David Iles

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences.

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

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.

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.

A RYR1 mutation associated with recessive congenital myopathy and dominant malignant hyperthermia in Asian families

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. Muscle Nerve, 2009

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.

Key gene regulatory sequences with distinctive ontological signatures associate with differentially endonuclease-accessible mouse sperm chromatin

Using a well-established endonuclease-based chromatin dissection procedure in conjunction with both experimental comparative genome hybridisation (CGH) array profiling and in silico data mining, we show that mouse spermatozoa contain chromatin that is sensitive and resistant to digestion with micrococcal nuclease (MNase). Sequences represented in the micrococcal nuclease digestion solubilised (MNDS) but not the MND insoluble (MNDI) chromatin are strongly enriched in chromosomal regions of high gene density. Furthermore, by fluorescence in situ hybridisation (FISH) analysis, we show that MNDS and MNDI DNAs occupy distinct domains of decondensed mouse sperm nuclei that may also retain abundant histones. More detailed in silico analysis of CGH probe location in relation to known promoters and sequences recognised by CCCTC binding factor (CTCF) shows a significant excess of both in MNDS chromatin. A functional analysis of gene promoters reveals strong ontological signatures for ion transport on methylated promoters associated with CTCF binding sequences in MNDS chromatin. Sensory perception is the only strong ontological signature present in MNDI chromatin, driven by promoters that are not associated with CTCF regardless of their methylation status.

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.

The Testis as a Conduit for Genomic Plasticity: an advanced interdisciplinary workshop

The premise for this unusual amalgamation of reproductive biologists, molecular geneticists and evolutionary biologists rested on the evidence-based assumption that reproductive tissues could be ideal environments for the expression and transmission of transposable elements that can move into new locations in the genome. These elements include DNA transposons and retrotransposons that, together, make up over 40% of the human genome. The testis may be a particularly good niche for their expression because of the unique dynamic of spermatogenesis, where the methylation-demethylation status of germ cell DNA is at its most plastic. Hence windows of opportunity can arise that may release transposable elements from the tight regulatory control of expression imposed on them by bulk DNA methylation. As the testis is where most mutations become embedded in the germline, the meeting included a number of keynote presentations that aimed to examine the potential for transposable elements to heritably alter the genome and effect variation independently of the usual Mendelian mechanisms. In essence, could the testis be one of the favoured sites where genomic plasticity makes its mark?

Nucleosomal chromatin in the mature sperm of Drosophila melanogaster

During spermiogenesis in mammals and many other vertebrate classes, histone-containing nucleosomes are replaced by protamine toroids, which can repackage chromatin at a 10 to 20-fold higher density than in a typical somatic nucleus. However, recent evidence suggests that sperm of many species, including human and mouse retain a small compartment of nucleosomal chromatin, particularly near genes important for embryogenesis. As in mammals, spermiogenesis in the fruit fly, Drosophila melanogaster has also been shown to undergo a programmed substitution of nucleosomes with protamine-like proteins. Using chromatin immunoprecipitation (ChIP) and whole-genome tiling array hybridization (ChIP-chip), supported by immunocytochemical evidence, we show that in a manner analogous to nucleosomal chromatin retention in mammalian spermatozoa, distinct domains packaged by the canonical histones H2A, H2B, H3 and H4 are present in the fly sperm nucleus. We also find evidence for the retention of nucleosomes with specific histone H3 trimethylation marks characteristic of chromatin repression (H3K9me3, H3K27me3) and active transcription (H3K36me3). Raw and processed data from the experiments are available at GEO, accession GSE52165.

In search of the acute oxygen sensor: Functional proteomics and acute regulation of large-conductance, calcium-activated potassium channels by hemeoxygenase-2

Detecting and reacting to acute perturbation in the partial pressure of atmospheric oxygen (pO2), particularly hypoxia, is a fundamental adaptive mechanism which is conserved throughout the animal kingdom. In mammals, a number of cellular systems respond, often co-operatively as oxygen availability becomes compromised, with the express aim of maximising oxygen uptake by the lungs and of optimising its delivery to the metabolically most active tissues. Thus, during hypoxia, ventilation rate and depth are increased to maximize air flow across the gaseous exchange surface, local lung perfusion rates become rapidly matched to local alveolar ventilation and systemic arteriolar dilatation ensures that tissue and cerebral blood flow become swiftly optimized.