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Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism which has directed research interest into oxidative phosphorylation (OXPHOS) and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ~16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA sequencing data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared to wild-type (WT) controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated OXPHOS in mutant muscle. Furthermore, we observed a gene-dose specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

BackgroundWe aimed to identify rare (minor allele frequency ≤1%), potentially pathogenic non-synonymous variants in a well-characterised cohort with a clinical history of exertional heat illness (EHI) or exertional rhabdomyolysis (ER). The genetic link between malignant hyperthermia (MH) and EHI was investigated due to their phenotypic overlap.MethodsThe coding regions of 38 genes relating to skeletal muscle calcium homeostasis or exercise intolerance were sequenced in 64 patients (mostly military personnel) with a history of EHI, or ER and who were phenotyped using skeletal muscle in vitro contracture tests. We assessed the pathogenicity of variants using prevalence data, in silico analysis, phenotype and segregation evidence and by review of the literature.ResultsWe found 51 non-polymorphic, potentially pathogenic variants in 20 genes in 38 patients. Our data indicate that RYR1 p.T3711M (previously shown to be likely pathogenic for MH susceptibility) and RYR1 p.I3253T are likely pathogenic for EHI. PYGM p.A193S was found in 3 patients with EHI, which is significantly greater than the control prevalence (p=0.000025). We report the second case of EHI in which a missense variant at CACNA1S p.R498 has been found. Combinations of rare variants in the same or different genes are implicated in EHI.ConclusionWe confirm a role of RYR1 in the heritability of EHI as well as ER but highlight the likely genetic heterogeneity of these complex conditions. We propose defects, or combinations of defects, in skeletal muscle calcium homeostasis, oxidative metabolism and membrane excitability are associated with EHI.

The ryanodine receptor mediates intracellular calcium ion release with excitation of nerve and muscle cells. Ryanodine receptor missense variants cause a number of myopathologies, such as malignant hyperthermia, and have been linked with various neuropathologies, including Alzheimer’s disease. We characterized the consequences of ryanodine receptor variants in vivo. Eight Caenorhabditis elegans strains, with ryanodine receptor modifications equivalent to human myopathic RYR1 variants, were generated by genome editing. In humans, these variants are rare and confer sensitivity to the inhalational anaesthetic halothane when heterozygous. Increased sensitivity to halothane was found in both homozygous and heterozygous C. elegans. Close analysis revealed distinct subtle locomotion defects, due to the different single amino acid residue changes, even in the absence of the external triggering agent. Distinct pre- and postsynaptic consequences of the variants were characterized through the responses to cholinergic pharmacological agents. The range of phenotypes reflects the complexity of the regulatory inputs to the ryanodine receptor and the criticality of the calcium ion channel opening properties, in different cell types and with age. Ryanodine receptors with these single amino acid residue changes still function as calcium ion channels, but with altered properties which are likely to have subtle consequences for human carriers of such variants. The long-term consequences of subtly altered calcium ion signalling could be cumulative and may be focussed in the smaller nerve cells rather than the more robust muscle cells. It was important to assess phenotypes in vivo to properly appreciate consequences for a whole organism.

BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasitePlasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population.Significance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

Background: Variants in RYR1 are associated with the majority of cases of malignant hyperthermia (MH), a form of heat illness pharmacogenetically triggered by general anesthetics, and they have also been associated with exertional heat illness (EHI). CACNA1S has also been implicated in MH. The authors applied a targeted next-generation sequencing approach to identify variants in RYR1 and CACNA1S in a cohort of unrelated patients diagnosed with MH susceptibility. They also provide the first comprehensive report of sequencing of these two genes in a cohort of survivors of EHI. Methods: DNA extracted from blood was genotyped using a “long” polymerase chain reaction technique, with sequencing on the Illumina GAII® or MiSeq® platforms (Illumina Inc., USA). Variants were assessed for pathogenicity using bioinformatic approaches. For further follow-up, DNA from additional family members and up to 211 MH normal and 556 MH-susceptible unrelated individuals was tested. Results: In 29 MH patients, the authors identified three pathogenic and four novel RYR1 variants, with a further five RYR1 variants previously reported in association with MH. Three novel RYR1 variants were found in the EHI cohort (n = 28) along with two more previously reported in association with MH. Two other variants were reported previously associated with centronuclear myopathy. The authors found one and three rare variants of unknown significance in CACNA1S in the MH and EHI cohorts, respectively. Conclusions: Targeted next-generation sequencing proved efficient at identifying diagnostically useful and potentially implicated variants in RYR1 and CACNA1S in MH and EHI.

