Minor Mutations Research Articles

Leber hereditary optic neuropathy “plus” with the m.14487 T>C mutation as the causality of hemidystonia: A Case Report

Introduction: LHON complicated with extraocular symptoms is called LHON plus. We describe a case of Leber hereditary optic neuropathy (LHON) plus with a rare mutation, that also caused dystonia. Case Presentation: An 18-year-old male patient developed symptoms of dystonia at the age of 15 years. Two years later, he noticed decreased visual acuity and central scotoma in the left eye. One month later the same symptoms occurred in the right eye. Although the optic discs in both eyes revealed mildly redness and edematous change, no abnormal findings were detected on fluorescence fundus angiography and orbital magnetic resonance imaging. Mitochondrial deoxyribonucleic acid (mtDNA) sequencing detected the m.14487 T>C mutation. From clinical course and fundus findings, the case was diagnosed LHON. The optic nerve gradually atrophied and central scotoma remained. Conclusion: The m.14487 T>C mutation is one of the causative mutations in patients with dystonia or Leigh encephalopathy and a minor mutation in patients with LHON. However, in the present case, ocular symptoms were more severe than systematic symptoms and the disease course was consistent with LHON. For the above reasons, this case can be diagnosed as LHON plus. Whole mtDNA sequencing is important in diagnosing LHON if none of the three major mutations are detected.

An extensive in silico analysis of missense mutations of the human AIMP2 gene

HLD17 (Hypomyelinating Leukodystrophy 17) is an inherited white matter disorder characterized by insufficient myelin production due to biallelic loss of function mutations in the aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) gene. In silico analysis of SNVs (single nucleotide variants) in the AIMP2 gene is an efficient and cost-effective method for analyzing and predicting the impact of mutations on protein function and disease pathophysiology.The study used dbSNP and Ensembl databases to obtain data on 343 nonsynonymous single nucleotide variants (nsSNVs) in the human AIMP2 gene. Six prediction algorithm tools were used to assess the effects of these nsSNVs on AIMP2's functions and structures. Results showed that 18 nsSNVs were located within functional domains, while 10 nsSNVs led to decreased protein stability. The structural and functional properties of the AIMP2 protein were investigated using databases such as Predict Protein, Mutpred2, and HOPE. ConSurf analysis provided information about conserved nsSNVs. GeneMANIA and STRING software tools were used to predict interactions between gene-gene and protein-protein, respectively. Phyre2 and I-TASSER web servers were used to predict the 3D structures of wild-type and mutant proteins.In addition, having the challenge of probable post-translational modification sites in the AIMP2, we made predictions using various bioinformatics tools. Consecuently, three minor mutations (L138Q, V161E, and I188N) and five major mutations (C23S, D121G, I122S, P128S, and W268S) were found to affect the AIMP2 protein's structure or function. Anyway, These mutations need to be further studied and confirmed through experimental investigation and GWAS studies.

Metabolic Labeling and Digital Microfluidic Single-Cell Sequencing for Single Bacterial Genotypic-Phenotypic Analysis.

Accurate assessment of phenotypic and genotypic characteristics of bacteria can facilitate comprehensive cataloguing of all the resistance factors for better understanding of antibiotic resistance. However, current methods primarily focus on individual phenotypic or genotypic profiles across different colonies. Here, a Digital microfluidic-based automated assay for whole-genome sequencing of single-antibiotic-resistant bacteria is reported, enabling Genotypic and Phenotypic Analysis of antibiotic-resistant strains (Digital-GPA). Digital-GPA can efficiently isolate and sequence antibiotic-resistant bacteria illuminated by fluorescent D-amino acid (FDAA)-labeling, producing high-quality single-cell amplified genomes (SAGs). This enables identifications of both minor and major mutations, pinpointing substrains with distinctive resistance mechanisms. Digital-GPA can directly process clinical samples to detect and sequence resistant pathogens without bacterial culture, subsequently provide genetic profiles of antibiotic susceptibility, promising to expedite the analysis of hard-to-culture or slow-growing bacteria. Overall, Digital-GPA opens a new avenue for antibiotic resistance analysis by providing accurate and comprehensive molecular profiles of antibiotic resistance at single-cell resolution.

