Sergey Nejentsev

Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes

The Wellcome Trust Case Control Consortium (WTCCC) primary genome-wide association (GWA) scan on seven diseases, including the multifactorial autoimmune disease type 1 diabetes (T1D), shows associations at P < 5 × 10−7 between T1D and six chromosome regions: 12q24, 12q13, 16p13, 18p11, 12p13 and 4q27. Here, we attempted to validate these and six other top findings in 4,000 individuals with T1D, 5,000 controls and 2,997 family trios independent of the WTCCC study. We confirmed unequivocally the associations of 12q24, 12q13, 16p13 and 18p11 (Pfollow-up ≤ 1.35 × 10−9; Poverall ≤ 1.15 × 10−14), leaving eight regions with small effects or false-positive associations. We also obtained evidence for chromosome 18q22 (Poverall = 1.38 × 10−8) from a GWA study of nonsynonymous SNPs. Several regions, including 18q22 and 18p11, showed association with autoimmune thyroid disease. This study increases the number of T1D loci with compelling evidence from six to at least ten.

Rare Variants of IFIH1, a Gene Implicated in Antiviral Responses, Protect Against Type 1 Diabetes

Genome-wide association studies (GWASs) are regularly used to map genomic regions contributing to common human diseases, but they often do not identify the precise causative genes and sequence variants. To identify causative type 1 diabetes (T1D) variants, we resequenced exons and splice sites of 10 candidate genes in pools of DNA from 480 patients and 480 controls and tested their disease association in over 30,000 participants. We discovered four rare variants that lowered T1D risk independently of each other (odds ratio = 0.51 to 0.74; P = 1.3 × 10–3 to 2.1 × 10–16) in IFIH1 (interferon induced with helicase C domain 1), a gene located in a region previously associated with T1D by GWASs. These variants are predicted to alter the expression and structure of IFIH1 [MDA5 (melanoma differentiation-associated protein 5)], a cytoplasmic helicase that mediates induction of interferon response to viral RNA. This finding firmly establishes the role of IFIH1 in T1D and demonstrates that resequencing studies can pinpoint disease-causing genes in genomic regions initially identified by GWASs.

Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A

The major histocompatibility complex (MHC) on chromosome 6 is associated with susceptibility to more common diseases than any other region of the human genome, including almost all disorders classified as autoimmune. In type 1 diabetes the major genetic susceptibility determinants have been mapped to the MHC class II genes HLA-DQB1 and HLA-DRB1 (refs 1–3), but these genes cannot completely explain the association between type 1 diabetes and the MHC region. Owing to the region’s extreme gene density, the multiplicity of disease-associated alleles, strong associations between alleles, limited genotyping capability, and inadequate statistical approaches and sample sizes, which, and how many, loci within the MHC determine susceptibility remains unclear. Here, in several large type 1 diabetes data sets, we analyse a combined total of 1,729 polymorphisms, and apply statistical methods—recursive partitioning and regression—to pinpoint disease susceptibility to the MHC class I genes HLA-B and HLA-A (risk ratios >1.5; Pcombined = 2.01 × 10-19 and 2.35 × 10-13, respectively) in addition to the established associations of the MHC class II genes. Other loci with smaller and/or rarer effects might also be involved, but to find these, future searches must take into account both the HLA class II and class I genes and use even larger samples. Taken together with previous studies, we conclude that MHC-class-I-mediated events, principally involving HLA-B*39, contribute to the aetiology of type 1 diabetes.

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866, published online 17 October; see Perspective by Conley and Fruman) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders. Gene sequencing of unrelated patients with recurrent airway infections identifies a common underlying mutation. [Also see Perspective by Conley and Fruman] Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Evolution and transmission of drug-resistant tuberculosis in a Russian population

The molecular mechanisms determining the transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole-genome sequencing of 1,000 prospectively obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. Multidrug-resistant (MDR) genotypes were found in 48% of isolates overall and in 87% of the major clades. The most common rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a new intragenic compensatory substitution was identified. The proportion of MDR cases with extensively drug-resistant (XDR) tuberculosis was 16% overall, with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confers clinical resistance without compromising fitness and transmissibility, showing that, in addition to weaknesses in the tuberculosis control program, biological factors drive the persistence and spread of MDR and XDR tuberculosis in Russia and beyond.

Genetic Association and Expression Studies Indicate a Role of Toll-Like Receptor 8 in Pulmonary Tuberculosis

Despite high rates of exposure, only 5–10% of people infected with Mycobacterium tuberculosis will develop active tuberculosis (TB) disease, suggesting a significant role for genetic variation in the human immune response to this infection. Here, we studied TB association and expression of 18 genes involved in the Toll-like receptor (TLR) pathways. Initially, we genotyped 149 sequence polymorphisms in 375 pulmonary TB patients and 387 controls from Indonesia. We found that four polymorphisms in the TLR8 gene on chromosome X showed evidence of association with TB susceptibility in males, including a non-synonymous polymorphism rs3764880 (Met1Val; P = 0.007, odds ratio (OR) = 1.8, 95% c.i. = 1.2–2.7). We genotyped these four TLR8 polymorphisms in an independent collection of 1,837 pulmonary TB patients and 1,779 controls from Russia and again found evidence of association in males (for rs3764880 P = 0.03, OR = 1.2, 95% c.i. = 1.02–1.48). Combined evidence for association is P = 1.2×10−3–6×10−4. In addition, a quantitative PCR analysis indicated that TLR8 transcript levels are significantly up-regulated in patients during the acute phase of disease (P = 9.36×10−5), relative to baseline levels following successful chemotherapy. A marked increase in TLR8 protein expression was also observed directly in differentiated macrophages upon infection with M. bovis bacille Calmette-Guérin (BCG). Taken together, our results provide evidence, for the first time, of a role for the TLR8 gene in susceptibility to pulmonary TB across different populations.