Background. Missense variants in the ryanodine receptor 1 gene (RYR1) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. Methods We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. Results We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. Conclusions Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

© 2014 Elsevier B.V. Foetal death, or abortion at term, in sheep is of major significance to the livestock industry, accounting for more than ¢24million lost per annum. We have investigated whether there is a genetic component to abortion within two flocks of pedigree Charollais sheep, one followed from 1989 to 2006, the other from 1992 to 2006. Abortion occurred at a rate of 5.74-8.78% per annum against a total mortality rate of 14-24%. By model covariate analysis we have shown that 15.5% aborting ewes went on to have one or more abortions and that this risk increased with parity (p = 0.006). Heritability estimates were approximately 0.08 as calculated by SOLAR, pedigreemm and ASReml3, with sire and dam components of 0.046 and 0.048, respectively. Where the lamb was aborted, heritability estimates were highly variable according to the method employed, 0.046-0.378, with sex of the lamb being a significant covariate. This variability indicated one or more underlying, significant factors that were not measured in these analyses, potentially including infectious agents that may be involved. Nevertheless, the ASReml3 estimate (0.179) resolved to 0.074 variance attributable to the sire and 0.092 attributable to the dam, which, while not significant, was suggestive that genetic variants passed by the dam to the lamb may be of more weight than that from the sire in determining whether a lamb will abort.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhoea in pigs and humans. The duration and severity of diarrhoea can be controlled using zinc supplementation, typically pharmacological levels of zinc oxide in pigs. In this study, IPEC J2 cells were used as an in vitro model of intestinal ETEC infection, with separate and simultaneous zinc treatment. Genomic analysis identified increased expression of a variety of innate immune response genes (NF-κB targets) in response to ETEC exposure, and several stress response genes in response to zinc exposure, provided as ZnO. Expression of genes involved in the innate immune response was reduced when cells were simultaneously exposed to ZnO, and it is suggested that ZnO treatment inhibits the induction of NF-κB in response to pathogens, possibly through up-regulated heat shock proteins. A similar response in vivo with consequent down-regulation in the inflammatory response would reduce further pathogen invasion, maintain normal gut function and maintain growth.

Summary The sheep has worldwide agricultural importance, yet the genetic control of the immune responses underlying susceptibility or resistance to ovine disease is little understood. Here, we identify six novel polymorphisms in the ovine immune response genes interferon-γ (IFNG), tumour necrosis factor-α (TNF), interleukin-1β (IL1B) and interleukin-4 (IL4) in pedigree Charollais flocks. We confirm the presence of previously reported polymorphisms in IFNG and IL1B in Charollais. Restriction fragment length polymorphism (RFLP) genotyping assays have been developed for four polymorphisms, IFNGg.168C>T, IFNGg.285A>G, IL1Bg.689C>T and TNFg.3UTRA>G, and a Taqman genotyping assay has been developed for IL4g.485C>T. The previously described IL2g.647C>T polymorphism is adapted for RFLP analysis. Allele frequencies are described in Charollais, Lleyn and Suffolk cross sheep. Polymorphisms are typed in both Charollais ewes and lambs and analysed against abortion phenotypes. A subset of animals have also been analysed for the presence of Toxoplasma gondii, an abortion-causing protozoan. The IFNGg.168T allele is shown to be associated with increased risk of a ewe having an abortion, while the IFNGg.285G allele is associated with increased risk of a lamb being aborted. These assays provide tools for the investigation of the genetic basis of other phenotypes in sheep, including infectious disease susceptibility.

