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.

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.

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.

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.

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.

Heritability of human hookworm infection in Papua New Guinea.

Hookworms infect approximately 740 million humans worldwide and are an important cause of morbidity. The present study examines the role of additive genetic effects in determining the intensity of hookworm infection in humans, and whether these effects vary according to the sex of the host. Parasitological and epidemiological data for a population of 704 subjects in Papua New Guinea were used in variance components analysis. The 'narrow-sense' heritability of hookworm infection was estimated as 0.15+/-0.04 (P<0.001), and remained significant when controlling for shared environmental (household) effects. Allowing the variance components to vary between the sexes of the human host consistently revealed larger additive genetic effects in females than in males, reflected by heritabilities of 0.18 in females and 0.08 in males in a conservative model. Household effects were also higher in females than males, although the overall household effect was not significant. The results indicate that additive genetic effects are an important determinant of the intensity of human hookworm infection in this population. However, despite similar mean and variance of intensity in each sex, the factors responsible for generating variation in intensity differ markedly between males and females.

Association between SLC11A1 polymorphisms and susceptibility to different clinical forms of tuberculosis in the Peruvian population.

Polymorphism in SLC11A1 has been implicated in host susceptibility to tuberculosis. We have studied associations between INT4, D543N, and 3'UTR polymorphisms of SLC11A1 and different clinical forms of TB. Analysis used 507 patients with pulmonary TB, 123 with extra pulmonary TB and 513 controls. INT4 and D543N showed allelic association with pulmonary TB (P=0.02 and 0.03 respectively). INT4-D543N-3'UTR haplotypes showed an association with pulmonary TB (P=0.03). No association of SLC11A1 with miliary TB was observed, and a possible association of D543N to the pleural form (P=0.08) was suggested. These results support association between SLC11A1 and TB, particularly to the common pulmonary form.

Transforming growth factor-beta is the major mediator of natural suppressor cells derived from normal bone marrow.

We previously reported that murine bone marrow cells activated by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) had potent nonspecific natural suppressor (NS) cell activity. In the present study, we demonstrated that these activated NS cells released a soluble factor (or factors) capable of nonspecifically inhibiting T cell mitogenic responses. Consistent with the properties of transforming growth factor-beta (TGF-beta), treatment of the NS supernates with heat failed to denature the factor, and in fact significantly increased its suppressive activity. The NS suppressor factor strongly inhibited proliferation of the TGF-beta-sensitive tumor cell line, A549. Cytokine activation of suppressive activity correlated with the production of a 10- to 13-kDa protein, consistent with the size of TGF-beta and rIL-3 induced a sevenfold increase in TGF-beta transcription. Finally, neutralizing anti-TGF-beta antibody inhibited the suppressive activity of the supernates, indicating that TGF-beta was responsible for most, if not all, of the suppression expressed by these bone marrow NS cells.

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.

The circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man.

The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH.

Plasma transport of the 20,000-dalton variant of human growth hormone (20K): evidence for a 20K-specific binding site.

The 20,000-dalton variant of human GH (20K) is known to circulate in blood, but few details are known about its plasma transport. A substantial portion of 20K appears to be protein bound despite its low affinity for the receptor-related GH-binding protein (BP). We, therefore, investigated the binding pattern of 20K in human plasma. Radioiodinated 20K was incubated with plasma or plasma fractions, and the mixture was fractionated by gel filtration. Saturation/Scatchard analysis was performed with both 20K and 22,000-dalton GH (22K). The majority (approximately 80%) of protein-bound 20K was complexed with a low affinity BP (Ka = approximately 2 x 10(5) M-1), with the remainder complexed with the high affinity2 receptor-related BP. The binding of 20K to the low affinity BP was specific for 20K; it may involve a 20K-specific binding site on the previously described low affinity plasma BP (peak I) or a separate 20K-specific BP. 22K did not interact with this binding site/BP. Analysis of the 20K-BP complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing suggested, but did not prove, the existence of a separate BP for 20K. We conclude that 20K in plasma binds primarily to a low affinity, 20K-specific binding site (possibly a new, third GH-BP), with only a small fraction bound to the GH receptor-related BP. This binding pattern is markedly different from that of 22K, which is predominantly bound to the receptor-related BP. The existence of a 20K-specific binding site/BP suggests an as yet unrecognized role for this GH variant.

