Extra Mutations Research Articles

Enhanced neutralization of SARS-CoV-2 variant BA.2.86 and XBB sub-lineages by a tetravalent COVID-19 vaccine booster

Emerging SARS-CoV-2 sub-lineages like XBB.1.5, XBB.1.16, EG.5, HK.3 (FLip), and XBB.2.3 and the variant BA.2.86 have recently been identified. Understanding the efficacy of current vaccines on these emerging variants is critical. We evaluate the serum neutralization activities of participants who received COVID-19 inactivated vaccine (CoronaVac), those who received the recently approved tetravalent protein vaccine (SCTV01E), or those who had contracted a breakthrough infection with BA.5/BF.7/XBB virus. Neutralization profiles against a broad panel of 30 sub-lineages reveal that BQ.1.1, CH.1.1, and all the XBB sub-lineages exhibit heightened resistance to neutralization compared to previous variants. However, despite their extra mutations, BA.2.86 and the emerging XBB sub-lineages do not demonstrate significantly increased resistance to neutralization over XBB.1.5. Encouragingly, the SCTV01E booster consistently induces higher neutralizing titers against all these variants than breakthrough infection does. Cellular immunity assays also show that the SCTV01E booster elicits a higher frequency of virus-specific memory B cells. Our findings support the development of multivalent vaccines to combat future variants.

SARS-CoV-2 emerging Omicron subvariants with a special focus on BF.7 and XBB.1.5 recently posing fears of rising cases amid ongoing COVID-19 pandemic

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron versions have been the sole one circulating for quite some time. Subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 of the Omicron emerged over time and through mutation, with BA.1 responsible for the most severe global pandemic between December 2021 and January 2022. Other Omicron subvariants such as BQ.1, BQ.1.1, BA.4.6, BF.7, BA.2.75.2, XBB.1 appeared recently and could cause a new wave of increased cases amid the ongoing COVID-19 pandemic. There is evidence that certain Omicron subvariants have increased transmissibility, extra spike mutations, and ability to overcome protective effects of COVID-19 neutralizing antibodies through immunological evasion. In recent months, the Omicron BF.7 subvariant has been in the news due to its spread in China and a small number of other countries, raising concerns about a possible rebound in COVID-19 cases. More recently, the Omicron XBB.1.5 subvariant has captured international attention due to an increase in cases in the United States. As a highly transmissible sublineage of Omicron BA.5, as well as having a shorter incubation time and the potential to reinfect or infect immune population, BF.7 has stronger infection ability. It appears that the regional immunological landscape is affected by the amount and timing of previous Omicron waves, as well as the COVID-19 vaccination coverage, which in turn determines whether the increased immune escape of BF.7 and XBB.1.5 subvariants is sufficient to drive new infection waves. Expanding our understanding of the transmission and efficacy of vaccines, immunotherapeutics, and antiviral drugs against newly emerging Omicron subvariants and lineages, as well as bolstering genomic facilities for tracking their spread and maintaining a constant vigilance, and shedding more light on their evolution and mutational events, would help in the development of effective mitigation strategies. Importantly, reducing the occurrence of mutations and recombination in the virus can be aided by bolstering One health approach and emphasizing its significance in combating zoonosis and reversal zoonosis linked with COVID-19. This article provides a brief overview on Omicron variant, its recently emerging lineages and subvairants with a special focus on BF.7 and XBB.1.5 as much more infectious and highly transmissible variations that may once again threaten a sharp increase in COVID-19 cases globally amid the currently ongoing pandemic, along with presenting salient mitigation measures.

Initial introduction and spread of the SARS-CoV-2 AY.4.2.1 Delta variant in Bulgaria, a genomic insight.

The evolution of the emerging SARS‐CoV‐2 variants carrying mutations in the spike protein raises concerns about the possibility of accelerated transmission in the ever‐evolving COVID‐19 pandemic worldwide. AY.4.2, a sublineage of the Delta variant, was considered a variant under investigation (VUI) and also gained the nickname “Delta Plus,” due to its extra mutations, Y145H and A222V. In this study, using genomic epidemiology, we provide the first insights into the introduction of AY.4.2 in Bulgaria and the AY.4.2.1 sublineage that found larger dissemination only in Bulgaria and the United Kingdom.

Is the Mutation Rate Lower in Genomic Regions of Stronger Selective Constraints?

A study of the plant Arabidopsis thaliana detected lower mutation rates in genomic regions where mutations are more likely to be deleterious, challenging the principle that mutagenesis is blind to its consequence. To examine the generality of this finding, we analyze large mutational data from baker's yeast and humans. The yeast data do not exhibit this trend, whereas the human data show an opposite trend that disappears upon the control of potential confounders. We find that the Arabidopsis study identified substantially more mutations than reported in the original data-generating studies and expected from Arabidopsis' mutation rate. These extra mutations are enriched in polynucleotide tracts and have relatively low sequencing qualities so are likely sequencing errors. Furthermore, the polynucleotide “mutations” can produce the purported mutational trend in Arabidopsis. Together, our results do not support lower mutagenesis of genomic regions of stronger selective constraints in the plant, fungal, and animal models examined.