Phocine distemper virus (PDV) has caused two mass mortalities of European harbour seals (Phoca vitulina) in recent decades. Levels of mortality varied considerably among European populations in both the 1988 and 2002 epidemics, with higher mortality in continental European populations in comparison to UK populations. High levels of genetic differentiation at neutral makers among seal populations allow for the possibility that there could be potential genetic differences at functional loci that may account for some of the variation in mortality. Recent genome sequencing of carnivore species and development of genomic tools have now made it possible to explore the possible contribution of variation in candidate genes from harbour seals in relation to the differential mortality patterns. We assessed variation in eight genes (CD46, IFNG, IL4, IL8, IL10, RARa, SLAM and TLR2) encoding key proteins involved in host cellular interactions with Morbilliviruses and the relationship of variants to disease status. This work constitutes the first genetic association study for Morbillivirus disease susceptibility in a non-model organism, and for a natural mortality event. We found no variation in harbour seals from across Europe in the protein coding domains of the viral receptors SLAM and CD46, but SNPs were present in SLAM intron 2. SNPs were also present in IL8 p2 and RARa exon 1. There was no significant association of SLAM or RARa polymorphisms with disease status implying no role of these genes in determining resistance to PDV induced mortality, that could be detected with the available samples and the small number of polymorphisms indentified. However there was significant differentiation of allele frequencies among populations. PDV and other morbilliviruses are important models for wildlife epidemiology, host switches and viral evolution. Despite a negative result in this case, full sequencing of pinniped and other 'non-model' carnivore genomes will help in refining understanding the role of host genetics in disease susceptibility for these viruses.

Pharmacological levels of zinc oxide (ZnO) incorporated into the post-weaning piglet diet reduce the incidence of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) K88. The mechanism for this is not understood. Here, Intestinal Porcine Epithelial Cells (IPEC) J2 were used as an in vitro model of the porcine intestine. ZnO reduced IPEC J2 viability at concentrations >= 200 mu M, and ETEC adhesion to the host cell was unaffected by ZnO. Characterisation of the metabolism of IPEC J2 cells and ETEC established the effects of ZnO treatment on the metabolic profile of both. Although 100 mu M ZnO did not inhibit growth of either host or pathogen in fully supplemented media, metabolic profiles were significantly altered. Glucose and mannose were essential energy sources for IPEC J2 cells in the presence of ZnO, as the ability to utilise other sources was compromised. The increase in specificity of requirements to support respiration in ETEC was more pronounced, in particular the need for cysteine as a nitrogen source. These findings indicate that ZnO impacts on both host cell and pathogen metabolism and may provide insight into the mechanism for diarrhoea reduction. (C) 2010 Elsevier B.V. All rights reserved.

Racial variation, twin studies, segregation analyses, linkage and association studies all suggest that genetic factors play an important role in predisposition to tuberculosis. Many previous studies have been performed with pulmonary TB patients, as the most prevalent form of clinical TB (nearly 95%), and very few of them have considered extrapulmonary TB. The present study evaluates the effects of variation in eight candidate genes (LTA, TNF, IL1B, IL1RN, IL10, TGFB1, TIRAP and P2X7) with pulmonary, pleural, miliary and other extrapulmonary forms of TB in a Peruvian population from the North of Lima. 626 TB cases and 513 healthy controls were enrolled in this study. LTA(+368) and IL10(-592) were associated with different clinical forms of TB (P

In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms.

Previous studies have established a genetic component for susceptibility to malaria. Here we use a pedigree based approach, and transmission disequilibrium testing (TDT), to identify immune response genes that influence susceptibility to Plasmodium falciparum malarial phenotypes (parasite density and frequency of clinical episodes) in a Tanzanian population. Evidence for association was observed between markers in the TNF gene cluster and both the malarial phenotypes. There was weaker evidence for associations between HLA-DRB1*04, HLA-DRB1*10, and loci in the TCRBV region with parasite density. There was no evidence for association with polymorphisms in the IL10 promoter, IL1 gene cluster, or from the IL4/IL13 region.