Potential emergence of terbinafine resistance by squalene epoxidase gene mutations: An 18-month cohort study of onychomycosis patients in the United States.

There is a concerning rise in antifungal-resistant dermatophytosis globally, with resistance to terbinafine conferred by point mutations in the squalene epoxidase (SQLE) gene. Report changes in the prevalence and profile of SQLE mutations in onychomycosis patients in the United States. A longitudinal cohort study of toenail samples was collected from suspected onychomycosis patients over an 18-month period from 2022 to 2023. Samples were submitted from across the United States and subjected to multiplex real-time polymerase chain reactions for dermatophyte detection, with further screening of SQLE mutations at four known hotspots (393Leu, 397Phe, 415Phe and 440His). A total of 62,056 samples were submitted (mean age: 57.5 years; female: 60.4%). Dermatophytes were detected in 38.5% of samples, primarily Trichophyton rubrum complex (83.6%) and T. mentagrophytes complex (10.7%). A survey of SQLE mutations was carried out in 22,610 dermatophyte samples; there was a significant increase in the prevalence of SQLE mutations between the first quarter of 2022 and the second quarter of 2023 (29.0 to 61.9 per 1000 persons). The Phe397Leu substitution was the predominant mutation; Phe415Ser and His440Tyr have also emerged which were previously reported as minor mutations in skin samples. The temporal change in mutation rates can be primarily attributed to the Phe415Ser substitution. Samples from elderly patients (>70 years) are more likely to be infected with the T.mentagrophytes complex including strains harbouring the Phe415Ser substitution. The prevalence of SQLE mutations among onychomycosis patients with Trichophyton infections may be underestimated. Older individuals may have a higher risk.

Dominance of Cotton leaf curl Multan virus-Rajasthan strain associated with third epidemic of cotton leaf curl disease in Pakistan

Cotton (Gossypium hirsutum) is an economically potent crop in many countries including Pakistan, India, and China. For the last three decades, cotton production is under the constant stress of cotton leaf curl disease (CLCuD) caused by begomoviruses/satellites complex that is transmitted through the insect pest, whitefly (Bemisia tabaci). In 2018, we identified a highly recombinant strain; Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Raj), associated with the Cotton leaf curl Multan betasatellite-Vehari (CLCuMuBVeh). This strain is dominant in cotton-growing hub areas of central Punjab, Pakistan, causing the third epidemic of CLCuD. In the present study, we have explored the CLCuD diversity from central to southern districts of Punjab (Faisalabad, Lodhran, Bahawalpur, Rahimyar Khan) and the major cotton-growing region of Sindh (Tandojam), Pakistan for 2 years (2020–2021). Interestingly, we found same virus (CLCuMuV-Raj) and associated betasatellite (CLCuMuBVeh) strain that was previously reported with the third epidemic in the central Punjab region. Furthermore, we found minor mutations in two genes of CLCuMuV-Raj C4 and C1 in 2020 and 2021 respectively as compared to its isolates in 2018, which exhibited virus evolution. Surprisingly, we did not find these mutations in CLCuMuV-Raj isolates identified from Sindh province. The findings of the current study represent the stability of CLCuMuV-Raj and its spread toward the Sindh province where previously Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Shahdadpur virus (CLCuShV) have been reported. The findings of the current study demand future research on CLCuD complex to explore the possible reasons for prevalence in the field and how the virus-host-vector compatible interaction can be broken to develop resistant cultivars.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