A second, lower affinity growth hormone-binding protein in human plasma.

In our previous description of the circulating GH-binding protein (GH-BP) we observed, in addition to the main GH-BP complex, a second macromolecular component designated peak I during gel filtration of [125I]GH-plasma mixtures. This component was not further characterized because of its small magnitude, seeming nonsaturability, and suspected artifactual nature. We have now characterized peak I as the complex of a low affinity BP with GH. To gain a better knowledge of the nature of peak I, whole plasma or plasma fractions containing isolated peak I-BP (ammonium sulfate precipitated or prepared by gel filtration of plasma) were incubated with monomeric [125I]GH and varying concentrations of unlabeled GH. The mixtures were then analyzed by Sephadex G-100 chromatography to separate free from protein-bound GH. Peak I-associated radioactivity was saturable at high concentrations of human GH, but not with animal GHs. Saturation/Scatchard analysis yielded an association constant of 10(5) M-1 and a maximum binding capacity of 15 mg/L plasma. Chemically cross-linked complexes of [125I]GH with isolated peak I BP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. These experiments yielded a mol wt of 124 kD and a pI of 7 for the cross-linked complex. This is in contradistinction to the previously reported high affinity GH-BP complex, which when cross-linked has a mol wt of 76 kD and a pI of 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal plasma growth hormone levels in man: new evidence for rhythmicity of growth hormone secretion.

Circulating GH levels in man fluctuate widely due to pulsatile GH secretion by the pituitary gland. During much of the time, plasma GH is undetectable by current assays. This is punctuated by occasional secretory episodes, resulting in plasma GH peaks of varying height. The principal diurnal secretory event for GH is that associated with early slow wave sleep, but little is known about the prevailing level and dynamics of GH during the day. We used a new ultrasensitive immunoradiometric assay for GH (Boots-Celltech IRMA; limit of detection, 20 ng/L) to measure plasma GH in the previously undetectable range and to assess its diurnal pattern. Plasma GH was measured every 20 min over a 24-h period in 12 normal subjects (6 men and 6 women, aged 20-47 yr) under physiological conditions. Time series analysis of plasma GH patterns was performed by the Cluster algorithm, autocorrelation, and spectral analysis. Plasma GH, as measured by IRMA, was detectable at all time points and ranged from 40-19,695 ng/L. Dynamic fluctuations occurred within and above the previously undetectable range, with amplitudes varying over 3 orders of magnitude. Women had significantly higher overall GH levels, higher peak amplitudes, and higher valley levels/nadirs than men. GH pulses occurred with an average frequency of about 13/day in both sexes, with a dominant, but not strictly periodic, 2-h rhythmicity. We conclude that in man pulsatile GH secretion occurs throughout the day, and that it is oscillatory rather than episodic. This neurosecretory pattern has eluded recognition heretofore because of the lack of assay sensitivity. Women of reproductive age have higher pulse amplitudes and a higher baseline but equal pulse frequency compared to men. Previous estimates of integrated GH concentrations and GH production rates were too high by a factor of 2 due to overestimation of GH levels in the undetectable range.

Absence of the plasma growth hormone-binding protein in Laron-type dwarfism.

We recently described a specific, high affinity binding protein (BP) for growth hormone (GH) in normal human plasma. Little is known about the source, regulation and biological role of this BP. Because its specificity is similar to that of the GH receptor, we considered the possibility that it represented a circulating receptor subunit or fragment. Laron-type dwarfism is a rare disorder characterized by severe growth failure, resistance to GH, and GH receptor deficiency. To probe the relationship between the receptor and the circulating binding protein, we measured the binding of GH to plasma from a child with Laron-type dwarfism and compared it with that in plasma of normal children and adults. Normal plasma samples were supplemented with unlabeled GH to the endogenous GH level in the plasma of the Laron patient to yield comparable saturation of the BP. After incubation of plasma with radiolabeled GH, bound GH was separated from free GH by gel filtration on Sephadex G-100. There was no detectable binding (less than 1.5%) of GH in the plasma of the Laron-type dwarf, whereas in normal children the bound GH fraction averaged 24.0 +/- 6.1% (mean +/- SD). Thus, the GH-BP is either absent or functionally abnormal in Laron-type dwarfism. This finding suggests that the circulating GH-BP is related to and/or may be derived from the GH receptor. Alternatively, it is possible that the BP plays an as yet poorly understood pivotal role in the promotion of somatic growth.