The Great Lakes lie within a region of East Africa with very high human genetic diversity, home of many ethno-linguistic groups usually assumed to be the product of a small number of major dispersals. However, our knowledge of these dispersals relies primarily on the inferences of historical, linguistics and oral traditions, with attempts to match up the archaeological evidence where possible. This is an obvious area to which archaeogenetics can contribute, yet Uganda, at the heart of these developments, has not been studied for mitochondrial DNA (mtDNA) variation. Here, we compare mtDNA lineages at this putative genetic crossroads across 409 representatives of the major language groups: Bantu speakers and Eastern and Western Nilotic speakers. We show that Uganda harbours one of the highest mtDNA diversities within and between linguistic groups, with the various groups significantly differentiated from each other. Despite an inferred linguistic origin in South Sudan, the data from the two Nilotic-speaking groups point to a much more complex history, involving not only possible dispersals from Sudan and the Horn but also large-scale assimilation of autochthonous lineages within East Africa and even Uganda itself. The Eastern Nilotic group also carries signals characteristic of West-Central Africa, primarily due to Bantu influence, whereas a much stronger signal in the Western Nilotic group suggests direct West-Central African ancestry. Bantu speakers share lineages with both Nilotic groups, and also harbour East African lineages not found in Western Nilotic speakers, likely due to assimilating indigenous populations since arriving in the region ~3000 years ago., This study was supported by Fundacao para a Ciencia e Tecnologia (FCT) and co-financed by the European Social Fund (Human Potential Thematic Operational Programme) through grants SFRH/BPD/76207/2011 (VG) and SFRH/BPD/81986/2011 (RP). PS is supported by FCT, European Social Fund, Programa Operacional Potencial Humano and the FCT Investigator Programme (IF/01641/2013) and acknowledges FCT/MEC for support to CBMA through Portuguese funds (PIDDAC)-PEst-OE/BIA/UI4050/2014. AS is supported by "Ministerio de Ciencia e Innovacion" (SAF2011-26983), the Plan Galego IDT (EM 2012/045) and the grant from the Sistema Universitario Gallego-Modalidad REDES (2012-PG226) from the Xunta de Galicia. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology, under the project "PEst-C/SAU/LA0003/2013". NORTE-07-0162-FEDER-00018-Contributos para o reforco da capacidade do IPATIMUP enquanto actor do sistema regional de inovacao and NORTE-07-0162-FEDER-000067 Reforco e consolidacao da capacidade infraestrutural do IPATIMUP para o sistema regional de inovacao, both supported by Programa Operacional Regional do Norte (ON. 2-O Novo Norte), through FEDER funds under the Quadro de Referencia Estrategico Nacional (QREN). We would like to acknowledge the contribution of Magdalen Awor, Fr. Germano Serra and Iva Gomes for collecting the Karamoja samples. We thank Eric Bridgeland for collecting the Kabale samples, Dr Narcis Kabatereine (Vector Control Division, Uganda Ministry of Health) for the Piida samples, and Abigail Enaburekhan, Sadie Anderson- Mann, Rebecca Cole and Steven Groom for preliminary work on the Kabale and Piida sequences.

The RYR1 gene encodes the skeletal muscle isoform ryanodine receptor and is fundamental to the process of excitation-contraction coupling and skeletal muscle calcium homeostasis. Mapping to chromosome 19q13.2, the gene comprises 106 exons and encodes a protein of 5,038 amino acids. Mutations in the gene have been found in association with several diseases: the pharmacogenetic disorder, malignant hyperthermia (MH); and three congenital myopathies, including central core disease (CCD), multiminicore disease (MmD), and in an isolated case of a congenital myopathy characterized on histology by cores and rods. The majority of gene mutations reported are missense changes identified in cases of MH and CCD. In vitro analysis has confirmed that alteration of normal calcium homeostasis is a functional consequence of some of these changes. Genotype-phenotype correlation studies performed using data from MH and CCD patients have also suggested that mutations may be associated with a range of disease severity phenotypes. This review aims to summarize the current understanding of RYR1 mutations reported in association with MH and CCD and the present viewpoint on the use of mutation data to aid clinical diagnosis of these conditions.

Here we report the results from a genome-wide linkage scan to identify genes and chromosomal regions that influence quantitative immune response traits, using multi-case leprosy and tuberculosis families from north-eastern Brazil. Total plasma IgE, antigen-specific IgG to Mycobacterium leprae soluble antigen (MLSA), M. tuberculosis soluble antigen (MTSA) and M. tuberculosis purified protein derivative (PPD), and antigen-specific lymphocyte proliferation (stimulation index or SI) and interferon-gamma (IFN-gamma) release to MLSA and PPD, were measured in 16 tuberculosis (184 individuals) and 21 leprosy (177 individuals) families. The individuals were genotyped at 382 autosomal microsatellite markers across the genome. The adjusted immune-response phenotypes were analysed using a variety of variance components and regression-based methods. These analyses highlighted a number of practical issues and problems with regard to implementation of the methods and, interestingly, differences were observed between several standard statistical and genetic analysis packages used. From this we determined that, for this set of traits in these pedigrees, significant p values for linkage using variance components analysis, supported by significance using the Visscher-Hopper modification of the Haseman-Elston method, provided the most compelling evidence for linkage. Using these criteria, linkage (5.8 x 10(-5) < p < 0.008) was seen for: total plasma IgE on chromosome 2; IgG to MLSA on chromosomes 8, 17 and 21; IgG to PPD on chromosome 12; SI to PPD on chromosome 1; IFN-gamma to MLSA on chromosomes 6, 7, 10, 12 and 14; and IFN-gamma to PPD on chromosomes 1, 16 and 19.