BackgroundThe successful detection of drug-resistance mutations (DRMs) in HIV-1 infected patients has improved the management of HIV infection. Next-generation sequencing (NGS) to detect low-frequency mutations is predicted to be useful for efficiently testing minority drug resistance mutations, which could contribute to virological failure. This study employed Sanger sequencing and NGS to detect and compare minority and majority drug resistance mutations in HIV-1 strains in treatment-naive patients from Ghana. MethodFrom a previous study, 20 antiretroviral therapy (ART)-naive participants were selected for a cross-sectional study. Sanger sequencing and NGS techniques were used to detect the majority and minority HIV drug resistance (HIVDR) mutations, respectively, in the protease (PR) and partial reverse transcriptase (RT) genes. NGS detected mutations at 1 % and 5 % frequencies and Sanger sequencing at ≥20 % frequencies. The sequences obtained from NGS and Sanger sequencing platforms were submitted to the Stanford HIV drug resistance database for subtyping, mutation identification, and interpretations. ResultsSequences from the twenty participants where the CRF02_AG was the predominant strain (16, 80 %) were analyzed. NGS detected 25 mutations in the RT and PR genes, compared to 21 mutations by Sanger sequencing. Minority DRMs were detected at the prevalence of 55.0 % with NGS against 35 % DRMs by Sanger sequencing. One of the patients had eight different HIVDR variants, with two minority variants. These mutations were directed against PI (K20I and D30DN), NNRTI (Y181C, M23LM and V108I) and NRTI (K65R, M184I, and D67N). ConclusionThe study affirms the usefulness of genomic sequencing for drug resistance testing in HIV. It further shows that Sanger sequencing alone may not be adequate to detect mutations and that NGS capacity should be developed and deployed in the Ghanaian clinical settings for patients living with HIV.

92P Minor RAS mutations as predictors of survival in recurrent or metastatic colorectal cancer: A retrospective study in a single institution

92P Minor RAS mutations as predictors of survival in recurrent or metastatic colorectal cancer: A retrospective study in a single institution

Exploring Drug-Resistant Mutations in Protease Inhibitors and Subtype Distribution Among HIV-1 Positive Patients in Lorestan Province, Iran

Background: Human immunodeficiency virus type 1 (HIV-1) is a major global public health concern, affecting millions worldwide. Despite significant advancements in antiretroviral therapy, the development of drug-resistant HIV-1 variants continues to challenge effective treatment. A multitude of antiretroviral drugs have been developed through intensive efforts by experts across various fields, including retrovirology, medicinal chemistry, enzymology, computational modeling, and structural biology. Objectives: This study focuses on drug-resistant mutations in protease inhibitors (PIs) and the distribution of HIV-1 subtypes in Lorestan province, Iran. Methods: A total of 59 patients were categorized into two groups: Recipients of antiretroviral therapy (ART) and drug-naive individuals. Genotypic resistance testing was performed using nested PCR, followed by sequencing and analysis of the PCR product to identify drug-resistance mutations and determine the viral subtype. Results: Among the ART recipients, 11 (78%) exhibited major mutations, while 3 (22%) had minor mutations specifically in PIs. The most commonly observed major protease inhibitor (PI) mutations were D30N (27.2%) and V32I (27.2%), followed by G48A (18.1%), L90M (18.1%), and L76V (9%). The most frequent minor PI mutations recorded were K20R (40%), L10I (20%), F53I (20%), and V11I (20%). No drug resistance was detected in drug-naive patients. Lopinavir (LPV) and nelfinavir (NFV) exhibited the highest levels of resistance, while saquinavir (SQV) and fosamprenavir (FPV) showed the highest levels of susceptibility. All participants were found to be infected with CRF35_AD, the dominant HIV-1 subtype in Iran. Conclusions: This study represents the first attempt in the region to analyze drug-resistant mutations in PIs among ART-experienced patients in Lorestan province. The findings contribute to ongoing efforts aimed at controlling the spread of drug-resistant HIV-1 strains.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

Abstract 1655: Targeting lipid metabolism disrupts KRAS oncogenesis in pancreatic cancer

