Discovery and replication of dopamine-related gene effects on caudate volume in young and elderly populations (N1198) using genome-wide search

The caudate is a subcortical brain structure implicated in many common neurological and psychiatric disorders. To identify specific genes associated with variations in caudate volume, structural magnetic resonance imaging and genome-wide genotypes were acquired from two large cohorts, the Alzheimer's Disease NeuroImaging Initiative (ADNI; N=734) and the Brisbane Adolescent/Young Adult Longitudinal Twin Study (BLTS; N=464). In a preliminary analysis of heritability, around 90% of the variation in caudate volume was due to genetic factors. We then conducted genome-wide association to find common variants that contribute to this relatively high heritability. Replicated genetic association was found for the right caudate volume at single-nucleotide polymorphism rs163030 in the ADNI discovery sample (P=2.36 × 10 -6) and in the BLTS replication sample (P=0.012). This genetic variation accounted for 2.79 and 1.61% of the trait variance, respectively. The peak of association was found in and around two genes, WDR41 and PDE8B, involved in dopamine signaling and development. In addition, a previously identified mutation in PDE8B causes a rare autosomal-dominant type of striatal degeneration. Searching across both samples offers a rigorous way to screen for genes consistently influencing brain structure at different stages of life. Variants identified here may be relevant to common disorders affecting the caudate.

Serum cholesterol and variant in cholesterol-related gene CETP predict white matter microstructure

Several common genetic variants influence cholesterol levels, which play a key role in overall health. Myelin synthesis and maintenance are highly sensitive to cholesterol concentrations, and abnormal cholesterol levels increase the risk for various brain diseases, including Alzheimer's disease. We report significant associations between higher serum cholesterol (CHOL) and high-density lipoprotein levels and higher fractional anisotropy in 403 young adults (23.8 ± 2.4years) scanned with diffusion imaging and anatomic magnetic resonance imaging at 4Tesla. By fitting a multi-locus genetic model within white matter areas associated with CHOL, we found that a set of 18 cholesterol-related, single-nucleotide polymorphisms implicated in Alzheimer's disease risk predicted fractional anisotropy. We focused on the single-nucleotide polymorphism with the largest individual effects, CETP (rs5882), and found that increased G-allele dosage was associated with higher fractional anisotropy and lower radial and mean diffusivities in voxel-wise analyses of the whole brain. A follow-up analysis detected white matter associations with rs5882 in the opposite direction in 78 older individuals (74.3 ± 7.3years). Cholesterol levels may influence white matter integrity, and cholesterol-related genes may exert age-dependent effects on the brain.

Identifying candidate gene effects by restricting search space in a multivariate genetic analysis of white matter microstructure

Several genetic variants are thought to influence white matter (WM) integrity, measured with diffusion tensor imaging (DTI). Voxel based methods can test genetic associations, but heavy multiple comparisons corrections are required to adjust for searching the whole brain and for all genetic variants analyzed. Thus, genetic associations are hard to detect even in large studies. Using a recently developed multi-SNP analysis, we examined the joint predictive power of a group of 18 cholesterol-related single nucleotide polymorphisms (SNPs) on WM integrity, measured by fractional anisotropy. To boost power, we limited the analysis to brain voxels that showed significant associations with total serum cholesterol levels. From this space, we identified two genes with effects that replicated in individual voxel-wise analyses of the whole brain. Multivariate analyses of genetic variants on a reduced anatomical search space may help to identify SNPs with strongest effects on the brain from a broad panel of genes.

Failure to launch: The self-regulating Md -MYB10R6 gene from apple is active in flowers but not leaves of Petunia

Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10R6, are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10R6 transgene (MYB10–R6pro:MYB10:MYB10term) activated anthocyanin synthesis when transiently expressed in Antirrhinumroseadorsea petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of ‘Mitchell’ petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.