Preimplantation genetic diagnosis (PGD) of single gene defects following assisted conception typically involves removal of single cells from preimplantation embryos and analysis using highly sensitive PCR amplification methods taking stringent precautions to prevent contamination from foreign or previously amplified DNA. Recently, whole genome amplification has been achieved from small quantities of genomic DNA by isothermal amplification with bacteriophage 29 DNA polymerase- and exonuclease-resistant random hexamer primers. Here we report that isothermal whole genome amplification from single and small numbers of lymphocytes and blastomeres isolated from cleavage stage embryos yielded microgram quantities of amplified DNA, and allowed analysis of 20 different loci, including the DeltaF508 deletion causing cystic fibrosis and polymorphic repeat sequences used in DNA fingerprinting. As with analysis by PCR-based methods, some preferential amplification or allele drop-out at heterozygous loci was detected with single cells. With 2-5 cells, amplification was more consistent and with 10 or 20 cells results were indistinguishable from genomic DNA. The use of isothermal whole genome amplification as a universal first step marks a new era for PGD since, unlike previous PCR-based methods, sufficient DNA is amplified for diagnosis of any known single gene defect by standard methods and conditions.

Cytokine and proliferative responses to Necator americanus infection were measured in a treatment-reinfection study of infected subjects from an area of Papua New Guinea where N. americanus is highly endemic. Before treatment, most subjects produced detectable interleukin (IL)-4 (97%), IL-5 (86%), and interferon (IFN)- gamma (64%) in response to adult N. americanus antigen. Pretreatment IFN- gamma responses were negatively associated with hookworm burden, decreasing by 18 pg/mL for each increase of 1000 eggs/gram (epg) (n=75; P.01). Mean IFN- gamma responses increased significantly after anthelmintic treatment, from 166 to 322 pg/mL (n=42; P.01). The intensity of reinfection was significantly negatively correlated with pretreatment IL-5 responses, decreasing by 551 epg for each 100 pg/mL increase in production of IL-5 (n=51; P.01). These data indicate that there is a mixed cytokine response in necatoriasis, with worm burden-associated suppression of IFN- gamma responses to adult N. americanus antigen. Resistance to reinfection is associated with the parasite-specific IL-5 response.

The region of conserved synteny on mouse chromosome 11/human 17q11-q21 is known to carry a susceptibility gene(s) for intramacrophage pathogens. The region is rich in candidates including NOS2A, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL5/RANTES, CCR7, STAT3 and STAT5A/5B. To examine the region in man, we studied 92 multicase tuberculosis (627 individuals) and 72 multicase leprosy (372 individuals) families from Brazil. Multipoint nonparametric analysis (ALLEGRO) using 16 microsatellites shows two peaks of linkage for leprosy at D17S250 (Z(lr) score 2.34; P=0.01) and D17S1795 (Z(lr) 2.67; P=0.004) and a single peak for tuberculosis at D17S250 (Z(lr) 2.04; P=0.02). Combined analysis shows significant linkage (peak Z(lr) 3.38) at D17S250, equivalent to an allele sharing LOD score 2.48 (P=0.0004). To determine whether one or multiple genes contribute, 49 informative single nucleotide polymorphisms were typed in candidate genes. Family-based allelic association testing that was robust to family clustering demonstrated significant associations with tuberculosis susceptibility at four loci separated by intervals (NOS2A-8.4 Mb-CCL18-32.3 kb-CCL4-6.04 Mb-STAT5B) up to several Mb. Stepwise conditional logistic regression analysis using a case/pseudo-control data set showed that the four genes contributed separate main effects, consistent with a cluster of susceptibility genes across 17q11.2.

