular, vaginal and rectal swab samples from sheep presenting with a range of diseases and in a comparison to C. pecorum sequence types (STs) from other hosts, one ST (ST 23) was recognised as a globally distributed strain associated with ovine and bovine diseases such as polyarthritis and encephalomyelitis. A second ST (ST 69) presently only described in Australian animals, was detected in association with ovine as well as koala chlamydial infections. The majority of vaginal and rectal C. pecorum STs from animals with NCD and/or anatomical sites with no clinical signs of disease in diseased animals, clustered together in a separate group, by both analyses. Furthermore, 8/13 detected STs were novel. This study provides a platform for strain selection for further research into the pathogenic potential of C. pecorum in animals and highlights targets for potential strain-specific diagnostic test development.

Polymorphisms in a desaturase 2 ortholog associate with cuticular hydrocarbon and male mating success variation in a natural population of Drosophila serrata

Elucidating the nature of genetic variation underlying both sexually selected traits and the fitness components of sexual selection is essential to understanding the broader consequences of sexual selection as an evolutionary process. To date, there have been relatively few attempts to connect the genetic variance in sexually selected traits with segregating DNA sequence polymorphisms. We set out to address this in a well-characterized sexual selection system - the cuticular hydrocarbons (CHCs) of Drosophila serrata - using an indirect association study design that allowed simultaneous estimation of the genetic variance in CHCs, sexual fitness and single nucleotide polymorphism (SNP) effects in an outbred population. We cloned and sequenced an ortholog of the D. melanogaster desaturase 2 gene, previously shown to affect CHC biosynthesis in D. melanogaster, and associated 36 SNPs with minor allele frequencies > 0.02 with variance in CHCs and sexual fitness. Three SNPs had significant multivariate associations with CHC phenotype (q-value < 0.05). At these loci, minor alleles had multivariate effects on CHCs that were weakly associated with the multivariate direction of sexual selection operating on these traits. Two of these SNPs had pleiotropic associations with male mating success, suggesting these variants may underlie responses to sexual selection due to this locus. There were 15 significant male mating success associations (q-value < 0.1), and interestingly, we detected a nonrandom pattern in the relationship between allele frequency and direction of effect on male mating success. The minor-frequency allele usually reduced male mating success, suggesting a positive association between male mating success and total fitness at this locus.

Single nucleotide polymorphisms in clinics: Fantasy or reality for cancer?

Single nucleotide polymorphisms (SNPs) have been classically used for dissecting various human complex disorders using candidate gene studies. During the last decade, large scale SNP analysis i.e. genome-wide association studies (GWAS) have provided an agnostic approach to identify possible genetic loci associated with heterogeneous disease such as cancer susceptibility, prognosis of survival or drug response. Further, the advent of new technologies, including microarray based genotyping as well as high throughput next generation sequencing has opened new avenues for SNPs to be used in clinical practice. It is speculated that the utility of SNPs to understand the mechanisms, biology of variable drug response and ultimately treatment individualization based on the individual’s genome composition will be indispensable in the near future. In the current review, we discuss the advantages and disadvantages of the clinical utility of genetic variants in disease risk-prediction, prognosis, clinical outcome and pharmacogenomics. The lessons and challenges for the utility of SNP based biomarkers are also discussed, including the need for additional functional validation studies.

Common variants in the HLA-DRB1-HLA-DQA1 HLA class II region are associated with susceptibility to visceral leishmaniasis

To identify susceptibility loci for visceral leishmaniasis, we undertook genome-wide association studies in two populations: 989 cases and 1,089 controls from India and 357 cases in 308 Brazilian families (1,970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, and combined analysis across the three cohorts for rs9271858 at this locus showed P combined = 2.76 × 10 -17 and odds ratio (OR) = 1.41, 95% confidence interval (CI) = 1.30-1.52. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion, the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species. © 2013 Nature America, Inc. All rights reserved.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.

Novel ANKH amino terminus mutation (Pro5Ser) associated with early-onset calcium pyrophosphate disease with associated phosphaturia

This report describes a 32-year-old woman presenting since childhood with progressive calcium pyrophosphate disease (CPPD), characterized by severe arthropathy and chondrocalcinosis involving multiple peripheral joints and intervertebral disks. Because ANKH mutations have been previously described in familial CPPD, the proband's DNA was assessed at this locus by direct sequencing of promoter and coding regions and revealed 3 sequence variants in ANKH. Sequences of exon 1 revealed a novel isolated nonsynonymous mutation (c.13 C>T), altering amino acid in codon 5 from proline to serine (CCG>TCG). Sequencing of parental DNA revealed an identical mutation in the proband's father but not the mother. Subsequent clinical evaluation demonstrated extensive chondrocalcinosis and degenerative arthropathy in the proband's father. In summary, we report a novel mutation, not previously described, in ANKH exon 1, wherein serine replaces proline, in a case of early-onset severe CPPD associated with metabolic abnormalities, with similar findings in the proband's father.

