Martin L. Hibberd

Normal gut microbiota modulates brain development and behavior

Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior.

Genomewide association study of leprosy.

BACKGROUND The narrow host range of Mycobacterium leprae and the fact that it is refractory to growth in culture has limited research on and the biologic understanding of leprosy. Host genetic factors are thought to influence susceptibility to infection as well as disease progression. METHODS We performed a two-stage genomewide association study by genotyping 706 patients and 1225 controls using the Human610-Quad BeadChip (Illumina). We then tested three independent replication sets for an association between the presence of leprosy and 93 single-nucleotide polymorphisms (SNPs) that were most strongly associated with the disease in the genomewide association study. Together, these replication sets comprised 3254 patients and 5955 controls. We also carried out tests of heterogeneity of the associations (or lack thereof) between these 93 SNPs and disease, stratified according to clinical subtype (multibacillary vs. paucibacillary). RESULTS We observed a significant association (P<1.00x10(-10)) between SNPs in the genes CCDC122, C13orf31, NOD2, TNFSF15, HLA-DR, and RIPK2 and a trend toward an association (P=5.10x10(-5)) with a SNP in LRRK2. The associations between the SNPs in C13orf31, LRRK2, NOD2, and RIPK2 and multibacillary leprosy were stronger than the associations between these SNPs and paucibacillary leprosy. CONCLUSIONS Variants of genes in the NOD2-mediated signaling pathway (which regulates the innate immune response) are associated with susceptibility to infection with M. leprae.

Genome-wide association identifies multiple ulcerative colitis susceptibility loci

Ulcerative colitis is a chronic, relapsing inflammatory condition of the gastrointestinal tract with a complex genetic and environmental etiology. In an effort to identify genetic variation underlying ulcerative colitis risk, we present two distinct genome-wide association studies of ulcerative colitis and their joint analysis with a previously published scan, comprising, in aggregate, 2,693 individuals with ulcerative colitis and 6,791 control subjects. Fifty-nine SNPs from 14 independent loci attained an association significance of P < 10−5. Seven of these loci exceeded genome-wide significance (P < 5 × 10−8). After testing an independent cohort of 2,009 cases of ulcerative colitis and 1,580 controls, we identified 13 loci that were significantly associated with ulcerative colitis (P < 5 × 10−8), including the immunoglobulin receptor gene FCGR2A, 5p15, 2p16 and ORMDL3 (orosomucoid1-like 3). We confirmed association with 14 previously identified ulcerative colitis susceptibility loci, and an analysis of acknowledged Crohns disease loci showed that roughly half of the known Crohns disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 109 virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.

Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease

BACKGROUND The reasons why meningococcal disease develops in only a small proportion of individuals carrying the causative bacteria are unknown. Differences in host responses to bacterial colonisation are thought to be involved, since people with deficiencies in the terminal components of the complement pathway, or of properdin, are susceptible to meningococcal disease. We postulate that genetic variants of mannose-binding lectin (MBL), a plasma opsonin that initiates another pathway of complement activation, might similarly cause susceptibility to meningococcal disease. METHODS The frequency of variants of the MBL gene was ascertained in children with meningococcal disease and controls from two independent studies; one hospital-based (194 patients and 272 controls [patients with non-infectious disorders]), and one community-based (72 patients and 110 controls [healthy individuals]), by means of PCR and restriction-enzyme digestion, with confirmation by DNA sequencing. FINDINGS The proportion of people homozygous for MBL-variant alleles was higher in patients with meningococcal disease than in controls in the hospital study (15 [7.7%] vs four [1.5%]; odds ratio 6.5 [95% CI 2.0-27.2]) and in the community study (six [8.3%] vs three [2.7%]; 4.5 [0.9-29.1]). The population attributable fraction of cases attributable to MBL variants (homozygous and heterozygous) was 32%. INTERPRETATION The MBL pathway is a critical determinant of meningococcal-disease susceptibility, and genetic variants of MBL might account for a third of all disease cases.

4G/5G promoter polymorphism in the plasminogen-activator-inhibitor-1 gene and outcome of meningococcal disease

BACKGROUND Intravascular coagulation with infarction of skin, digits, and limbs is a characteristic feature of meningococcal sepsis. Children with meningococcal sepsis have higher than normal concentrations of plasminogen activator inhibitor 1 (PAI-1) in plasma. Combined with the widespread venous thrombosis, this finding suggests an impairment of fibrinolysis. A common functional insertion/deletion (4G/5G) polymorphism exists in the promoter region of the PAI-1 gene. We tested the hypothesis that children with the 4G/4G genotype produce higher concentrations of PAI-1, develop more severe coagulopathy, and are at greater risk of death during meningococcal sepsis. METHODS The relation between meningococcal disease outcome, PAI-1 concentration, and PAI-1 genotype was investigated in 175 children with meningococcal disease (37 from Rotterdam, the Netherlands, and 138 from London, UK) and 226 controls (137 from Rotterdam, 89 from London). PAI-1 concentrations in plasma were measured by ELISA, and the 4G/5G PAI-1 polymorphism was detected by PCR and hybridisation. FINDINGS Concentrations of PAI-1 on admission correlated with presentation (sepsis or meningitis) and outcome. The median PAI-1 concentration in children who died was substantially higher than that in survivors (2448 [IQR 1115-3191] vs 370 [146-914] ng/mL; p<0.0001). Patients with the 4G/4G genotype had significantly higher PAI-1 concentrations than those with the 4G/5G or 5G/5G genotype (1051 [550-2440] vs 436 [198-1225] ng/mL; p=0.03), and had an increased risk of death (relative risk 2.0 [1.0-3.8] for the two cohorts combined, and 4.8 [1.8-13] for the London cohort). INTERPRETATION A genetic predisposition to produce high concentrations of PAI-1 is associated with poor outcome of meningococcal sepsis. This finding suggests that impaired fibrinolysis is an important factor in the pathophysiology of meningococcal sepsis.