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle. Susceptibility to MH is only apparent after exposure to volatile anesthetics and/or depolarizing muscle relaxants. CCD patients present with diffuse muscular weakness but are also at risk of MH. Mutations in RYR1 (19q13.1), encoding a skeletal muscle calcium release channel (ryanodine receptor), account for the majority of MH and CCD cases. Fifteen RYR1 N-terminal mutations are considered causative of MH susceptibility, five of which are also associated with CCD. In the first extensive UK population survey, eight of 15 mutations were detected in 85 out of 297 (29%) unrelated MH susceptible cases, with G2434R detected in 53 cases (18%). Mutation type was shown to affect significantly MH phenotypes (in vitro contracture test (IVCT) response to caffeine, halothane, and ryanodine). RYR1 mutations associated with both CCD and MH (R163C, R2163H, R2435H) had more severe caffeine and halothane response phenotypes than those associated with MH alone. Mutations near the amino terminal (R163C, G341R) had a relatively greater effect on responses to caffeine than halothane, with a significantly increased caffeine:halothane tension ratio compared to G2434R of the central domain. All phenotypes were more severe in males than females, and were also affected by muscle specimen size and viability. Discordance between RYR1 genotype and IVCT phenotype was observed in seven families (nine individuals), with five false-positives and four false-negatives. This represents the most extensive study of MH patient clinical and genetic data to date and demonstrates that RYR1 mutations involved in CCD are those associated with one end of the spectrum of MH IVCT phenotypes.

Background Malignant hyperthermia (MH), central core disease (CCD), exertional heat stroke (EHS) and late-onset axial myopathy have been attributed to mutations in ryanodine receptor type 1 (RYR1). The RyR1 protein is over 5000 amino acid residues long, making manipulation of the mammalian gene difficult. The ryanodine receptor in Caenorhabditis elegans is UNC-68, which has 40% amino acid identity to the human protein.

• A more detailed analysis of in vitro contracture test (IVCT) data to better define phenotype for research purposes, and to assess the effects of specific mutations on the range of phenotypes. We have previously conducted an analysis of the effects of a number of ryanodine receptor type 1 (RYR1) mutations on IVCT and MH reaction phenotypes. • We have previously published preliminary work considering the possibility of genetic modifiers, and this is a topical area with the role of common versus rare variants as contributors to complex disorders being hotly debated. It will be possible to initiate small projects on the effects of previously identified single nucleotide variants and SNV-SNV interactions on phenotype. • To analyse observations such as discordancy in greater depth. • To assess the statistical power for large-scale studies such as a Genome Wide Association Scan.

Malignant hyperthermia (MH) is a potentially lethal disorder triggered in susceptible individuals on exposure to common anaesthetic agents. Crises reflect the consequences of disturbed skeletal muscle calcium homeostasis. MH is an autosomal dominant, genetically heterogeneous trait. Defects in a single major gene have been assumed to determine susceptibility status in individual families. However, in some pedigrees phenotypic and genotypic data are discordant. One explanation, in contrast to the current genetic model, is that susceptibility is dependent upon the effects of more than one gene. Using the transmission disequilibrium test we assessed the involvement of 8 MH candidate loci (RYR1, CACNA1S, CACNA2D1, MHS4 at 3q13.1, MHS6 at 5p, LIPE, DM1, dystrophin) by analysis of data from 130 MH nuclear families. Results suggested that variations in more than one gene may influence MH susceptibility in single families.

Amazonian localized cutaneous leishmaniasis (LCL) is caused by parasites of the subgenera Leishmania and Viannia. Respectively, these parasites may cause diffuse cutaneous leishmaniasis (DCL) and mucocutaneous leishmaniasis (MCL). This, together with differing skin test responses, suggests some species-specificity in cell mediated immunity. In this study, T cell responses (proliferative and interferon-gamma) to crude and defined antigens were examined in paired samples pre and post chemotherapy. Untreated L. (L.) amazonensis LCL patients showed lower responses to crude leishmanial antigens than the L. (V.) spp. group. L. (V.) braziliensis antigen was a more potent stimulator of T cell responses than L. (L.) amazonensis antigen in all patient groups. Few positive responses were seen to the L. (L.) amazonensis glycoprotein GP46. A substantial proportion of LCL patients did respond to the L. (L.) pifanoi amastigote antigens A2, and the surface membrane glycoprotein P8. DCL patients were poor responders to all leishmanial antigens, except GP46. In contrast, MCL patients were good responders to all antigens except GP46 and A2. A significant rise in the response to P8 and A2 antigen was seen post treatment across all LCL and MCL patients, indicating that these antigens might provide suitable vaccine candidates.

Background Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Methods and results Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0–6 copies per diploid genome (pdg) in Peru, between 0–12 pdg in !Xhosa samples and between 0–10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). Conclusions The case–control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations.

