Genetic epidemiology of malignant hyperthermia in the UK.

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<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

Assessing the pathogenicity of RYR1 variants in malignant hyperthermia.

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.

Transcriptional repression of IFNß1 by ATF2 confers melanoma resistance to therapy.

The resistance of melanoma to current treatment modalities represents a major obstacle for durable therapeutic response, and thus the elucidation of mechanisms of resistance is urgently needed. The crucial functions of activating transcription factor-2 (ATF2) in the development and therapeutic resistance of melanoma have been previously reported, although the precise underlying mechanisms remain unclear. Here, we report a protein kinase C-ɛ (PKCɛ)- and ATF2-mediated mechanism that facilitates resistance by transcriptionally repressing the expression of interferon-ß1 (IFNß1) and downstream type-I IFN signaling that is otherwise induced upon exposure to chemotherapy. Treatment of early-stage melanomas expressing low levels of PKCɛ with chemotherapies relieves ATF2-mediated transcriptional repression of IFNß1, resulting in impaired S-phase progression, a senescence-like phenotype and increased cell death. This response is lost in late-stage metastatic melanomas expressing high levels of PKCɛ. Notably, nuclear ATF2 and low expression of IFNß1 in melanoma tumor samples correlates with poor patient responsiveness to biochemotherapy or neoadjuvant IFN-α2a. Conversely, cytosolic ATF2 and induction of IFNß1 coincides with therapeutic responsiveness. Collectively, we identify an IFNß1-dependent, cell-autonomous mechanism that contributes to the therapeutic resistance of melanoma via the PKCɛ-ATF2 regulatory axis.

Novel polymorphisms in ovine immune response genes and their association with abortion.

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-1ß (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.

Genetic variation in RYR1 and malignant hyperthermia phenotypes.

BACKGROUND: Malignant hyperthermia (MH) is associated, in the majority of cases, with mutations in RYR1, the gene encoding the skeletal muscle ryanodine receptor. Our primary aim was to assess whether different RYR1 variants are associated with quantitative differences in MH phenotype. METHODS: The degree of in vitro pharmacological muscle contracture response and the baseline serum creatine kinase (CK) concentration were used to generate a series of quantitative phenotypes for MH. We then undertook the most extensive RYR1 genotype-phenotype correlation in MH to date using 504 individuals from 204 MH families and 23 RYR1 variants. We also determined the association between a clinical phenotype and both the laboratory phenotype and RYR1 genotype. RESULTS: We report a novel correlation between the degree of in vitro pharmacological muscle contracture responses and the onset time of the clinical MH response in index cases (P<0.05). There was also a significant correlation between baseline CK concentration and clinical onset time (P=0.039). The specific RYR1 variant was a significant determinant of the severity of each laboratory phenotype (P<0.0001). CONCLUSIONS: The MH phenotype differs significantly with different RYR1 variants. Variants leading to more severe MH phenotype are distributed throughout the gene and tend to lie at relatively conserved sites in the protein. Differences in phenotype severity between RYR1 variants may explain the variability in clinical penetrance of MH during anaesthesia and why some variants have been associated with exercise-induced rhabdomyolysis and heat stroke. They may also inform a mutation screening strategy in cases of idiopathic hyperCKaemia.

Epigenetic allele silencing and variable penetrance of malignant hyperthermia susceptibility.

BACKGROUND: Tissue-specific monoallelic silencing of the RYR1 gene has been proposed as an explanation for variable penetrance of dominant RYR1 mutations in malignant hyperthermia (MH). We examined the hypothesis that monoallelic silencing could explain the inheritance of an MH discordant phenotype in some instances. METHODS: We analysed parent-offspring transmission data from MH kindreds to assess whether there was any deviation from the expected autosomal dominant Mendelian inheritance pattern. We also evaluated informative single-nucleotide polymorphism (SNP) genotypes in a cohort of unrelated MH patients using genomic DNA (gDNA, prepared from leucocytes) and coding DNA (cDNA, prepared from skeletal muscle). Finally, we examined the segregation of specific mutations at the gDNA and cDNA level within MH families where positive RYR1 gDNA genotype/normal MH phenotype discordance had been observed. RESULTS: In 2113 transmissions from affected parents, there was a consistent parent-of-origin effect (P<0.001) with affected fathers having fewer affected daughters (20%, 95% CI 17-22%) than affected sons (25%, 95% CI 23-26%) or unaffected daughters (27%, 95% CI 25-30%). No discrepancies were observed between the RYR1 SNP genotypes recorded at the gDNA and cDNA levels. In 14 MH negative individuals from 11 discordant families, the familial mutation was detected in skeletal muscle cDNA in all cases. CONCLUSIONS: Epigenetic allele silencing may play a role in the inheritance of MH susceptibility, but this is unlikely to involve silencing of RYR1.

Analysis of RYR1 haplotype profile in patients with malignant hyperthermia.