A Review on Variant of Concern (Delta Variant of COVID 19)

The B.1.6179 Delta variant of the severe acute metabolic process syndrome corona virus (SARS –COV-2). The virus that causes corona virus unwellness 2019 (covid -19), has contributed to a surge in cases of Republic of India and has currently been detected across the world, as well as a notable increase in cases within the UK. Delta and (B.167.2.1/(Ay.1) could be a new variant of the SARS- CoV-2. corona virus came in existence because of a mutation within the delta strain of the virus (B.1.617.2 variant). It is technically successive generation of SARS-COV-2. This mutant of delta was initial detected in Europe march 2021.The delta virus that was initial detected in Republic of India (in February 2021) eventually became an enormous downside for the total world. However, the delta variant at the moment, is restricted to smaller areas within the country. Individual’s reportable symptoms like headaches, sore throats, fluid noses, and fever. The world health organization (WHO)is following this variant as a part of the delta variant as well as for different variants of concern with extra mutations. This review provides detail idea about delta virus its treatment, prevention and methods for reduction of sensitivity.

Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.

Emergence and spread of a sub-lineage of SARS-CoV-2 Alpha variant B.1.1.7 in Europe, and with further evolution of spike mutation accumulations shared with the Beta and Gamma variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution plays a significant role in shaping the dynamics of the coronavirus disease 2019 pandemic. To monitor the evolution of SARS-CoV-2 variants, through international collaborations, we performed genomic epidemiology analyses on a weekly basis with SARS-CoV-2 samples collected from a border region between Germany, Poland, and the Czech Republic in a global background. For identified virus mutant variants, active viruses were isolated and functional evaluations were performed to test their replication fitness and neutralization sensitivity against vaccine-elicited serum neutralizing antibodies. Thereby we identified a new B.1.1.7 sub-lineage carrying additional mutations of nucleoprotein G204P and open-reading-frame-8 K68stop. Of note, this B.1.1.7 sub-lineage is the predominant B.1.1.7 variant in several European countries such as Czech Republic, Austria, and Slovakia. The earliest samples belonging to this sub-lineage were detected in November 2020 in a few countries in the European continent, but not in the UK. We have also detected its further evolution with extra spike mutations D138Y and A701V, which are signature mutations shared with the Gamma and Beta variants, respectively. Antibody neutralization assay of virus variant isolations has revealed that the variant with extra spike mutations is 3.2-fold less sensitive to vaccine-elicited antibodies as compared to the other B.1.1.7 variants tested, indicating potential for immune evasion, but it also exhibited reduced replication fitness, suggesting lower transmissibility. The wide spread of this B.1.1.7 sub-lineage was related to the pandemic waves in early 2021 in various European countries. These findings about the emergence, spread, evolution, infection, and transmission abilities of this B.1.1.7 sub-lineage add to our understanding about the pandemic development in Europe and highlight the importance of international collaboration on virus mutant surveillance.

Distinct Modes of Ongoing Antigen Interactions Shape Intraclonal Dynamics in Splenic Marginal Zone Lymphoma