A candidate gene association study for growth performance in an improved Giant Freshwater Prawn (Macrobrachium rosenbergii) culture line

A candidate gene approach using type I single nucleotide polymorphism (SNP) markers can provide an effective method for detecting genes and gene regions that underlie phenotypic variation in adaptively significant traits. In the absence of available genomic data resources, transcriptomes were recently generated in Macrobrachium rosenbergii to identify candidate genes and markers potentially associated with growth. The characterisation of 47 candidate loci by ABI re-sequencing of four cultured and eight wild samples revealed 342 putative SNPs. Among these, 28 SNPs were selected in 23 growth-related candidate genes to genotype in 200 animals selected for improved growth performance in an experimental GFP culture line in Vietnam. The associations between SNP markers and individual growth performance were then examined. For additive and dominant effects, a total of three exonic SNPs in glycogen phosphorylase (additive), heat shock protein 90 (additive and dominant) and peroxidasin (additive), and a total of six intronic SNPs in ankyrin repeats-like protein (additive and dominant), rolling pebbles (dominant), transforming growth factor-β induced precursor (dominant), and UTP-glucose-1-phosphate uridylyltransferase 2 (dominant) genes showed significant associations with the estimated breeding values in the experimental animals (P =0.001−0.031). Individually, they explained 2.6−4.8 % of the genetic variance (R2=0.026−0.048). This is the first large set of SNP markers reported for M. rosenbergii and will be useful for confirmation of associations in other samples or culture lines as well as having applications in marker-assisted selection in future breeding programs.

N-ethyl-N-nitrosourea (ENU) induced mutations within the Klotho gene lead to ectopic calcification and reduced lifespan in mouse models

Ectopic calcification (EC), which is the pathological deposition of calcium and phosphate in extra-skeletal tissues, may be associated with hypercalcaemic and hyperphosphataemic disorders, or it may occur in the absence of metabolic abnormalities. In addition, EC may be inherited as part of several monogenic disorders and studies of these have provided valuable insights into the metabolic pathways regulating mineral metabolism. For example, studies of tumoural calcinosis, a disorder characterised by hyperphosphataemia and progressive EC, have revealed mutations of fibroblast growth factor 23 (FGF23), polypeptide N-acetyl galactosaminyltransferase 3 (GALNT3) and klotho (KL), which are all part of a phosphate-regulating pathway. However, such studies in humans are limited by the lack of available large families with EC, and to facilitate such studies we assessed the progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for EC. This identified two mutants with autosomal recessive forms of EC, and reduced lifespan, designated Ecalc1 and Ecalc2. Genetic mapping localized the Ecalc1 and Ecalc2 loci to a 11.0 Mb region on chromosome 5 that contained the klotho gene (Kl), and DNA sequence analysis identified nonsense (Gln203Stop) and missense (Ile604Asn) Kl mutations in Ecalc1 and Ecalc2 mice, respectively. The Gln203Stop mutation, located in KL1 domain, was severely hypomorphic and led to a 17-fold reduction of renal Kl expression. The Ile604Asn mutation, located in KL2 domain, was predicted to impair klotho protein stability and in vitro expression studies in COS-7 cells revealed endoplasmic reticulum retention of the Ile604Asn mutant. Further phenotype studies undertaken in Ecalc1 (kl203X/203X) mice demonstrated elevations in plasma concentrations of phosphate, FGF23 and 1,25-dihydroxyvitamin D. Thus, two allelic variants of Kl that develop EC and represent mouse models for tumoural calcinosis have been established. © 2015 Esapa et al.

The MS risk allele of CD40 is associated with reduced cell-membrane bound expression in antigen presenting cells: Implications for gene function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. © 2015 Field et al.