A robust model for read count data in exome sequencing experiments and implications for copy number variant calling

Motivation: Exome sequencing has proven to be an effective tool to discover the genetic basis of Mendelian disorders. It is well established that copy number variants (CNVs) contribute to the etiology of these disorders. However, calling CNVs from exome sequence data is challenging. A typical read depth strategy consists of using another sample (or a combination of samples) as a reference to control for the variability at the capture and sequencing steps. However, technical variability between samples complicates the analysis and can create spurious CNV calls. Results: Here, we introduce ExomeDepth, a new CNV calling algorithm designed to control for this technical variability. ExomeDepth uses a robust model for the read count data and uses this model to build an optimized reference set in order to maximize the power to detect CNVs. As a result, ExomeDepth is effective across a wider range of exome datasets than the previously existing tools, even for small (e.g. one to two exons) and heterozygous deletions. We used this new approach to analyse exome data from 24 patients with primary immunodeficiencies. Depending on data quality and the exact target region, we find between 170 and 250 exonic CNV calls per sample. Our analysis identified two novel causative deletions in the genes GATA2 and DOCK8. Availability: The code used in this analysis has been implemented into an R package called ExomeDepth and is available at the Comprehensive R Archive Network (CRAN). Contact: v.plagnol@ucl.ac.uk Supplementary Information: Supplementary data are available at Bioinformatics online.

Association of the vitamin D metabolism gene CYP27B1 with type 1 diabetes

OBJECTIVE—Epidemiological studies have linked vitamin D deficiency with the susceptibility to type 1 diabetes. Higher levels of the active metabolite 1α,25-dihydroxyvitamin D (1α,25(OH)2D) could protect from immune destruction of the pancreatic β-cells. 1α,25(OH)2D is derived from its precursor 25-hydroxyvitamin D by the enzyme 1α-hydroxylase encoded by the CYP27B1 gene and is inactivated by 24-hydroxylase encoded by the CYP24A1 gene. Our aim was to study the association between the CYP27B1 and CYP24A1 gene polymorphisms and type 1 diabetes. RESEARCH DESIGN AND METHODS—We studied 7,854 patients with type 1 diabetes, 8,758 control subjects from the U.K., and 2,774 affected families. We studied four CYP27B1 variants, including common polymorphisms −1260C>A (rs10877012) and +2838T>C (rs4646536) and 16 tag polymorphisms in the CYP24A1 gene. RESULTS—We found evidence of association with type 1 diabetes for CYP27B1 −1260 and +2838 polymorphisms, which are in perfect linkage disequilibrium. The common C allele of CYP27B1 −1260 was associated with an increased disease risk in the case-control analysis (odds ratio for the C/C genotype 1.22, P = 9.6 × 10−4) and in the fully independent collection of families (relative risk for the C/C genotype 1.33, P = 3.9 × 10−3). The combined P value for an association with type 1 diabetes was 3.8 × 10−6. For the CYP24A1 gene, we found no evidence of association with type 1 diabetes (multilocus test, P = 0.23). CONCLUSIONS—The present data provide evidence that common inherited variation in the vitamin D metabolism affects susceptibility to type 1 diabetes.

Microevolution of extensively drug-resistant tuberculosis in Russia.

Extensively drug-resistant (XDR) tuberculosis (TB), which is resistant to both first- and second-line antibiotics, is an escalating problem, particularly in the Russian Federation. Molecular fingerprinting of 2348 Mycobacterium tuberculosis isolates collected in Samara Oblast, Russia, revealed that 72% belonged to the Beijing lineage, a genotype associated with enhanced acquisition of drug resistance and increased virulence. Whole-genome sequencing of 34 Samaran isolates, plus 25 isolates representing global M. tuberculosis complex diversity, revealed that Beijing isolates originating in Eastern Europe formed a monophyletic group. Homoplasic polymorphisms within this clade were almost invariably associated with antibiotic resistance, indicating that the evolution of this population is primarily driven by drug therapy. Resistance genotypes showed a strong correlation with drug susceptibility phenotypes. A novel homoplasic mutation in rpoC, found only in isolates carrying a common rpoB rifampicin-resistance mutation, may play a role in fitness compensation. Most multidrug-resistant (MDR) isolates also had mutations in the promoter of a virulence gene, eis, which increase its expression and confer kanamycin resistance. Kanamycin therapy may thus select for mutants with increased virulence, helping preserve bacterial fitness and promoting transmission of drug-resistant TB strains. The East European clade was dominated by two MDR clusters, each disseminated across Samara. Polymorphisms conferring fluoroquinolone resistance were independently acquired multiple times within each cluster, indicating that XDR TB is currently not widely transmitted.

Common variants at 11p13 are associated with susceptibility to tuberculosis

After imputation of data from the 1000 Genomes Project into a genome-wide dataset of Ghanaian individuals with tuberculosis and controls, we identified a resistance locus on chromosome 11p13 downstream of the WT1 gene (encoding Wilms tumor 1). The strongest signal was obtained at the rs2057178 SNP (P = 2.63 × 10−9). Replication in Gambian, Indonesian and Russian tuberculosis case-control study cohorts increased the significance level for the association with this SNP to P = 2.57 × 10−11.