CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury

Background: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. Methods: Non-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry. Results: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed “leukocyte aggregates”. We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, “off-target” or unpredicted effects in targeting CD147. Conclusion: CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury

Comprehensive genetic diagnosis of tandem repeat expansion disorders with programmable targeted nanopore sequencing

More than 50 neurological and neuromuscular diseases are caused by short tandem repeat (STR) expansions, with 37 different genes implicated to date. We describe the use of programmable targeted long-read sequencing with Oxford Nanopore's ReadUntil function for parallel genotyping of all known neuropathogenic STRs in a single assay. Our approach enables accurate, haplotype-resolved assembly and DNA methylation profiling of STR sites, from a list of predetermined candidates. This correctly diagnoses all individuals in a small cohort (n = 37) including patients with various neurogenetic diseases (n = 25). Targeted long-read sequencing solves large and complex STR expansions that confound established molecular tests and short-read sequencing and identifies noncanonical STR motif conformations and internal sequence interruptions. We observe a diversity of STR alleles of known and unknown pathogenicity, suggesting that long-read sequencing will redefine the genetic landscape of repeat disorders. Last, we show how the inclusion of pharmacogenomic genes as secondary ReadUntil targets can further inform patient care

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis

Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families

Long read sequencing overcomes challenges in the diagnosis of SORD neuropathy

Biallelic mutations in sorbitol dehydrogenase (SORD) have been recently identified as a common cause of recessive axonal Charcot-Marie-Tooth neuropathy (CMT2). We aimed to assess a novel long-read sequencing approach to overcome current limitations in SORD neuropathy diagnostics due to the SORD2P pseudogene and the phasing of biallelic mutations in recessive disease. We conducted a screen of our Australian whole exome sequencing (WES) CMT cohort to identify individuals with homozygous or compound heterozygous SORD variants. Individuals detected with SORD mutations then underwent long-read sequencing, clinical assessment, and serum sorbitol analysis. An individual was detected with compound heterozygous truncating mutations in SORD exon 7, NM_003104.5:c.625C>T (p.Arg209Ter) and NM_003104.5:c.757del (p.Ala253GlnfsTer27). Subsequent Oxford Nanopore Tech (ONT) long-read sequencing was used to successfully differentiate SORD from the highly homologous non-functional SORD2P pseudogene and confirmed that the mutations were biallelic through haplotype-resolved analysis. The patient presented with axonal sensorimotor polyneuropathy (CMT2) and ulnar neuropathy without compression at the elbow. Burning neuropathic pain in the forearms and feet was also reported and was exacerbated by alcohol consumption and improved with alcohol cessation. UPLC-tandem mass spectrometry confirmed that the patient had elevated serum sorbitol levels (12.0 mg/L) consistent with levels previously observed in patients with biallelic SORD mutations. This represents a novel clinical presentation and expands the phenotype associated with biallelic SORD mutations causing CMT2. Our study is the first report of long-read sequencing for an individual with CMT and demonstrates the utility of this approach for clinical genomics

Epidemiological and Genomic analysis of a Sydney Hospital COVID-19 Outbreak

Abstract Australia’s early COVID-19 experience involved clusters in northern Sydney, including hospital and aged-care facility (ACF) outbreaks. We explore transmission dynamics, drivers and outcomes of a metropolitan hospital COVID-19 outbreak that occurred in the context of established local community transmission. A retrospective cohort analysis is presented, with integration of viral genome sequencing, clinical and epidemiological data. We demonstrate using genomic epidemiology that the hospital outbreak (n=23) was linked to a concurrent outbreak at a local aged care facility, but was phylogenetically distinct from other community clusters. Thirty day survival was 50% for hospitalised patients (an elderly cohort with significant comorbidities) and 100% for staff. Staff who acquired infection were unable to attend work for a median of 26.5 days (range 14-191); an additional 140 staff were furloughed for quarantine. Transmission from index cases showed a wide dispersion (mean 3.5 persons infected for every patient case and 0.6 persons infected for every staff case). One patient, who received regular nebulised medication prior to their diagnosis being known, acted as an apparent superspreader. No secondary transmissions occurred from isolated cases or contacts who were quarantined prior to becoming infectious. This analysis elaborates the wide-ranging impacts on patients and staff of nosocomial COVID-19 transmission and highlights the utility of genomic analysis as an adjunct to traditional epidemiological investigations. Delayed case recognition resulted in nosocomial transmission but once recognised, prompt action by the outbreak management team and isolation with contact and droplet (without airborne) precautions were sufficient to prevent transmission within this cohort. Our findings support current PPE recommendations in Australia but demonstrate the risk of administering nebulised medications when COVID-19 is circulating locally

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

ABSTRACT Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, we performed viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. Despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >98% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives

Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with C

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families