Recent studies have shown that mucocutaneous leishmaniasis (MCL), a severe and debilitating form of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis infection, is accompanied by high circulating levels of tumor necrosis factor (TNF)-alpha. Analysis of TNF polymorphisms in Venezuelan ACL patients and endemic unaffected controls demonstrates a high relative risk (RR) of 7.5 (P < 0.001) of MCL disease in homozygotes for allele 2 of a polymorphism in intron 2 of the TNF-beta gene, especially in females (RR = 9.5; P < 0.001) compared with males (RR = 4; P < 0.05). A significantly higher frequency (P < 0.05) of allele 2 at the -308-basepair TNF-alpha gene polymorphism was also observed in MCL patients (0.18) compared with endemic control subjects (0.069), again associated with a high relative risk of disease (RR = 3.5; P < 0.05) even in the heterozygous condition. Because both the TNF-alpha and TNF-beta polymorphisms have previously been linked with functional differences in TNF-alpha levels, these data suggest that susceptibility to the mucocutaneous form of disease may be directly associated with regulatory polymorphisms affecting TNF-alpha production.

Murine Nramp is a candidate for the macrophage resistance gene Ity/Lsh/Bcg. Sequence analysis of human NRAMP was undertaken to determine its role in man. A yeast artificial chromosome carrying NRAMP was subcloned and positive clones sequenced. The transcriptional start site was mapped using 5′ RACE PCR. Polymorphic variants were amplified by PCR. Linkage analysis was used to map NRAMP. NRAMP spans 12kb and has 15 exons encoding a 550 amino acid protein showing 85% identity (92% similarity) with Nramp. Two conserved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding domain, and in exon 3. Striking sequence similarities (57 and 53%) were observed with yeast mitochondrial proteins, SMF1 and SMF2, especially within putative functional domains: exon 6 encoding the second transmembrane spanning domain, site of the murine susceptibility mutation; and exon 11 encoding a conserved transport motif. No mutations comparable to the murine susceptibility mutation were found. The transcriptional initiation site mapped 148 bp 5′ of the translational initiation codon. 440bp of 5′ flanking sequence contained putative promoter region elements: 6 interferon-γ response elements, 3 W-elements, 3 NFκB binding sites and 1 AP-1 site. Nine purine-rich GGAA core motifs for the myeloid-specific PU. 1 transcription factor were identified, two combining with imperfect AP1-like sites to create PEA3 motifs. TATA, GC and CCAAT boxes were absent. A possible enhancer element containing the Z-DNA forming dinucleotide repeat t(gt), ac(gt), ac(gt), g was polymorphic (4 alleles; n=4,9,10,11), and was used to map NRAMP to 2q35. This analysis provides important resources to study the role of NRAMP in human disease.

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTIn−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.

This paper presents the first evaluation of the molecular epidemiology of Mycobacterium tuberculosis in Peru. We characterised 323 isolates using spoligotyping and mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTR) typing. We aimed to determine the levels of genetic diversity and genetic differentiation among and within Peruvian isolates and the epidemiological factors which may be driving patterns of population structure and evolution of M. tuberculosis in Peru. Our results compared to the fourth international spoligotyping database (SpolDB4) and MIRU-VNTRplus, show that the main M. tuberculosis families present are Latin American-Mediterranean, Haarlem, T, and Beijing. Bayesian clustering recovered 15 groups in the Peruvian M. tuberculosis isolates, among which two were composed mainly of orphans, implying the presence of native “Peruvian” strains not previously reported. Variable levels of association with drug resistance were observed, with Beijing genotypes not showing any association with multidrug resistance, while in other groups MIRU-VNTR loci 2, 23, 31, and 40 were found to be associated with the multidrug-resistant tuberculosis (MDR-TB) phenotype, suggesting that a linkage disequibrium between these MIRU and drug resistance loci may be present. Genetic differentiation was present among drug resistant and sensitive strains. Ethambutol appeared to be the main driver of differentiation, suggesting that strong selection pressure could have been exerted by drug treatment in Peru over recent years.