Abstract Background: KRAS mutations are chief genetic event in pancreatic cancer. Recently approved inhibitors, sotorasib and adagrasib specifically target KRASG12C, a minor mutation in pancreatic cancer. Further, patients developed acquired resistance by generating secondary KRAS mutations, posing an added obstacle for allele specific inhibitors, warranting alternative strategies to tackle KRAS-dependent cancers. KRAS forms proteolipid signaling platforms on plasma membrane (PM) termed nanoclusters. We have shown mutant KRAS nanoclusters selectively enrich phosphatidylserine (PS) lipid, being particularly receptive to unsaturated PS, suggesting lipid acyl chain metabolism is crucial for KRAS function. In cells, lysophosphatidylcholine acyltransferases 3 (LPCAT3) preferentially catalyzes synthesis of unsaturated PS. We tested the hypothesis that inhibition of LPCAT3 depletes unsaturated PS in the PM, perturbs the PS-dependent PM interactions and oncogenic functions of KRAS. Methods: LPCAT3 knockdown (KD) mediated by siRNA in doxycycline (Dox) inducible KRASG12D murine cells (iKRAS) and CRISPR-Cas9 in human pancreatic tumor line (MiaPaCa-2). LPCAT3 inhibition achieved using small molecule inhibitors (SMIs): HTS3 and HTS4. Shotgun lipidomics characterized re-modeling of PS lipidomes. PM distribution and nanoclustering of KRAS was evaluated by super-resolution electron microscopy (EM). Anti-tumor effect of LPCAT3 inhibition was examined in vitro by proliferation, colony formation and soft agar assays and cellular signaling by western blot. Results: LPCAT3KD decreased polyunsaturated PS levels in MiaPaCa-2 and mislocalized mutant KRASG12V on the PM with a 4-fold reduction in KRAS PM distribution (p<0.0013) and disrupted KRAS nanoclustering, without affecting that of HRASG12V in transformed MiaPaCa-2. LPCAT3KD reduced colony formation ability in MiaPaCa-2. LPCAT3 siRNA reduced KRAS-dependent MAPK signaling, and significantly reduced colony formation only upon Dox-induced expression of KRASG12D, while having no effect in uninduced iKRAS. An integrated knowledgebase tool canSAR.ai generated a druggability score of 77% suggesting LPCAT3 is highly druggable by chemistry-based assessment. Indeed, recently discovered LPCAT3 inhibitors, significantly reduced MAPK signaling, proliferation and colony formation in mutant KRAS-dependent tumor cells while having no significant effect in WT KRAS cells corroborating mutant KRAS specificity of LPCAT3. Conclusion: We demonstrate targeting unsaturated PS lipid metabolism by inhibiting LPCAT3 disrupts KRAS signaling platform and perturbs its oncogenic functions in pancreatic cancer cells. Since various KRAS mutants require unsaturated PS to propagate signaling, our approach targets diverse KRAS mutants, uniquely addressing drug resistance issue. Further, we will optimize the LPCAT3 inhibitors and study their efficacy in xenograft mouse models. Citation Format: Neha Arora, Hong Liang, Alex Reed, Jeffrey T. Chang, Benjamin F. Cravatt, Yong Zhou. Targeting lipid metabolism disrupts KRAS oncogenesis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1655.

HIV protease resistance mutations in patients receiving second-line antiretroviral therapy in Libreville, Gabon

