Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μM lead chloride and 0.05 μM lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 μM lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 μM. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μM and reached half-maximal inhibition of motility at about 50 μM. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke

Genetic factors have been implicated in stroke risk, but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) for ischemic stroke and its subtypes in 3,548 affected individuals and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 affected individuals and 6,281 controls. We replicated previous associations for cardioembolic stroke near PITX2 and ZFHX3 and for large vessel stroke at a 9p21 locus. We identified a new association for large vessel stroke within HDAC9 (encoding histone deacetylase 9) on chromosome 7p21.1 (including further replication in an additional 735 affected individuals and 28,583 controls) (rs11984041; combined P = 1.87 × 10 -11; odds ratio (OR) = 1.42, 95% confidence interval (CI) = 1.28-1.57). All four loci exhibited evidence for heterogeneity of effect across the stroke subtypes, with some and possibly all affecting risk for only one subtype. This suggests distinct genetic architectures for different stroke subtypes.

Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies. © 2011 Duncan et al.

A Mouse Model of Early-Onset Renal Failure Due to a Xanthine Dehydrogenase Nonsense Mutation

Chronic kidney disease (CKD) is characterized by renal fibrosis that can lead to end-stage renal failure, and studies have supported a strong genetic influence on the risk of developing CKD. However, investigations of the underlying molecular mechanisms are hampered by the lack of suitable hereditary models in animals. We therefore sought to establish hereditary mouse models for CKD and renal fibrosis by investigating mice treated with the chemical mutagen N-ethyl-N-nitrosourea, and identified a mouse with autosomal recessive renal failure, designated RENF. Three-week old RENF mice were smaller than their littermates, whereas at birth they had been of similar size. RENF mice, at 4-weeks of age, had elevated concentrations of plasma urea and creatinine, indicating renal failure, which was associated with small and irregularly shaped kidneys. Genetic studies using DNA from 10 affected mice and 91 single nucleotide polymorphisms mapped the Renf locus to a 5.8Mbp region on chromosome 17E1.3. DNA sequencing of the xanthine dehydrogenase (Xdh) gene revealed a nonsense mutation at codon 26 that co-segregated with affected RENF mice. The Xdh mutation resulted in loss of hepatic XDH and renal Cyclooxygenase-2 (COX-2) expression. XDH mutations in man cause xanthinuria with undetectable plasma uric acid levels and three RENF mice had plasma uric acid levels below the limit of detection. Histological analysis of RENF kidney sections revealed abnormal arrangement of glomeruli, intratubular casts, cellular infiltration in the interstitial space, and interstitial fibrosis. TUNEL analysis of RENF kidney sections showed extensive apoptosis predominantly affecting the tubules. Thus, we have established a mouse model for autosomal recessive early-onset renal failure due to a nonsense mutation in Xdh that is a model for xanthinuria in man. This mouse model could help to increase our understanding of the molecular mechanisms associated with renal fibrosis and the specific roles of XDH and uric acid. © 2012 Piret et al.

Whole-genome screening in ankylosing spondylitis: Evidence of non-MHC genetic-susceptibility loci

Ankylosing spondylitis (AS) is a common inflammatory arthritis predominantly affecting the axial skeleton. Susceptibility to the disease is thought to be oligogenic. To identify the genes involved, we have performed a genomewide scan in 185 families containing 255 affected sibling pairs. Two-point and multipoint nonparametric linkage analysis was performed. Regions were identified showing "suggestive" or stronger linkage with the disease on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q. The MHC locus was identified as encoding the greatest component of susceptibility, with an overall LOD score of 15.6. The strongest non-MHC linkage lies on chromosome 16q (overall LOD score 4.7). These results strongly support the presence of non-MHC genetic-susceptibility factors in AS and point to their likely locations.

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.

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

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.

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.

Identification of major loci controlling clinical manifestations of ankylosing spondylitis

Objective. To identify genomic regions linked with determinants of age at symptom onset, disease activity, and functional impairment in ankylosing spondylitis (AS). Methods. A whole genome linkage scan was performed in 188 affected sibling pair families with 454 affected individuals. Traits assessed were age at symptom onset, disease activity assessed by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and functional impairment assessed by the Bath Ankylosing Spondylitis Functional Index (BASFI). Parametric and nonparametric quantitative linkage analysis was performed using parameters defined in a previous segregation study. Results. Heritabilities of the traits studied in this data set were as follows: BASDAI 0.49 (P = 0.0001, 95% confidence interval [95% CI] 0.23-0.75), BASFI 0.76 (P = 10-7, 95% CI 0.49-1.0), and age at symptom onset 0.33 (P = 0.005, 95% CI 0.04-0.62). No linkage was observed between the major histocompatibility complex (MHC) and any of the traits studied (logarithm of odds [LOD] score <1.0). "Significant" linkage (LOD score 4.0) was observed between a region on chromosome 18p and the BASDAI. Age at symptom onset showed "suggestive" linkage to chromosome 11p (LOD score 3.3). Maximum linkage with the BASFI was seen at chromosome 2q (LOD score 2.9). Conclusion. In contrast to the genetic determinants of susceptibility to AS, clinical manifestations of the disease measured by the BASDAI, BASFI, and age at symptom onset are largely determined by a small number of genes not encoded within the MHC.