Antibody (IgG) responses to mycobacterial (BCG; PPD; Mycobacterium leprae soluble antigen, MLSA) and leishmanial (Leishmania mexicana LV4) antigens were measured in 208 initially PPD and leishmanin skin-test negative volunteers divided into four vaccine groups as follows: 68 received BCG plus killed promastigotes (group A), 47 received BCG alone (group B), 47 received killed promastigotes alone (group C), and 46 formed the diluent control (placebo, group D). Three vaccine doses were administered at 8-12 week intervals. Vaccinees were bled immediately prior to each vaccination, and again at 3- and 12-month follow-up. Skin tests were performed prevaccination, and again at the 3- and 12-month follow-up. Anti-BCG, anti-PPD and anti-MLSA IgG levels increased significantly in groups A and B receiving BCG. The presence of leishmanial antigen (with BCG) in the inoculum suppressed the IgG response to Mycobacterium tuberculosis/Mycobacterium bovis-related (PPD and BCG), but not M. leprae-related (MLSA)-related, antigens. A small but significant increase (relative to prevaccination level) in response to MLSA, but not to BCG or PPD was observed in the non-BCG-vaccinated groups. The background level of response to mycobacterial and leishmanial antigens was higher in the Venezuelan vaccinees than in non-endemic (British) volunteers. Responses to leishmanial antigen were not enhanced in the two vaccine groups receiving killed promastigotes (with/without BCG) compared with the BCG alone and placebo groups. Instead, all vaccine groups showed a pattern of response consistent with either (i) a response to the skin-test antigen or, more likely, (ii) seasonal endemic exposure to leishmanial antigen. Interestingly, this endemic response to leishmanial antigen was enhanced in the vaccine groups receiving BCG, despite the fact that group B received no leishmanial antigen in the vaccine inoculum. When prevaccination IgG levels (mean + 3 standard deviations) were used to determine a negative cut-off, a low percentage (< 38%) of vaccinees converted to responder status for either anti-mycobacterial or anti-leishmanial responses, and those who did would be classified as 'low-responder' status compared with titres observed in severe forms of disease. Hence, although there was evidence for a background endemic response to both leishmanial and mycobacterial antigens, there was no evidence that vaccination per se led to a potentially disease exacerbatory level of TH2-associated antibody response especially with respect to the anti-leishmanial response.(ABSTRACT TRUNCATED AT 400 WORDS)

The mycobacterial diseases leprosy and tuberculosis (TB) and the leishmaniases are characterized by a wide spectrum of disease phenotypes, and by the fact that the majority of individuals exposed to the causative organisms Mycobacterium leprae, M. tuberculosis and Leishmania sp. become infected but do not present with clinical disease. In order to determine whether a human homologue to the murine macrophage resistance gene Lsh/Ity/Bcg influences susceptibility to human disease, multicase families for all three diseases have been collected, and linkage analysis performed using a panel of markers in the region of human chromosome 2q33-q37 known to be conserved with the Lsh/Ity/Bcg-containing region of murine chromosome 1. Because of the paucity of available polymorphic markers/linkage information for 2q33-q37, data from 35 multicase leprosy, TB and visceral leishmaniasis families (310 individuals) were first pooled to produce a detailed RFLP map of the region. Peak LOD scores well in excess of 3 were observed for linkage between adjacent pairs of a more proximal (2q33-q35) set of markers CRYGP1, MAP2, FN1, TNP1, VIL1 and DES, and between adjacent pairs of a more distal (2q35-q37) set COL6A3, D2S55 and D2S3. These peak LOD scores and the corresponding values for theta were used in the MAP92 program to generate a multiple two-point map with gene order/map intervals (cM) of: CRYGP1-4.65-MAP2-3.45-FN1-5.95-TNP1-3.41-VIL1-3. 01- DES-20.14-COL6A-10.91-D2S55-3.67-D2S3. Although local support for the placement of loci in this order was weak (LOD < 2, except for DES-COL6A3 where LOD = 6.02), the map is consistent with the gene order for those loci (Cryg, Fn-1, Tp-1, Vil, Des, Col6a3) previously mapped in the mouse. Data from 17 multicase leprosy families (149 individuals) were further analysed for linkage between a putative disease susceptibility locus (DSL) controlling susceptibility to leprosy per se and each of the marker loci. Assuming 100% penetrance for the susceptibility allele, no positive LOD score was obtained for linkage between the DSL and any of the marker genes. Instead, the data provide convincing evidence (LOD scores < -2) that a DSL does not fall within 10-20 cM of CRYGP1, MAP2, TNP1, VIL1, DES or D2S55, or within 5-10 cM of FN1, COL6A3 or D2S3. This effectively excludes a putative DSL controlling susceptibility to leprosy per se from the entire region 2q33-q37.(ABSTRACT TRUNCATED AT 400 WORDS)