IntroductionIn 2022, the WHO reported that 29.8 million people around the world were living with HIV (PLHIV) and receiving antiretroviral treatment (ART), including 25‌ 375 people in Gabon (54% of all those living with HIV in the country). The literature reports a frequency of therapeutic failure with first-line antiretrovirals (ARVs) of between 20% and 82%. Unfortunately, data relating to the failure of second-line ARVs are scarce in Gabon. This study aims to determine the profiles of HIV drug resistance mutations related to protease inhibitors in Gabon.MethodologyPlasma from 84 PLHIV receiving ARVs was collected from 2019 to 2021, followed by RNA extraction, amplification, and sequencing of the protease gene. ARV resistance profiles were generated using the Stanford interpretation algorithm version 8.9-1 (https://hivdb.stanford.edu) and statistical analyses were performed using EpiInfo software version 7.2.1.0 (CDC, USA).ResultsOf 84 HIV plasma samples collected from 45 men and 39 women, 342 mutations were detected. Of these, 43.3% (148/342) were associated with nucleoside reverse transcriptase inhibitors (NRTIs), 30.4% (104/342) with non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 26.3% (90/342) with protease inhibitors (PIs). Most NRTI mutations were associated with thymidine analogues (TAMs) (50.7%; 75/148), including T215F/V (14.9%; 22/148), D67DN/E/G/N/T (10.1%; 15/148), M41L (9.5%; 14/148), and K70E/KN/S/R (9.5%; 14/148). Resistance mutations related to non-TAM NRTIs (33.1%; 49/148) were M184V (29.1%; 43/148), and L74I/V (8.1%; 12/148). NNRTI mutations were predominantly K103N/S (32.7%; 34/104), V108I (10.6%; 11/104), A98G (10.6%; 11/104), and P225H (9.6%; 10/104). Minor mutations associated with PIs (60.0%; 54/90) were predominantly K20I (15.6%; 14/90) and L10F/I/V (14.5%; 13/90). The major mutations associated with PIs (40.0%; 36/90) were M41L (12.2%; 11/90), I84V (6.7%; 06/90), and V82A (6.7%; 06/90). The four most prescribed therapeutic regimens were TDF + 3TC + LPV/r (20.3%; 17/84), ABC + DDI + LPV/r (17.9%; 15/84), TDF + FTC + LPV/r (11.9%; 10/84), and ABC + 3TC + LPV/r (11.9%; 10/84).ConclusionThis study revealed that HIV drug resistance mutations are common in Gabon. The major mutations associated with PIs were M41L, I84V, and V82A. There is a need for access to new NRTIs, NNRTIs, and PIs for a better therapeutic management of PLHIV in Gabon.

Clinical characteristics of patients treated with immune checkpoint inhibitors in EGFR-mutant non-small cell lung cancer: CS-Lung-003 prospective observational registry study

PurposeImmune checkpoint inhibitors (ICIs) are ineffective against epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC). This study aimed to investigate the clinical characteristics of patients who were treated or not treated with ICIs, and of those who benefit from immunotherapy in EGFR-mutant NSCLC.MethodsWe analyzed patients with unresectable stage III/IV or recurrent NSCLC harboring EGFR mutations using a prospective umbrella-type lung cancer registry (CS-Lung-003).ResultsA total of 303 patients who met the eligibility criteria were analyzed. The median age was 69 years; 116 patients were male, 289 had adenocarcinoma, 273 had major mutations, and 67 were treated with ICIs. The duration of EGFR-TKI treatment was longer in the Non-ICI group than in the ICI group (17.1 vs. 12.7 months, p < 0.001). Patients who received ICIs for more than 6 months were categorized into the durable clinical benefit (DCB) group (24 patients), and those who received ICIs for less than 6 months into the Non-DCB group (43 patients). The overall survival in the DCB group exhibited longer than the Non-DCB group (69.3 vs. 47.1 months), and an equivalent compared to that in the Non-ICI group (69.3 vs. 68.9 months). Multivariate analysis for time to next treatment (TTNT) of ICIs showed that a poor PS was associated with a shorter TTNT [hazard ratio (HR) 3.309; p < 0.001]. Patients who were treated with ICIs and chemotherapy combination were associated with a longer TTNT (HR 0.389; p = 0.003). In addition, minor EGFR mutation was associated with a long TTNT (HR 0.450; p = 0.046).ConclusionICIs were administered to only 22% of patients with EGFR-mutated lung cancer, and they had shorter TTNT of EGFR-TKI compared to other patients. ICI treatment should be avoided in EGFR mutated lung cancer with poor PS but can be considered for lung cancer with EGFR minor mutations. Pathological biomarker to predict long-term responders to ICI are needed.