This study represents a new approach to characterising patients at risk of malignant hyperthermia (MH) through the use of a recently published method for identifying high-risk haplotypes in candidate genes. We present analysis based upon the largest standardised and genotyped database of MH patients worldwide. We used unphased RYR1 SNP data directly to (1) assess RYR1 haplotype frequency differences between susceptible cases and control groups and (2) analyse population-based association via clustering of RYR1 haplotypes based on disease risk. Our results show a significant difference in RYR1 haplotype frequency between susceptible cases and UK Caucasian population controls. Furthermore we identify a high-risk cluster of haplotypes that is associated with the commonest UK MH mutation p.G2434R/c.7300G>A. These results demonstrate the applicability of this new and practical method for population based association analysis.

Factor XIII transglutaminase supports hematogenous tumor cell metastasis through a mechanism dependent on natural killer cell function.

BACKGROUND: Multiple studies suggest that the hemostatic and innate immune systems functionally cooperate in establishing the fraction of tumor cells that successfully form metastases. In particular, platelets and fibrinogen have been shown to support metastatic potential through a mechanism coupled to natural killer (NK) cell function. As the transglutaminase that ultimately stabilizes platelet/fibrin thrombi through the covalent crosslinking of fibrin, factor (F) XIII is another thrombin substrate that is likely to support hematogenous metastasis. OBJECTIVE: Directly define the role of FXIII in tumor growth, tumor stroma formation, and metastasis. METHODS: Tumor growth and metastatic potential were quantitatively and qualitatively evaluated in wild-type mice and gene-targeted mice lacking the catalytic FXIII-A subunit. RESULTS: Loss of FXIIIa function significantly diminished hematogenous metastatic potential in both experimental and spontaneous metastasis assays in immunocompetent mice. However, FXIII was not required for the growth of established tumors or tumor stroma formation. Rather, detailed analyses of the early fate of circulating tumor cells revealed that FXIII supports the early survival of micrometastases by a mechanism linked to NK cell function. CONCLUSIONS: Factor XIII is a significant determinant of metastatic potential and supports metastasis by impeding NK cell-mediated clearance of tumor cells. Given that these findings parallel previous observations in fibrinogen-deficient mice, an attractive hypothesis is that FXIII-mediated stabilization of fibrin/platelet thrombi associated with newly formed micrometastases increases the fraction of tumor cells capable of evading NK cell-mediated lysis.

Evidence for a cluster of genes on chromosome 17q11-q21 controlling susceptibility to tuberculosis and leprosy in Brazilians.

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.

Identification of three novel SEDL mutations, including mutation in the rare, non-canonical splice site of exon 4.

Spondyloepiphyseal dysplasia tarda (SEDT) is an X-linked recessive disorder, characterized by disproportionately short stature and degenerative joint disease, which manifests in the early teens. The gene responsible for SED tarda, SEDL, has been identified in Xp22. We report on three novel SEDL mutations. The first mutation is in the rare, non-canonical 5' splice site of intron 4 (IVS4+4T>C) in an Italian family. Reverse transcription-polymerase chain reaction (RT-PCR) analysis has revealed that this mutation causes alternative splicing of exon 5, and, as a consequence, inclusion of exon 4b sequence. This gives rise to an altered, truncated SEDL protein. We also describe two new deletions: one is a 4-bp deletion in exon 6 [333-336del(GAAT)], identified in a Slovak patient with SEDT, and one is a 1.335-kb deletion (in5/ex6del), found in a Belgian patient. The identification of these novel mutations in SEDL adds to the spectrum of 30 mutations previously identified. A short summary of all currently known SEDL gene mutations is presented.

Genetic analysis of multicase families of visceral leishmaniasis in northeastern Brazil: no major role for class II or class III regions of HLA.

Familial aggregation, high relative risk to siblings, and segregation analysis, suggest genetic control of visceral leishmaniasis in Brazil. Class II gene effects in mice, and high circulating tumour necrosis factor alpha in humans, provide reasons to target HLA. Fifteen polymorphic markers across 1.03 Mb (DQB1 to TNFa) were genotyped (87 multicase families; 638 individuals). Model-based parametric analyses using single-point combined segregation and linkage in COMDS, or multi-point linkage in ALLEGRO, failed to detect linkage. Model-free nonparametric affected sibling pair (SPLINK) or NPL(all) score (ALLEGRO) analyses also failed to detect linkage. Information content mapping confirmed sufficient marker information to detect linkage. Analysis of simulated data sets demonstrated that these families had 100% power to detect NPL(all) scores of 5 to 6 (>LOD4; P < 0.00001) over the range (7% to 61%) of age-related penetrances for a disease susceptibility gene. The extended transmission disequilibrium test (TDT) showed no consistent allelic associations between disease and the 15 loci. TDT also failed to detect significant associations between extended haplotypes and disease, consistent with failure to detect significant linkage disequilibrium across the region. Linkage disequilibrium between adjacent groups of markers (HLADQ/DR; 82-1/82-3/-238bpTNFA; LTA/62/TNFa) was not accompanied by significant global haplotype TDT associations with disease. The data suggest that class II/III regions of HLA do not contain major disease gene(s) for visceral leishmaniasis in Brazil.