A Genome-Wide Association Study Identifies Novel and Functionally Related Susceptibility Loci for Kawasaki Disease

Kawasaki disease (KD) is a pediatric vasculitis that damages the coronary arteries in 25% of untreated and approximately 5% of treated children. Epidemiologic data suggest that KD is triggered by unidentified infection(s) in genetically susceptible children. To investigate genetic determinants of KD susceptibility, we performed a genome-wide association study (GWAS) in 119 Caucasian KD cases and 135 matched controls with stringent correction for possible admixture, followed by replication in an independent cohort and subsequent fine-mapping, for a total of 893 KD cases plus population and family controls. Significant associations of 40 SNPs and six haplotypes, identifying 31 genes, were replicated in an independent cohort of 583 predominantly Caucasian KD families, with NAALADL2 (rs17531088, p combined = 1.13×10−6) and ZFHX3 (rs7199343, p combined = 2.37×10−6) most significantly associated. Sixteen associated variants with a minor allele frequency of >0.05 that lay within or close to known genes were fine-mapped with HapMap tagging SNPs in 781 KD cases, including 590 from the discovery and replication stages. Original or tagging SNPs in eight of these genes replicated the original findings, with seven genes having further significant markers in adjacent regions. In four genes (ZFHX3, NAALADL2, PPP1R14C, and TCP1), the neighboring markers were more significantly associated than the originally associated variants. Investigation of functional relationships between the eight fine-mapped genes using Ingenuity Pathway Analysis identified a single functional network (p = 10−13) containing five fine-mapped genes—LNX1, CAMK2D, ZFHX3, CSMD1, and TCP1—with functional relationships potentially related to inflammation, apoptosis, and cardiovascular pathology. Pair-wise blood transcript levels were measured during acute and convalescent KD for all fine-mapped genes, revealing a consistent trend of significantly reduced transcript levels prior to treatment. This is one of the first GWAS in an infectious disease. We have identified novel, plausible, and functionally related variants associated with KD susceptibility that may also be relevant to other cardiovascular diseases.

Assay of locus-specific genetic load implicates rare Toll-like receptor 4 mutations in meningococcal susceptibility

As the central component of the human endotoxin sensor, Toll-like receptor 4 (TLR4) functions in the early detection and response to Gram-negative infection. We therefore examined a large collection of patients with meningococcal sepsis, comparing the frequency of rare TLR4 coding changes to those in an ethnically matched control population. TLR2 sequences were also acquired and compared. Total nucleotide variation at TLR4 and TLR2 loci was assayed by using a novel computational method. A total of 3.01 megabases of coding sequence was captured at these loci from white subjects with or without meningococcal disease. Authentic mutations were found and high-quality, bidirectional coverage was measured across the coding region by using mutationseeker, a program specifically designed to assay locus-specific genetic load. Using a method that obviates the confounding effect of linkage disequilibrium, we observed that rare heterozygous missense mutations of TLR4 contribute to the development of systemic meningococcal disease among white populations of the southern United Kingdom (P = 0.02; odds ratio 8.2). When results from all white populations were pooled, an overwhelmingly significant excess of such mutations was observed among individuals with disease (P = 2 × 10−6; odds ratio 27.0). The common white TLR4 variant (TLR4B), synonymous TLR4 substitutions, and variant TLR2 alleles were not significantly over-represented among patients with systemic meningococcal infections. No single variant of TLR4 was significantly over-represented in the meningococcal population. Collectively, however, rare TLR4 coding variants were markedly over-represented. Sensing via TLR4 probably contributes to the early containment of meningococcal infection, and sensing defects create increased risk of disease.

LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets

The study of cell-population heterogeneity in a range of biological systems, from viruses to bacterial isolates to tumor samples, has been transformed by recent advances in sequencing throughput. While the high-coverage afforded can be used, in principle, to identify very rare variants in a population, existing ad hoc approaches frequently fail to distinguish true variants from sequencing errors. We report a method (LoFreq) that models sequencing run-specific error rates to accurately call variants occurring in <0.05% of a population. Using simulated and real datasets (viral, bacterial and human), we show that LoFreq has near-perfect specificity, with significantly improved sensitivity compared with existing methods and can efficiently analyze deep Illumina sequencing datasets without resorting to approximations or heuristics. We also present experimental validation for LoFreq on two different platforms (Fluidigm and Sequenom) and its application to call rare somatic variants from exome sequencing datasets for gastric cancer. Source code and executables for LoFreq are freely available at http://sourceforge.net/projects/lofreq/.

Genome-wide association study identifies variants in the CFH region associated with host susceptibility to meningococcal disease

Meningococcal disease is an infection caused by Neisseria meningitidis. Genetic factors contribute to host susceptibility and progression to disease, but the genes responsible for disease development are largely unknown. We report here a genome-wide association study for host susceptibility to meningococcal disease using 475 individuals with meningococcal disease (cases) and 4,703 population controls from the UK. We performed, in Western European and South European cohorts (consisting of 968 cases and 1,376 controls), two replication studies for the most significant SNPs. A cluster of complement factor SNPs replicated independently in both cohorts, including SNPs within complement factor H (CFH) (rs1065489 (p.936D<E), P = 2.2 × 10−11) and in CFH-related protein 3 (CFHR3)(rs426736, P = 4.6 × 10−13). N. meningitidis is known to evade complement-mediated killing by the binding of host CFH to the meningococcal factor H–binding protein (fHbp). Our study suggests that host genetic variation in these regulators of complement activation plays a role in determining the occurrence of invasive disease versus asymptomatic colonization by this pathogen.