Minor introns impact on hematopoietic malignancies

In the intricate orchestration of the central dogma, pre-mRNA splicing plays a crucial role in the post-transcriptional process that transforms DNA into mature mRNA. Widely acknowledged as a pivotal RNA processing step, it significantly influences gene expression and alters the functionality of gene product proteins. Although U2-dependent spliceosomes efficiently manage the removal of over 99% of introns, a distinct subset of essential genes undergo splicing with a different intron type, denoted as minor introns, using U12-dependent spliceosomes. Mutations in spliceosome component genes are now recognized as prevalent genetic abnormalities in cancer patients, especially those with hematologic malignancies. Despite the relative rarity of minor introns, genes containing them are evolutionarily conserved and play crucial roles in functions such as the RAS-MAPK pathway. Disruptions in U12-type minor intron splicing caused by mutations in snRNA or its regulatory components significantly contribute to cancer progression. Notably, recurrent mutations associated with myelodysplastic syndrome (MDS) in the minor spliceosome component ZRSR2 underscore its significance. Examination of ZRSR2-mutated MDS cells has revealed that only a subset of minor spliceosome-dependent genes, such as LZTR1, consistently exhibit missplicing. Recent technological advancements have uncovered insights into minor introns, raising inquiries beyond current understanding. This review comprehensively explores the importance of minor intron regulation, the molecular implications of minor (U12-type) spliceosomal mutations and cis-regulatory regions, and the evolutionary progress of studies on minor, aiming to provide a sophisticated understanding of their intricate role in cancer biology.

The 2.6 Å Structure of a Tulane Virus Variant with Minor Mutations Leading to Receptor Change.

Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA-a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution.

Validation of an HIV whole genome sequencing method for HIV drug resistance testing in an Australian clinical microbiology laboratory.

Detection of HIV drug resistance (HIVDR) is vital to successful anti-retroviral therapy (ART). HIVDR testing to determine drug-resistance mutations is routinely performed in Australia to guide ART choice in newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our clinical microbiology laboratory sought to validate a next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger-sequencing (SS)-based ViroSeq. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for whole-genome recovery and HIVDR-testing in a diagnostic setting. veSEQ-HIV displayed 100% and 98% accuracy in major and minor mutation detection, respectively, and 100% accuracy of subtyping (provided > 1000 mapped reads were obtained). Pairwise comparison exhibited low inter-and intrarun variability across the whole-genome (Jaccard index [J] = 0.993; J = 0.972) and the Pol gene (J = 0.999; J = 0.999), respectively. veSEQ-HIV met all our pre-set criteria based on WHO recommendations and successfully replaced ViroSeq in our laboratory. Scaling-down veSEQ-HIV to a limited batch size and sequencing on Illumina iSeq. 100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times. As HIVDR-testing moves from SS- to NGS-based methods and new ART drugs come to market (particularly those with targets outside the Pol region), whole-genome recovery using veSEQ-HIV provides a robust, cost-effective and "future-proof" NGS method for HIVDR-testing.

Higher Levels of SARS-CoV-2 Genetic Variation in Immunocompromised Patients: A Retrospective Case-Control Study.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection lasts longer in immunocompromised hosts than in immunocompetent patients. Prolonged infection is associated with a higher probability of selection for novel SARS-CoV-2 mutations, particularly in the spike protein, a critical target for vaccines and therapeutics. From December 2020 to September 2022, respiratory samples from 444 immunocompromised patients and 234 health care workers positive for SARS-CoV-2, diagnosed at 2 hospitals in Paris, France, were analyzed using whole-genome sequencing using Nanopore technology. Custom scripts were developed to assess the SARS-CoV-2 genetic diversity between the 2 groups and within the host. Most infections were SARS-CoV-2 Delta or Omicron lineages. Viral genetic diversity was significantly higher in infections of immunocompromised patients than those of controls. Minor mutations were identified in viruses sequenced from immunocompromised individuals, which became signature mutations for newer SARS-CoV-2 variants as the epidemic progressed. Two patients were coinfected with Delta and Omicron variants. The follow-up of immunocompromised patients revealed that the SARS-CoV-2 genome evolution differed in the upper and lower respiratory tracts. This study found that SARS-CoV-2 infection in immunocompromised patients is associated with higher genetic diversity, which could lead to the emergence of new SARS-CoV-2 variants with possible immune evasion or different virulence characteristics.

