Anja H. Schiemann

Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex.

ABSTRACT The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.

Organization and expression of the Australian sheep blowfly (Lucilia cuprina) hsp23, hsp24, hsp70 and hsp83 genes.

In this study we report the isolation and characterization of a heat shock protein 70 (hsp70) gene, the hsp83 gene and two genes that encode small Hsps (Lchsp23 and Lchsp24) from the Australian sheep blowfly, Lucilia cuprina, a major agricultural pest. Phylogenetic analyses indicate that the LcHsp23 protein is the orthologue of Drosophila melanogaster Hsp23 and LcHsp24 is the orthologue of Sarcophaga crassipalpis Hsp23. Quantitative reverse‐transcriptase PCR analysis showed that the basal level of Lchsp83 RNA is relatively high at all developmental stages and only moderately induced by heat shock. In contrast, Lchsp70 transcripts are present at low levels and strongly induced by heat shock at all stages. The basal levels of expression and degrees of heat induction of the Lchsp23 and Lchsp24 transcripts were more variable across the different developmental stages. Putative heat shock factor binding sites were identified in the Lchsp24, Lchsp70 and Lchsp83 gene promoters. The isolation of these hsp gene promoters will facilitate constitutive or conditional expression of a gene of interest in transgenic Lucilia.

Sequence capture and massively parallel sequencing to detect mutations associated with malignant hyperthermia

BACKGROUND Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder in which intracellular calcium homeostasis in the skeletal muscle of susceptible individuals is disrupted upon exposure to halogenated anaesthetics. While MH is linked to the ryanodine receptor (RYR1) on chromosome 19 and the α1S subunit of the voltage-dependent L-type calcium channel (CACNA1S) on chromosome 1, mutations have been found in only 50-70% of patients, and subsequently, there is a need for a more powerful screening tool. METHODS Genomic DNA capture and next-generation sequencing was used to screen 32 genes involved in excitation-contraction coupling, skeletal muscle calcium homeostasis, or immune response in two MH patients. Lymphoblastoid cell lines were used to functionally characterize candidate RYR1 mutations in one family. RESULTS Sequence analysis revealed two putative causative mutations in RYR1 in one patient. Segregation analysis and functional analysis support a causative role of the detected variants. The amount of Ca(2+) released after stimulation with 4-chloro-m-cresol from B lymphocytes of the MH-susceptible patients in the family was significantly greater compared with that of Ca(2+) released from cells of an MH-negative family member. In the other patient, no causative mutations were identified in the 32 genes screened. CONCLUSIONS In this study, we successfully demonstrate the use of genomic DNA capture and next-generation sequencing for identification of putative mutations causing MH. We also suggest that whole exome sequencing may be necessary to identify MH causing mutations in patients where no mutations in RYR1 and CACNA1S have been identified thus far.

Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility.

Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca(2+) release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca(2+) release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca(2+) release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.

Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages

ABSTRACT Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, PE1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant PE1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the PE1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.

Functional analysis of RYR1 variants linked to malignant hyperthermia

ABSTRACT Malignant hyperthermia manifests as a rapid and sustained rise in temperature in response to pharmacological triggering agents, e.g. inhalational anesthetics and the muscle relaxant suxamethonium. Other clinical signs include an increase in end-tidal CO2, increased O2 consumption, as well as tachycardia, and if untreated a malignant hyperthermia episode can result in death. The metabolic changes are caused by dysregulation of skeletal muscle Ca2+ homeostasis, resulting from a defective ryanodine receptor Ca2+ channel, which resides in the sarcoplasmic reticulum and controls the flux of Ca2+ ions from intracellular stores to the cytoplasm. Most genetic variants associated with susceptibility to malignant hyperthermia occur in the RYR1 gene encoding the ryanodine receptor type 1. While malignant hyperthermia susceptibility can be diagnosed by in vitro contracture testing of skeletal muscle biopsy tissue, it is advantageous to use DNA testing. Currently only 35 of over 400 potential variants in RYR1 have been classed as functionally causative of malignant hyperthermia and thus can be used for DNA diagnostic tests. Here we describe functional analysis of 2 RYR1 variants (c. 7042_7044delCAG, p.ΔGlu2348 and c.641C>T, p.Thr214Met) that occur in the same malignant hyperthermia susceptible family. The p.Glu2348 deletion, causes hypersensitivity to ryanodine receptor agonists using in vitro analysis of cloned human RYR1 cDNA expressed in HEK293T cells, while the Thr214Met substitution, does not appear to significantly alter sensitivity to agonist in the same system. We suggest that the c. 7042_7044delCAG, p.ΔGlu2348 RYR1 variant could be added to the list of diagnostic mutations for susceptibility to malignant hyperthermia.

Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia.

BACKGROUND:Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. More than 300 variants in the ryanodine receptor 1 (RYR1) have been associated with MH; however, only 31 have been identified as causative. To confirm a mutation in RYR1 as being causative for MH, segregation of the potential mutation in at least 2 unrelated families with MH susceptibility must be demonstrated and functional assays must show abnormal calcium release compared with wild-type RYR1. METHODS:We used “Hot-spot” DNA screening to identify mutations in RYR1 in 3 New Zealand families. B-lymphoblastoid cells were used to compare the amount of calcium released on stimulation with 4-chloro-m-cresol between wild-type RYR1 cells and cells carrying the new variants in RYR1. RESULTS:We identified a known RYR1 mutation (R2355W) in 2 families and another more recently identified (V2354M) mutation in another family. Both mutations segregated with MH susceptibility in the respective families. Cell lines carrying a mutation in RYR1 showed increased sensitivity to 4-chloro-m-cresol. CONCLUSIONS:We propose that R2355W is confirmed as being an MH-causative mutation and suggest that V2354M is a RYR1 mutation likely to cause MH.

Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia

BACKGROUND Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. OBJECTIVE We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. METHODS Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. RESULTS The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. CONCLUSIONS The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

Targeted exon capture and NGS to investigate an undefined myopathy reveal RYR1 variants

Background The family under investigation consists of parents and two daughters, one being the proband. The mother and the proband have elongated facial features. The father and second daughter appear normal. The older daughter presented for elective tonsillectomy aged 8 years. She had severe masseter spasm after suxamethonium. The rest of the procedure was carried out under total intravenous anaesthesia. No blood gas analysis could be done, but a creatine kinase next day was significantly elevated (2934). This led to study of both parents. There was no family history of malignant hyperthermia but an undefined myopathy was suspected in mother and daughter. Both mother and father were diagnosed malignant hyperthermia (MH) susceptible by in vitro contracture test (IVCT). This prompted a DNA analysis for variants associated with MH.