Prevalence of persistent SARS-CoV-2 in a large community surveillance study.

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.

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.

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burden, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. By analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior exposure, viral burden was 44% lower among Alpha variant infections, compared to those with the predecessor strain, B.1.177. Vaccination reduced viral burden by 67%, and among vaccinated individuals, viral burden was 286% higher among Delta variant, compared to Alpha variant, infections. In addition, viral burden increased by 17% for every 10-year age increment of the infected individual. In summary, within-host viral burden increases with age, is reduced by vaccination, and is influenced by the interplay of vaccination status and viral variant.

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.].

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.

Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages.

Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.

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 boundaries-a 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.

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.

Determination of the mutation spectrum of the EXT1/EXT2 genes in British Caucasian patients with multiple osteochondromas, and exclusion of six candidate genes in EXT negative cases.

We describe here the spectrum and distribution of mutations in the EXT1 and EXT2 genes in the largest reported British Caucasian multiple osteochondromas (MO) population. Furthermore, we report for the first time the screening of the EXT1 and EXT2 promoters, 5'UTRs, and 3'UTRs, and exclude six potential MO candidate genes in individuals without a detectable mutation within the coding region of EXT1 and EXT2. The coding exons of EXT1 and EXT2 were screened in 72 unrelated probands affected with MO. Forty-six different mutations were identified in 56 probands, of which 29 were novel. Mutation in the EXT1 and EXT2 genes each accounted for 50% of the mutations identified. Of the 72 probands, 42 were of British Caucasian descent, which when added to the 41 British Caucasian families previously reported from our total cohort, gave a total of 83 families. This cohort's proportional frequency for EXT1/EXT2 mutation was 53%/47%. We also validated the technique of high-resolution melting analysis in a blind study using 27 unique EXT1 or EXT2 mutations. This technique was found to be sensitive with a detection rate of 100% regarding heterozygote detection for EXT mutation scanning. Furthermore, this technique has a very high throughput and is very cost-effective.

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

ATP2C1, encoding the human secretory pathway Ca(2+)-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 Ca(2+) and/or Mn(2+) 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.

Netherton syndrome: disease expression and spectrum of SPINK5 mutations in 21 families.

Netherton syndrome is a severe autosomal recessive skin disorder characterized by congenital erythroderma, a specific hair-shaft abnormality, and atopic manifestations with high IgE levels. Recently, we identified SPINK5, which encodes the serine protease inhibitor Kazal-type 5 protein (LEKTI), as the defective gene in Netherton syndrome. Here we describe the intron-exon organization of the gene and characterize the SPINK5 mutations in patients from 21 families of different geographic origin, using denaturing high performance liquid chromatography and direct sequencing. We identified 18 mutations, of which 13 were novel and seven (39%) were recurrent. The majority of the mutations were clustered between exons 1-8 and exons 21-26. They comprised four nonsense mutations (22%), eight frameshift insertions or deletions (44%), and six splice-site defects (33%). All mutations predict the formation of premature termination codons. Northern blot analysis showed variable reduction of SPINK5 mutant transcript levels, suggesting variable efficiency of nonsense-mediated mRNA decay. Seven patients were homozygotes, eight were compound heterozygotes, and five were heterozygotes with only one identifiable SPINK5 mutation. Five mutations, one of which resulted in perinatal lethal disease in three families, were associated with certain ethnic groups. We also describe 45 intragenic polymorphisms in the patients studied. The clinical features of erythroderma, trichorrhexis invaginata, and atopic manifestations were present in the majority of affected individuals and ichthyosis linearis circumflexa was seen in 12 out of 24 patients. Interfamilial and intrafamilial variation in disease severity was observed, with no clear correlation between mutations and phenotype, suggesting that the degree of severity may be affected by other factors.

Genetic variation in COL17A1 and the development of bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering disease of the skin characterized by autoantibody attack on collagen XVII. OBJECTIVES: To characterize the genetic complexity of COL17A1, the gene which encodes for the autoantigen collagen XVII. The data will be used to determine whether there is an association between polymorphisms and haplotypes of COL17A1 and genetic susceptibility to development of BP. METHODS: The genetic complexity in COL17A1 was deduced by screening and then sequencing the gene. Haplotypes were constructed from the resulting polymorphisms using the statistical programme PHASE. The linkage disequilibrium (D') between the polymorphisms was deduced from haplotypic data using the statistical programme GOLD. Association of the polymorphisms and haplotypes was tested for, in a cohort of BP patients and controls. RESULTS: Screening of COL17A1 for genetic variation was carried out in 29 individuals of North European caucasoid origin, and it revealed 19 single-nucleotide polymorphisms in approximately 14.7 kb of sequence. These variants resulted in 60 different haplotypes in 191 individuals, of which 13 occurred above 1% in the population. D' between the variants was found to be extensive, have a low correlation with physical distance and to extend over 33.8 kb. No association was found with any of the polymorphisms or haplotypes and development of BP, when tested for, in a cohort of patients and controls. CONCLUSION: This study provides an extensive description of the genetic variation in COL17A1 and shows no association of the genetic variants with susceptibility to BP.

ProtocadherinX/Y, a candidate gene-pair for schizophrenia and schizoaffective disorder: a DHPLC investigation of genomic sequence.

Protocadherin X and Protocadherin Y (PCDHX and PCDHY) are cell-surface adhesion molecules expressed predominantly in the brain. The PCDHX/Y gene-pair was generated by an X-Y translocation approximately 3 million years ago (MYA) that gave rise to the Homo sapiens-specific region of Xq21.3 and Yp11.2 homology. Genes within this region are expected to code for sexually dimorphic human characteristics, including, for example, cerebral asymmetry a dimension of variation that has been suggested is relevant to psychosis. We examined differences in patients with schizophrenic or schizoaffective psychosis in the genomic sequence of PCDHX and PCDHY in coding and adjacent intronic sequences using denaturing high performance liquid chromatography (DHPLC). Three coding variants were detected in PCDHX and two in PCDHY. However, neither the coding variants nor the intronic polymorphisms could be related to psychosis within families. Low sequence variation suggests selective pressure against sequence change in modern humans in contrast to the structural chromosomal and sequence changes including fixed X-Y differences that occurred in this region earlier in hominid evolution. Our findings exclude sequence variation in PCDHX/Y as relevant to the aetiology of psychosis. However, we note the unusual status of this region with respect to X-inactivation. Further investigation of the epigenetic control of PCDHX/Y in relation to psychosis is warranted.