Investigation of the individual genetic evolution of SARS-CoV-2 in a small cluster during the rapid spread of the BF.5 lineage in Tokyo, Japan.

There has been a decreasing trend in new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and fatalities worldwide. The virus has been evolving, indicating the potential emergence of new variants and uncertainties. These challenges necessitate continued efforts in disease control and mitigation strategies. We investigated a small cluster of SARS-CoV-2 Omicron variant infections containing a common set of genomic mutations, which provided a valuable model for investigating the transmission mechanism of genetic alterations. We conducted a study at a medical center in Japan during the Omicron surge (sub-lineage BA.5), sequencing the entire SARS-CoV-2 genomes from infected individuals and evaluating the phylogenetic tree and haplotype network among the variants. We compared the mutations present in each strain within the BA.5 strain, TKYnat2317, which was first identified in Tokyo, Japan. From June 29th to July 4th 2022, nine healthcare workers (HCWs) tested positive for SARS-CoV-2 by real-time PCR. During the same period, five patients also tested positive by real-time PCR. Whole genome sequencing revealed that the infected patients belonged to either the isolated BA.2 or BA.5 sub-lineage, while the healthcare worker infections were classified as BF.5. The phylogenetic tree and haplotype network clearly showed the specificity and similarity of the HCW cluster. We identified 12 common mutations in the cluster, including I110V in nonstructural protein 4 (nsp4), A1020S in the Spike protein, and H47Y in ORF7a, compared to the BA.5 reference. Additionally, one case had the extra nucleotide-deletion mutation I27* in ORF10, and low frequencies of genetic alterations were also found in certain instances. The results of genome sequencing showed that the nine HCWs shared a set of genetic mutations, indicating transmission within the cluster. Minor mutations observed in five HCW individuals suggested the emergence of new virus variants. Five amino acid substitutions occurred in nsp3, which could potentially affect virus replication or immune escape. Intra-host evolution also generated additional mutations. The cluster exhibited a mild disease course, with individuals in this case, recovering without requiring any medical treatments. Further investigation is needed to understand the relationship between the genetic evolution of the virus and the symptoms.

Frequency of Atypical Mutations in the Spike Glycoprotein in SARS-CoV-2 Circulating from July 2020 to July 2022 in Central Italy: A Refined Analysis by Next Generation Sequencing.

In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/μL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

Full-spectrum HIV drug resistance mutation detection by high-resolution complete pol gene sequencing.

Drug resistance mutation testing is a key element for HIV clinical management, informing effective treatment regimens. However, resistance screening in current clinical practice is limited in reporting linked cross-class resistance mutations and minority variants, both of which may increase the risk of virological failure. To address these limitations, we obtained 358 full-length pol gene sequences from 52 specimens of 20 HIV infected individuals by combining microdroplet amplification, unique molecular identifier (UMI) labeling, and long-read high-throughput sequencing. We conducted a rigorous assessment of the accuracy of our pipeline for precision drug resistance mutation detection, verifying that a sequencing depth of 35 high-throughput reads achieved complete, error-free pol gene sequencing. We detected 26 distinct drug resistance mutations to Protease Inhibitors (PIs), Nucleoside Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and Integrase Strand Transfer Inhibitors (INSTIs). We detected linked cross-class drug resistance mutations (PI+NRTI, PI+NNRTI, and NRTI+NNRTI) that confer cross-resistance to multiple drugs in different classes. Fourteen different types of minority mutations were also detected with frequencies ranging from 3.2% to 19%, and the presence of these mutations was verified by Sanger reference sequencing. We detected a putative transmitted drug resistance mutation (TDRM) in one individual that persisted for over seven months from the first sample collected at the acute stage of infection prior to seroconversion. Our comprehensive drug resistance mutation profiling can advance clinical practice by reporting mutation linkage and minority variants to better guide antiretroviral therapy options.