Association and linkage of leprosy phenotypes with HLA class II and tumour necrosis factor genes.

Previous analyses indicate major gene control of susceptibility to leprosy per se and the HLA class II region has been implicated in determining susceptibility and control of clinical phenotype. Segregation analysis using data from 76 Brazilian leprosy multi-case pedigrees (1166 individuals) supported a two locus model as the best fit: a recessive major gene and a recessive modifier gene(s) (single locus vs two locus model, P = 0.0007). Combined segregation and linkage analysis to the major locus, showed strong linkage to HLA class II (HLA-DQB1 P = 0.000002, HLA-DQA1 P = 0.000002, HLA-DRB1 P = 0.0000003) and tumour necrosis factor genes (TNF P = 0.00002, LTA P = 0.003). Extended transmission disequilibrium testing, using multiple affected family members, demonstrated that the common allele TNF*1 of the -308 promoter region polymorphism showed linkage and/or association with disease per se, at a high level of significance (P < 0.0001). Two locus transmission disequilibrium testing suggested susceptibility (TNF*1/LTA*2) and protective (TNF*2/LTA*2) haplotypes in the class iii region. Taken together the segregation and HLA analyses suggest the possibility of more than one susceptibility locus in the MHC.

Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs.

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78-88%) 0-135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93-100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

Tissue cytokine responses in canine visceral leishmaniasis.

To elucidate the local tissue cytokine response of dogs infected with Leishmania chagasi, cytokine mRNA levels were measured in bone marrow aspirates from 27 naturally infected dogs from Brazil and were compared with those from 5 uninfected control animals. Interferon-gamma mRNA accumulation was enhanced in infected dogs and was positively correlated with humoral (IgG1) but not with lymphoproliferative responses to Leishmania antigen in infected dogs. Increased accumulation of mRNA for interleukin (IL)-4, IL-10, and IL-18 was not observed in infected dogs, and mRNA for these cytokines did not correlate with antibody or proliferative responses. However, infected dogs with detectable IL-4 mRNA had significantly more severe symptoms. IL-13 mRNA was not detectable in either control or infected dogs. These data suggest that clinical symptoms are not due to a deficiency in interferon-gamma production. However, in contrast to its role in human visceral leishmaniasis, IL-10 may not play a key immunosuppressive role in dogs.

Evaluation of functional outcomes (speech, swallowing and voice) in patients attending speech pathology after head and neck cancer treatment(s): development of a multi-centre database.

Since April 1997, in Melbourne, Australia, speech pathologists have collaborated to establish a prospective database of functional outcomes of speech, swallowing and voice for patients undergoing head and neck cancer treatments. Staff at eight acute care hospitals, all of which offer speech pathology for head and neck cancer services in Victoria, are contributing data, collated centrally, in an agreed pro forma. Early results are given (after 12 months' data collection). The implications for clinically-based research, and the future potential for benchmarking outcomes--by expansion of the rehabilitation database beyond the current participating sites--is discussed. This paper outlines the rationale of establishing the database is multicentered, and explores some of the complexities involved, including the challenges inherent in long-term accurate data collection in the head and neck cancer patient population. This work represents the development of an appropriate, usable tool for data collection on functional outcomes.

Multiple interacting gene products may influence susceptibility to malignant hyperthermia.

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.

Evidence that genetic susceptibility to Mycobacterium tuberculosis in a Brazilian population is under oligogenic control: linkage study of the candidate genes NRAMP1 and TNFA.

SETTING: A study of multicase tuberculosis pedigrees from Northern Brazil. OBJECTIVE: To determine the model of inheritance for genetic susceptibility to tuberculosis, and to test the hypothesis that TNFA and NRAMP1 are candidate susceptibility genes. DESIGN: The study sample included 98 pedigrees, 704 individuals and 205 nuclear families. Segregation analyses were performed using the programs POINTER and COMDS. Combined segregation and linkage analysis was carried out within COMDS. Non-parametric linkage analyses were performed using BETA. RESULTS: A sporadic model for disease distribution in families was strongly rejected, as were polygenic and multifactorial models. A codominant single gene model provided the best fit (P < 0.001) to the data using POINTER. COMDS extended the analysis to compare single-gene and two-gene models. A general two-locus model for disease control was marginally favoured (0.01 < P < 0.05) over the codominant single-gene model. No evidence was found for linkage between susceptibility to disease per se and the TNF gene cluster. Weak linkage was observed using COMDS for genes (IL8RB, P = 0.039; D2S1471, P = 0.025) tightly linked (< 150 kb) to NRAMP1, but not for NRAMP1 itself. CONCLUSIONS: Tuberculosis susceptibility in this region of Brazil is under oligogenic control. Although a minor role for TNFA and NRAMP1 cannot be excluded, our data suggest that neither is a major gene involved in this oligogenic control.

Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis.

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.