Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethiON platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans

Cellular interference in craniofrontonasal syndrome: Males mosaic for mutations in the x-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundariesa process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5 untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69. The 5 UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females. © The Author 2013. Published by Oxford University Press. All rights reserved

Lineage replacement and evolution captured by 3 years of the United Kingdom Coronavirus (COVID-19) Infection Survey

The Office for National Statistics Coronavirus (COVID-19) Infection Survey (ONS-CIS) is the largest surveillance study of SARS-CoV-2 positivity in the community, and collected data on the United Kingdom (UK) epidemic from April 2020 until March 2023 before being paused. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing the sequenced samples collected by the ONS-CIS during this period. We observed a series of sweeps or partial sweeps, with each sweeping lineage having a distinct growth advantage compared to their predecessors, although this was also accompanied by a gradual fall in average viral burdens from June 2021 to March 2023. The sweeps also generated an alternating pattern in which most samples had either S-gene target failure (SGTF) or non-SGTF over time. Evolution was characterized by steadily increasing divergence and diversity within lineages, but with step increases in divergence associated with each sweeping major lineage. This led to a faster overall rate of evolution when measured at the between-lineage level compared to within lineages, and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens

A blood atlas of COVID-19 defines hallmarks of disease severity and specificity.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism, and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design, and personalized medicine approaches for COVID-19

Molecular characterisation of hereditary multiple exostoses

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Hailey-Hailey disease: identification of novel mutations in ATP2C1 and missense mutation A528P on protein expression levels

ATP2C1, encoding the human secretory pathway Ca2+-ATPase (hSPCA1), was recently identified as the defective gene in Hailey–Hailey disease (HHD), an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion in the suprabasal layers of the epidermis. In this study, we used denaturing high-performance liquid chromatography to screen all 28 exons and flanking intron boundaries of ATP2C1 for mutations in 9 HHD patients. Nine different mutations were identified. Five of these mutations, including one nonsense, one deletion, two splice-site, and one missense mutation, have not been previously reported. Recently, functional analysis of a series of site-specific mutants, designed to mimic missense mutations found in ATP2C1, uncovered specific defects in Ca2+ and/or Mn2+ transport and protein expression in mutant hSPCA1 polypeptides. In order to investigate the molecular and physiological basis of HHD in the patient carrying missense mutation A528P, located in the putative nucleotide binding domain of the molecule, site-directed mutagenesis was employed to introduce this mutation into the wild-type ATP2C1 (hSPCA1) sequence. Functional analyses of HHD-mutant A528P demonstrated a low level of protein expression, despite normal levels of mRNA and correct targeting to the Golgi, suggesting instability or abnormal folding of the mutated hSPCA1 polypeptides. Analogous to conclusions drawn from our previous studies, these results further support the theory of haploinsufficiency as a prevalent mechanism for the dominant inheritance of HHD, by suggesting that the level of hSPCA1 in epidermal cells is critical

Correction: Viral burden is associated with age, vaccination, and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection-related sampling bias.

[This corrects the article DOI: 10.1371/journal.ppat.1011461.]