Genome-wide study of familial juvenile hyperuricaemic (gouty) nephropathy (FJHN) indicates a new locus, FJHN3, linked to chromosome 2p22.1-p21

Familial juvenile hyperuricaemic (gouty) nephropathy (FJHN), is an autosomal dominant disease associated with a reduced fractional excretion of urate, and progressive renal failure. FJHN is genetically heterogeneous and due to mutations of three genes: uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1beta (HNF-1β) on chromosomes 16p12, 1q32.1, and 17q12, respectively. However, UMOD, REN or HNF-1β mutations are found in only ~45% of FJHN probands, indicating the involvement of other genetic loci in ~55% of probands. To identify other FJHN loci, we performed a single nucleotide polymorphism (SNP)-based genome-wide linkage analysis, in six FJHN families in whom UMOD, HNF-1β and REN mutations had been excluded. Parametric linkage analysis using a 'rare dominant' model established linkage in five of the six FJHN families, with a LOD score >+3, at 0% recombination, between FJHN and SNPs at chromosome 2p22.1-p21. Analysis of individual recombinants in two unrelated affected individuals defined a ~5.5 Mbp interval, flanked telomerically by SNP RS372139 and centromerically by RS896986 that contained the locus, designated FJHN3. The interval contains 28 genes, and DNA sequence analysis of the most likely candidate, solute carrier family 8 member 1 (SLC8A1), did not identify any abnormalities in the FJHN3 probands. FJHN3 is likely located within a ~5.5 Mbp interval on chromosome 2p22.1-p21, and identifying the genetic abnormality will help to further elucidate mechanisms predisposing to gout and renal failure.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Epidemiology of ankylosing spondylitis: IGAS 2009

The International Genetics of Ankylosing Spondylitis (IGAS) meeting was held in Houston, Texas, July 25, 2009. Sixteen investigators from Asia, Australia, Europe, and North and South America presented the status of their respective cohorts of patients with ankylosing spondylitis (AS). They also reviewed a proposal to examine their patients by single-nucleotide polymorphism (SNP) genotyping on an Illumina Infinium microarray SNP genotyping chip in a case-control cohort exceeding 12,000 samples. This chip will type 200,000 SNP selected from the most strongly associated variants identified in genome-wide association studies of inflammatory diseases, including inflammatory bowel disease, psoriasis, and ankylosing spondylitis.

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with diseaseIRGM for Crohns disease, HLA for Crohns disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetesalthough in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases. © 2010 Macmillan Publishers Limited. All rights reserved.

Investigating the genetic association between ERAP1 and ankylosing spondylitis

A strong association between ERAP1 and ankylosing spondylitis (AS) was recently identified by the Wellcome Trust Case Control Consortium and the Australo-Anglo-American Spondylitis Consortium (WTCCC-TASC) study. ERAP1 is highly polymorphic with strong linkage disequilibrium evident across the gene. We therefore conducted a series of experiments to try to identify the primary genetic association(s) with ERAP1. We replicated the original associations in an independent set of 730 patients and 1021 controls, resequenced ERAP1 to define the full extent of coding polymorphisms and tested all variants in additional association studies. The genetic association with ERAP1 was independently confirmed; the strongest association was with rs30187 in the replication set (P = 3.4 × 103). When the data were combined with the original WTCCC-TASC study the strongest association was with rs27044 (P = 1.1 × 10-9). We identified 33 sequence polymorphisms in ERAP1, including three novel and eight known non-synonymous polymorphisms. We report several new associations between AS and polymorphisms distributed across ERAP1 from the extended case-control study, the most significant of which was with rs27434 (P = 4.7 × 10-7). Regression analysis failed to identify a primary association clearly; we therefore used data from HapMap to impute genotypes for an additional 205 non-coding SNPs located within and adjacent to ERAP1. A number of highly significant associations (P < 5 × 10-9) were identified in regulatory sequences which are good candidates for causing susceptibility to AS, possibly by regulating ERAP1 expression. © 2009 The Author(s).