Polymorphisms in the receptor tyrosine kinase MERTK gene are associated with Multiple Sclerosis susceptibility

Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million people worldwide. The TAM family of receptor tyrosine kinases (TYRO3, AXL and MERTK) have been implicated as important players during demyelination in both animal models of MS and in the human disease. We therefore conducted an association study to identify single nucleotide polymorphisms (SNPs) within genes encoding the TAM receptors and their ligands associated with MS. Analysis of genotype data from a genome-wide association study which consisted of 1618 MS cases and 3413 healthy controls conducted by the Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene) revealed several SNPs within the MERTK gene (Chromosome 2q14.1, Accession Number NG_011607.1) that showed suggestive association with MS. We therefore interrogated 28 SNPs in MERTK in an independent replication cohort of 1140 MS cases and 1140 healthy controls. We found 12 SNPs that replicated, with 7 SNPs showing p-values of less than 10-5 when the discovery and replication cohorts were combined. All 12 replicated SNPs were in strong linkage disequilibrium with each other. In combination, these data suggest the MERTK gene is a novel risk gene for MS susceptibility. © 2011 Ma et al.

The interleukin 1 gene cluster contains a major susceptibility locus for ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly heritable inflammatory arthropathy. Although the gene HLA-B27 is almost essential for the inheritance of the condition, it alone is not sufficient to explain the pattern of familial recurrence of the disease. We have previously demonstrated suggestive linkage of AS to chromosome 2q13, a region containing the interleukin 1 (IL-1) family gene cluster, which includes several strong candidates for involvement in the disease. In the current study, we describe strong association and transmission of IL-1 family gene cluster single-nucleotide polymorphisms and haplotypes with AS.

Matters Arising: Zhu et al, "A novel gene variation of TNFα associated with ankylosing spondylitis: A reconfirmed study"

In a recently published study1 involving 79 ankylosing spondylitis (AS) patients and 132 unrelated healthy blood donors, Zhu et al report association of a tumour necrosis factor α (TNFα) single nucleotide polymorphism (SNP), rs1799724, with AS.1 I have many concerns with this paper and its conclusions...

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 (IL1) gene family members with ankylosing spondylitis (AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms (SNPs) and the IL1RN.VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN.4, and IL1RN.VNTR was observed with AS (p<0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN.4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR (both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 (D′ 0.4 to 0.9, p<0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding.

Gene expression profiling reveals a downregulation in immune-associated genes in patients with AS

Objective: To identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with healthy individuals. Methods: RNA was extracted from PBMCs collected from 18 patients with active disease and 18 gender-matched and age-matched controls. Expression profiles of these cells were determined using microarray. Candidate genes with differential expressions were confirmed in the same samples using quantitative reverse transcription-PCR (qRT-PCR). These genes were then validated in a different sample cohort of 35 patients with AS and 18 controls by qRT-PCR. Results: Microarray analysis identified 452 genes detected with 485 probes which were differentially expressed between patients with AS and controls. Underexpression of NR4A2, tumour necrosis factor AIP3 (TNFAIP3) and CD69 was confirmed. These genes were further validated in a different sample group in which the patients with AS had a wider range of disease activity. Predictive algorithms were also developed from the expression data using receiver-operating characteristic curves, which demonstrated that the three candidate genes have ∼80% power to predict AS according to their expression levels. Conclusions: The findings show differences in global gene expression patterns between patients with AS and controls, suggesting an immunosuppressive phenotype in the patients. Furthermore, downregulated expression of three immune-related genes was confirmed. These candidate genes were also shown to be strong predictive markers for AS.

Ankylosing spondylitis is associated with the anthrax toxin receptor 2 gene (ANTXR2)