Effects of growth hormone-binding proteins on serum growth hormone measurements.

GH-binding proteins (GH-BPs) in human blood theoretically may interfere with measurements of immunoreactive GH by forming complexes with GH and competing with antibody reagents for ligand. Indeed, results of serum GH obtained by immunoassays are known to differ markedly depending on the assay employed. To assess the potential role of circulating GH-BPs in this phenomenon, we systematically examined their effect on GH measurement in four RIAs and two immunoradiometric assays. In all except one RIA, the effect of the BPs on tracer binding to antibody was mildly inhibitory. In both immunoradiometric assays, BPs increased the nonspecific association of the tracer with the solid phase reagent. However, these effects were minor at the BP concentrations realistically encountered in practice. Furthermore, the impact of BPs on GH standard curves and final results was negligible because BP effects fall into the category of nonspecific or zero dose counts, which are subtracted during data reduction. We conclude that GH-BPs are only a minor disturbance in GH immunoassays, which is completely compensated for by conventional assay design. Disparities among results yielded by different assays are probably not due to BP interference.

Diurnal pattern of plasma growth hormone-binding protein in man.

The identification of specific GH-binding proteins (GH-BP) in human plasma, one of which is a fragment of the GH receptor, has added new complexity to the state of GH in the circulation. A major proportion of GH circulates in complexed form, which differs in kinetics and possibly bioactivity from free GH. Little is known about the regulation of the GH-BP, their constancy or variation in plasma, or plasma factors affecting GH binding. Consequently, the temporal pattern of bound and free GH in plasma is also unknown. Knowledge about possible spontaneous variability in GH-BP levels/activity is required for physiological investigations and comparative studies among different populations. To address these issues, we measured GH-binding activity in plasma every hour over a 24-h period in six normal adults (three men and three women). A standardized GH binding assay, employing incubation of plasma with [125I]GH and separation of bound from free GH by gel filtration, was used. GH-BP activity showed no significant diurnal variation in any subject. The average GH-BP activity was similar in all subjects, although statistically significant differences were found between some subjects. No age- or sex-related differences were identified. We conclude that in normal man plasma GH-BP activity is constant throughout the day, thereby implying 1) constancy of binding protein (and possibly GH receptor) concentration, and 2) absence of significant fluctuations in potential binding inhibitors/enhancers in plasma.

Plasma transport of human growth hormone in vivo.

We have previously shown that a substantial part of human GH is complexed with GH-binding proteins (BPs) when GH is incubated with plasma in vitro. The proportion of GH bound in vivo, however, is unknown and may differ because of factors that cannot be assessed in vitro, such as binding to tissue receptors, distribution of GH outside the vascular compartment, and fluctuating GH and possibly BP levels. Accordingly, we studied the plasma transport characteristics of GH in vivo in six normal men. Monomeric, natural sequence human GH (Humatrope, Eli Lilly Co.) was injected iv in a dose designed to yield physiological plasma levels. Endogenous GH was suppressed before injection with oral glucose administration. Fifteen minutes after injection, plasma was obtained and immediately analyzed by zonal and frontal analysis in gel chromatography, followed by GH measurement in the fractions by RIA. The results obtained were very similar to those derived from in vitro studies, regardless of which analytical method was used. Frontal analysis at 37 C, which most directly reflects the true bound fraction, showed that 38.8 +/- 4.7% (mean +/- SD) of GH was bound to BPs at plasma GH levels ranging from 32-59 micrograms/L, indistinguishable from in vitro results. [When allowance was made for partial BP saturation, the fraction bound at low GH levels (greater than 7 micrograms/L) was calculated as 45.5 +/- 7.5%.] There was evidence for binding to both high and low affinity BPs in the expected proportions. In contrast to complex formation between GH and BPs, no evidence was obtained for conversion of the monomeric GH to oligomeric forms. We conclude that in vitro predictions about binding of GH to BPs in human plasma are representative of in vivo conditions. Shortly after a GH pulse, almost half of plasma GH circulates in complexed form, primarily bound to the high affinity (receptor-related) BP. Aggregation of GH in plasma does not occur (at least within a 15-min period), suggesting that the pituitary is the predominant, if not sole, source of circulating GH oligomers.