BackgroundScrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.MethodologyA clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.Key resultsDevelopment of a standardised growth assay to quantify bacterial replication and viability in vitro.Quantitative comparison of different DNA extraction methods.Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.ConclusionsHere we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.
Background Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. Methods Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon–intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon–intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. Results We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. Conclusions Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases.
Pathogenic variants in glutamate receptor, ionotropic, NMDA‐1 (GRIN1) cause an autosomal dominant or recessive neurodevelopmental disorder with global developmental delay, with or without seizures (AD or AR GRIN1‐NDD). Here, we describe a novel homozygous canonical splice site variant in GRIN1 in a 12‐month‐old boy with early infantile epileptic encephalopathy and severe global developmental delay. This represents only the second family with a homozygous predicted‐null variant in GRIN1 reported to date. We review the published literature on AR GRIN1‐NDD and find that the phenotype in our patient is much more severe than those seen with homozygous missense variants. A similarly severe phenotype of intractable epilepsy and infantile death has only been reported in one other family with a homozygous nonsense variant in GRIN1. We, therefore, propose that biallelic predicted‐null variants in GRIN1 can cause a markedly more severe clinical phenotype than AR GRIN1‐NDD caused by missense variants.
ABL1 is a proto-oncogene encoding a nonreceptor tyrosine kinase, best known in the somatic BCR-ABL fusion gene associated with chronic myeloid leukaemia. Recently, germline missense variants in ABL1 have been found to cause an autosomal dominant developmental syndrome with congenital heart disease, skeletal malformations and characteristic facies. Here, we describe a series of six new unrelated individuals with heterozygous missense variants in ABL1 (including four novel variants) identified via whole exome sequencing. All the affected individuals in this series recapitulate the phenotype of the ABL1 developmental syndrome and additionally we affirm that hearing impairment is a common feature of the condition. Four of the variants cluster in the myristoyl-binding pocket of ABL1, a region critical for auto-inhibitory regulation of the kinase domain. Bio-informatic analysis of transcript-wide conservation and germline/somatic variation reveals that this pocket region is subject to high missense constraint and evolutionary conservation. Functional work to investigate ABL1 kinase activity in vitro by transient transfection of HEK293T cells with variant ABL1 plasmid constructs revealed increased phosphorylation of ABL1-specific substrates compared to wild-type. The increased tyrosine kinase activity was suppressed by imatinib treatment. This case series of six new patients with germline heterozygous ABL1 missense variants further delineates the phenotypic spectrum of this condition and recognises microcephaly as a common finding. Our analysis supports an ABL1 gain-of-function mechanism due to loss of auto-inhibition, and demonstrates the potential for pharmacological inhibition using imatinib.
Genomic variants which disrupt splicing are a major cause of rare genetic disease. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project, and assess the contribution of non-canonical splicing variants to rare genetic diseases. We show that splicing branchpoints are highly constrained by purifying selection, and harbour damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. We use phenotype matching and RNA studies to confirm a new diagnosis for six individuals to date. In summary, we demonstrate the clinical value of examining non-canonical splicing variants in participants with unsolved rare diseases.
Purpose: Identifying pathogenic non-coding variants in individuals with developmental disorders (DD) is challenging due to the large search space. It is common to find a single protein-altering variant in a recessive gene in DD patients, but the prevalence of pathogenic non-coding second hits in trans with these is unknown. Methods: In 4,073 genetically undiagnosed rare disease trio probands from the 100,000 Genomes project, we identified rare heterozygous loss-of-function (LoF) or ClinVar pathogenic variants in recessive DD-associated genes. Using stringent region-specific filtering, we identified rare non-coding variants on the other haplotype. Identified genes were clinically evaluated for phenotypic fit, and where possible, we performed functional testing using RNA-sequencing. Results: We found 2,430 probands with one or more rare heterozygous pLoF or ClinVar pathogenic variants in recessive DD-associated genes, for a total of 3,761 proband-variant pairs. For 1,366 (36.3%) of these pairs, we identified at least one rare non-coding variant in trans. After stringent bioinformatic filtering and clinical review, five were determined to be a good clinical fit (in ALMS1, NPHP3, LAMA2, IGHMBP2 and GAA). Conclusion: We developed a pipeline to systematically identify and annotate compound heterozygous coding/non-coding genotypes. Using this approach we uncovered new diagnoses and conclude that this mechanism is a rare cause of DDs.
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