Bayesian refinement of association signals for 14 loci in 3 common diseases

To further investigate susceptibility loci identified by genome-wide association studies, we genotyped 5,500 SNPs across 14 associated regions in 8,000 samples from a control group and 3 diseases: type 2 diabetes (T2D), coronary artery disease (CAD) and Graves' disease. We defined, using Bayes theorem, credible sets of SNPs that were 95% likely, based on posterior probability, to contain the causal disease-associated SNPs. In 3 of the 14 regions, TCF7L2 (T2D), CTLA4 (Graves' disease) and CDKN2A-CDKN2B (T2D), much of the posterior probability rested on a single SNP, and, in 4 other regions (CDKN2A-CDKN2B (CAD) and CDKAL1, FTO and HHEX (T2D)), the 95% sets were small, thereby excluding most SNPs as potentially causal. Very few SNPs in our credible sets had annotated functions, illustrating the limitations in understanding the mechanisms underlying susceptibility to common diseases. Our results also show the value of more detailed mapping to target sequences for functional studies. © 2012 Nature America, Inc. All rights reserved.

Mutations in the gene encoding IFT dynein complex component WDR34 cause jeune asphyxiating thoracic dystrophy

Bidirectional (anterograde and retrograde) motor-based intraflagellar transport (IFT) governs cargo transport and delivery processes that are essential for primary cilia growth and maintenance and for hedgehog signaling functions. The IFT dynein-2 motor complex that regulates ciliary retrograde protein transport contains a heavy chain dynein ATPase/motor subunit, DYNC2H1, along with other less well functionally defined subunits. Deficiency of IFT proteins, including DYNC2H1, underlies a spectrum of skeletal ciliopathies. Here, by using exome sequencing and a targeted next-generation sequencing panel, we identified a total of 11 mutations in WDR34 in 9 families with the clinical diagnosis of Jeune syndrome (asphyxiating thoracic dystrophy). WDR34 encodes a WD40 repeat-containing protein orthologous to Chlamydomonas FAP133, a dynein intermediate chain associated with the retrograde intraflagellar transport motor. Three-dimensional protein modeling suggests that the identified mutations all affect residues critical for WDR34 protein-protein interactions. We find that WDR34 concentrates around the centrioles and basal bodies in mammalian cells, also showing axonemal staining. WDR34 coimmunoprecipitates with the dynein-1 light chain DYNLL1 in vitro, and mining of proteomics data suggests that WDR34 could represent a previously unrecognized link between the cytoplasmic dynein-1 and IFT dynein-2 motors. Together, these data show that WDR34 is critical for ciliary functions essential to normal development and survival, most probably as a previously unrecognized component of the mammalian dynein-IFT machinery.

Association of STAT3 and TNFRSF1A with ankylosing spondylitis in Han Chinese

Objectives: Recent association studies by the Australo-Anglo-American Spondyloarthritis Consortium (TASC) in Caucasian European populations from Australia, North America and the UK have identified a number of genes as being associated with ankylosing spondylitis (AS). A candidate gene study in a Han Chinese population was performed based on these findings to identify associated genes in this population. Methods: A case-control study was performed in a Han Chinese population of patients with AS (n=775) and controls (n=1587) from Shanghai and Nanjing. All patients met the modified New York criteria for AS. The cases and controls were genotyped for 115 single nucleotide polymorphisms (SNPs) tagging IL23R, ERAP1, STAT3, JAK2, TNFRSF1A and TRADD, as well as other confirmation SNPs from the TASC study, using the Sequenom iPlex and the ABI OpenArray platforms. Statistical analysis of genotyped SNPs was performed using the Cochran - Armitage test for trend and meta-analysis was performed using METAL. SNPs in AS-associated genes in this study were then imputed using MaCH, and association with AS tested by logistic regression. Results: SNPs in TNFRSF1A (rs4149577, p=8.2×10-4), STAT3 (rs2293152, p=0.0015; rs1053005, p=0.017) and ERAP1 (rs27038, p=0.0091; rs27037, p=0.0092) were significantly associated with AS in Han Chinese. Association was also observed between AS and the intergenic region 2p15 (rs10865331, p=0.023). The lack of association between AS and IL23R in Han Chinese was confirmed (all SNPs p>0.1). Conclusions: The study results demonstrate for the first time that genetic polymorphisms in STAT3, TNFRSF1A and 2p15 are associated with AS in Han Chinese, suggesting common pathogenic mechanisms for the disease in Chinese and Caucasian European populations. Furthermore, previous findings demonstrating that ERAP1, but not IL23R, is associated with AS in Chinese patients were confirmed.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition. © The Author 2014.

Methylenetetrahydrofolate reductase CpG islands: Epigenotyping

Background Determination of the differential DNA methylation patterns of methylenetetrahydrofolate reductase (MTHFR) that are associated with differential MTHFR activity is important to understand the pathogenesis of ischemic stroke. However, to date, no data are available on the differential DNA methylation profiles of Kelantanese Malays. Therefore, we developed a rapid and efficient serial pyrosequencing assay to determine differential DNA methylation profiles of MTHFR, which help to further our understanding of the pathogenesis of ischemic stroke. The developed assay also served as the validation platform for our previous computational epigenetic research on MTHFR. Methods Polymerase chain reaction primers were designed and validated to specifically amplify the cytosine that is followed by guanine residues (CpGs) A and B regions. Prior epigenotyping on 110 Kelantanese Malays, the serial pyrosequencing assays for the CpGs A and B regions were validated using five validation controls. The mean values of the DNA methylation profiles of CpGs A and B were calculated. Results The mean DNA methylation levels for CpGs A and B were 0.984 ± 0.582 and 2.456 ± 1.406, respectively. The CpGs 8 and 20 showed the highest (5.581 ± 4.497) and the lowest (0.414 ± 2.814) levels of DNA methylation at a single-base resolution. Conclusion We have successfully developed and validated a pyrosequencing assay that is fast and can yield high-quality pyrograms for DNA methylation analysis and is therefore applicable to high throughput study. Using this newly developed pyrosequencing assay, the MTHFR DNA methylation profiles of 110 Kelantanese Malays were successfully determined. It also validated our computational epigenetic research on MTHFR.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

VEGF-mediated cell survival in non-small-cell lung cancer: implications for epigenetic targeting of VEGF receptors as a therapeutic approach

Aims: To evaluate the potential therapeutic utility of histone deacetylase inhibitors (HDACi) in targeting VEGF receptors in non-small-cell lung cancer. Materials & methods: Non-small-cell lung cancer cells were screened for the VEGF receptors at the mRNA and protein levels, while cellular responses to various HDACi were examined. Results: Significant effects on the regulation of the VEGF receptors were observed in response to HDACi. These were associated with decreased secretion of VEGF, decreased cellular proliferation and increased apoptosis which could not be rescued by addition of exogenous recombinant VEGF. Direct remodeling of the VEGFR1 and VEGFR2 promoters was observed. In contrast, HDACi treatments resulted in significant downregulation of the Neuropilin receptors. Conclusion: Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for lung cancer patients in the clinical setting.