A genome-wide screen for susceptibility loci in ankylosing spondylitis

Objective. To localize the regions containing genes that determine susceptibility to ankylosing spondylitis (AS). Methods. One hundred five white British families with 121 affected sibling pairs with AS were recruited, largely from the Royal National Hospital for Rheumatic Diseases AS database. A genome-wide linkage screen was undertaken using 254 highly polymorphic microsatellite markers from the Medical Research Council (UK) (MRC) set. The major histocompatibility complex (MHC) region was studied more intensively using 5 microsatellites lying within the HLA class III region and HLA-DRB1 typing. The Analyze package was used for 2-point analysis, and GeneHunter for multipoint analysis. Results. When only the MRC set was considered, 11 markers in 7 regions achieved a P value of ≤0.01. The maximum logarithm of odds score obtained was 3.8 (P = 1.4 x 10-5) using marker D6S273, which lies in the HLA class III region. A further marker used in mapping of the MHC class III region achieved a LOD score of 8.1 (P = 1 x 10-9). Nine of 118 affected sibling pairs (7.6%) did not share parental haplotypes identical by descent across the MHC, suggesting that only 31% of the susceptibility to AS is coded by genes linked to the MHC. The maximum non-MHC LOD score obtained was 2.6 (P = 0.0003) for marker D16S422. Conclusion. The results of this study confirm the strong linkage of the MHC with AS, and provide suggestive evidence regarding the presence and location of non-MHC genes influencing susceptibility to the disease.

Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom

Objective. To undertake a systematic wholegenome screen to identify regions exhibiting genetic linkage to rheumatoid arthritis (RA). Methods. Two hundred fifty-two RA-affected sibling pairs from 182 UK families were genotyped using 365 highly informative microsatellite markers. Microsatellite genotyping was performed using fluorescent polymerase chain reaction primers and semiautomated DNA sequencing technology. Linkage analysis was undertaken using MAPMAKER/SIBS for single-point and multipoint analysis. Results. Significant linkage (maximum logarithm of odds score 4.7 [P = 0.000003] at marker D6S276, 1 cM from HLA-DRB1) was identified around the major histocompatibility complex (MHC) region on chromosome 6. Suggestive linkage (P < 7.4 × 10-4) was identified on chromosome 6q by single- and multipoint analysis. Ten other sites of nominal linkage (P < 0.05) were identified on chromosomes 3p, 4q, 7p, 2 regions of 10q, 2 regions of 14q, 16p, 21q, and Xq by single-point analysis and on 3 sites (1q, 14q, and 14q) by multipoint analysis. Conclusion. Linkage to the MHC region was confirmed. Eleven non-HLA regions demonstrated evidence of suggestive or nominal linkage, but none reached the genome-wide threshold for significant linkage (P = 2.2 × 10-5). Results of previous genome screens have suggested that 6 of these regions may be involved in RA susceptibility.

Dissection of class III major histocompatibility complex haplotypes associated with rheumatoid arthritis

Objective. We have previously identified a single-nucleotide polymorphism (SNP) haplotype involving the lymphotoxin α (LTA) and tumor necrosis factor (TNF) loci (termed haplotype LTA-TNF2) on chromosome 6 that shows differential association with rheumatoid arthritis (RA) on HLA-DRB1*0404 and *0401 haplotypes, suggesting the presence of additional non-HLA-DRB1 RA susceptibility genes on these haplotypes. To refine this association, we performed a case-control association study using both SNPs and microsatellite markers in haplotypes matched either for HLA-DRB1*0404 or for HLA-DRB1*0401. Methods. Fourteen SNPs lying between HLA-DRB1 and LTA were genotyped in 87 DRB1*04-positive families. High-density microsatellite typing was performed using 24 markers spanning 2,500 kb centered around the TNF gene in 305 DRB1*0401 or *0404 cases and 400 DRB1*0401 or *0404 controls. Single-marker, 2-marker, and 3-marker minihaplotypes were constructed and their frequencies compared between the DRB1*0401 and DRB1*0404 matched case and control haplotypes. Results. Marked preservation of major histocompatibility complex haplotypes was seen, with chromosomes carrying LTA-TNF2 and either DRB1*0401 or DRB1*0404 both carrying an identical SNP haplotype across the 1-Mb region between TNF and HLA-DRB1. Using microsatellite markers, we observed two 3-marker minihaplotypes that were significantly overrepresented in the DRB1*0404 case haplotypes (P = 0.00024 and P = 0.00097). Conclusion. The presence of a single extended SNP haplotype between LTA-TNF2 and both DRB1*0401 and DRB1*0404 is evidence against this region harboring the genetic effects in linkage disequillbrium with LTA-TNF2. Two RA-associated haplotypes on the background of DRB1*0404 were identified in a 126-kb region surrounding and centromeric to the TNF locus.