Objectives ANTXR2 variants have been associated with ankylosing spondylitis (AS) in two previous genome-wide association studies (GWAS) (p∼9×10-8). However, a genome-wide significant association (p<5×10-8) was not observed. We conducted a more comprehensive analysis of ANTXR2 in an independent UK sample to confirm and refine this association. Methods A replication study was carried out with 2978 cases and 8365 controls. Then, these were combined with non-overlapping samples from the two previous GWAS in a meta-analysis. Human leukocyte antigen (HLA)-B27 stratification was also performed to test for ANTXR2-HLA-B27 interaction. Results Out of nine single nucleotide polymorphisms (SNP) in the study, five SNPs were nominally associated (p<0.05) with AS in the replication dataset. In the meta-analysis, eight SNPs showed evidence of association, the strongest being with rs12504282 (OR=0.88, p=6.7×10-9). Seven of these SNPs showed evidence for association in the HLA-B27-positive subgroup, but none was associated with HLA-B27-negative AS. However, no statistically significant interaction was detected between HLA-B27 and ANTXR2 variants. Conclusions ANTXR2 variants are clearly associated with AS. The top SNPs from two previous GWAS (rs4333130 and rs4389526) and this study (rs12504282) are in strong linkage disequilibrium (r2≥0.76). All are located near a putative regulatory region. Further studies are required to clarify the role played by these ANTXR2 variants in AS.

The chromosome 16q region associated with ankylosing spondylitis includes the candidate gene tumour necrosis factor receptor type 1-associated death domain (TRADD)

Objective: To replicate and refine the reported association of ankylosing spondylitis (AS) with two nonsynonymous single nucleotide polymorphisms (nsSNPs) on chromosome 16q22.1. Methods: Firstly, 730 independent UK patients with AS were genotyped for rs9939768 and rs6979 and allele frequencies were compared with 2879 previously typed historic disease controls. Secondly, the two data sets were combined in meta-analyses. Finally, 5 tagging SNPs, located between rs9939768 and rs6979, were analysed in 1604 cases and 1020 controls. Results: The association of rs6979 with AS was replicated, p=0.03, OR=1.14 (95% CI 1.01 to 1.28), and a trend for association with rs9939768 detected, p=0.06, OR=1.25 (95% CI 0.99 to 1.57). Meta-analyses revealed association of both SNPs with AS, p=0.0008, OR=1.31 (95% CI 1.12 to 1.54) and p=0.0009, OR=1.15 (95% CI 1.06 to 1.23) for rs9939768 and rs6979, respectively. New associations with rs9033 and rs868213 (p=0.00002, OR=1.23 (95% CI 1.12 to 1.36) and p=0.00002 OR=1.45 (95% CI 1.22 to 1.72), respectively, were identified. Conclusions: The region on chromosome 16 that has been replicated in the present work is interesting as the highly plausible candidate gene, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD), is located between rs9033 and rs868213. It will require additional work to identify the primary genetic association(s) with AS.

Prospective meta-analysis of interleukin 1 gene complex polymorphisms confirms associations with ankylosing spondylitis

Objectives: The aim of the current study was to determine the contribution of interleukin (IL) 1 gene cluster polymorphisms previously implicated in susceptibility for ankylosing spondylitis (AS) to AS susceptibility in different populations worldwide. Methods: Nine polymorphisms in the IL1 gene cluster members IL1A (rs2856836, rs17561 and rs1894399), IL1B (rs16944), IL1F10 (rs3811058) and IL1RN (rs419598, the IL1RA VNTR, rs315952 and rs315951) were genotyped in 2675 AS cases and 2592 healthy controls recruited in 12 different centres in 10 countries. Association of variants with AS was tested by Mantel-Haenszel random effects analysis. Results: Strong association was observed with three single nucleotide polymorphisms (SNPs) in the IL1A gene (rs2856836, rs17561, rs1894399, p = 0.0036, 0.000019 and 0.0003, respectively). There was no evidence of significant heterogeneity of effects between centres, and no evidence of non-combinability of findings. The population attributable risk fraction of these variants in Caucasians is estimated at 4-6%. Conclusions: This study confirms that IL1A is associated with susceptibility to AS. Association of the other IL1 gene complex members could not be excluded in specific populations. Prospective meta-analysis is a useful tool in confirmation studies of genes associated with complex genetic disorders such as AS, providing sufficiently large sample sizes to produce robust findings often not achieved in smaller individual cohorts.