Almost one-third of all splenic marginal zone lymphoma (SMZL) cases express B cell receptor immunoglobulin (BcR IG) encoded by the IGHV1-2*04 gene. Such cases display a distinctive profile of genomic aberrations (e.g. higher incidence of NOTCH2 and KLF2 mutations) and a more aggressive clinical course compared to SMZL cases utilizing other IGHV genes. Such skewing of the BcR IG gene repertoire implicates antigen selection in SMZL ontogeny. Although the supportive evidence is compelling, it mostly derives from low-throughput approaches, which are inherently limited in their capacity to capture the complexity of the BcR IG gene repertoire. This hinders the comprehensive assessment of the subclonal architecture of SMZL that could offer insight into the dynamics of antigen-IG interactions. Here, we sought to overcome this limitation through a high-throughput immunogenetic investigation of SMZL, focusing on the detailed characterization of somatic hypermutation (SHM) and intraclonal diversification (ID) profiles. Our study included 22 cases utilizing the IGHV1-2*04 gene and 36 cases utilizing other IGHV genes. IGHV-IGHD-IGHJ (IGH) gene rearrangements were PCR-amplified and libraries were sequenced on the Illumina MiSeq platform. Data was analyzed with the IMGT/HighV-QUEST and TRIP software as well as a novel bioinformatics/biostatistics pipeline. Clonotypes were defined as unique combinations of a given IGHV gene+VH CDR3 amino acid (aa) sequence. Only IGH gene rearrangement sequences assigned to the dominant clonotypes of each case were assessed. In detail, all nucleotide variants (nt vars, i.e. all sequences clustered in the same dominant clonotype yet displaying distinct SHM profiles) were identified and further analyzed. Starting from the most abundant nt var, a network was built representing its connections with all other nt vars. For this analysis, we introduce the terms ‘most relevant pathway’ (MRP) corresponding to the pathway including connected nt vars with the highest total number of IGH sequences; and ‘longest mutational pathways’ (LMP) corresponding to the pathways with the highest number of nt vars (Fig. 1). Different graph metrics assessed the impact of ID in different SMZL subgroups: the first one focuses on the ‘most relevant pathway’ and quantifies SHM convergence [ratio of the total number of IGH sequences corresponding to the nt vars of this pathway to the number of IGH sequences in the most abundant nt var]; while the second refers to the length of the ‘longest mutational pathways’.Cases lacking additional connected nt vars [length of the LMP=1; 3 IGHV1-2*04 cases and 4 non-IGHV1-2*04 cases] were excluded. Consequently, the analysis included 19 IGHV1-2*04 cases and 32 non-IGHV1-2*04 cases. Significant differences were noted in the SHM and ID profiles between groups; the IGHV1-2*04 group had significantly (p<0.01) higher convergence values ranging from 0.009 to 1.243 (median: 0.102), as opposed to the non-IGHV1-2*04 group (range: 0.002-1.13, median: 0.014), overall suggesting that stronger selective pressures act in SMZL cases expressing the IGHV1-2*04 versus others. Moreover, IGHV1-2*04 cases displayed significantly (p<0.01) longer mutational pathways (length range: 2-6, median: 3) compared to the other group (range: 2-5, median: 2), alluding to more pronounced ID arising due to ongoing SHM.Finally, all mutations leading to aa changes were analyzed in the context of ID networks. More recurrent aa mutations were identified amongst cases with higher levels of convergence. For instance, the VH FR2 M39I change, one of the most prominent recurrent SHMs in the IGHV1-2*04 group, was found in the most abundant nt var in 13/19 IGHV1-2*04 cases, while it was identified in nt vars with extra mutations in another 3 cases. Of interest, it was present at the end of the mutational pathways in these 16 cases, whilst in the other group it was present only in one case using the IGHV1-2*02 gene, and absent in the rest (p<0.01).In conclusion, in the first large-scale high-throughput immunogenetic analysis of SMZL we provide strong evidence for more pronounced antigenic pressure in cases utilizing IGHV1-2*04 versus other IGHV genes. Our findings highlight a unique subclonal architecture for IGHV1-2*04 SMZL and corroborate the hypothesis that this group may represent a distinct molecular variant of SMZL. [Display omitted] DisclosuresRossi: Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Verastem: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Cellestia: Honoraria, Research Funding. Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

DMD/BMD - GENETICS: EP.115 RNA-seq analysis for Dystrophinopathy

Dystrophinopathy, including Duchenne and Becker muscular dystrophies (DMD/BMD), is the most common hereditary neuromuscular disease caused by mutations in the DMD gene. It is known that the deletions/duplications spanning one or more exons are the cause of the disease in approximately 70% of patients while the small mutations involving one or at most several nucleotides are responsible for the remaining 30%. Nowadays, a combination of multiplex ligation-dependent probe amplification (MLPA) and targeted resequencing are widely used for the diagnosis of dystrophinopathy. Nevertheless, around 1% of patients still remain undiagnosed and seem to carry deep intronic mutations creating an extra exon or mutations produced by chromosomal rearrangement, leading to abnormal transcript. To decipher such mutations, we performed RNA-seq on 23 undiagnosed DMD/BMD cases in whom no variant was identified albeit dystrophin was deficient on muscle biopsy. We also analyzed 6 DMD/BMD cases who had a missense mutation in DMD gene as we suspected aberrant splicing may be caused by the mutation in these cases. By RNA-seq, abnormal splicing events were seen in 11 of 23 undiagnosed cases, in whom we further confirmed the presence of intronic mutations that led to pseudo exon production. The remaining 12 cases showed low expression of DMD transcript but without obvious abnormal splicing. In addition, in 2 of 6 cases with missense mutation, skipping of the exon harboring the mutation was detected. The variants were located the last or second to last exon in 2 cases. The remaining 4 cases showed normal expression of DMD transcript. Still 12 cases remained unsolved even by RNA-seq. Combination of whole genome sequencing and long-read sequencing will be performed for these cases. Not only RNA-seq analysis complements the routine genetic testing of dystrophinopathy but may help uncover the actual pathomechanisms of “missense” variants.

Abstract 39: Real-time spatial and temporal monitoring on tumor subclonal evolution

Abstract Many types of cancer are found to exhibit a high level of intrasample genetic heterogeneity, as a result of the microevolution process. As genomic aberrations accumulate within tumor cells, a subset of the cells may gain a selective advantage and form an outgrowth within the tumor. When chemotherapeutic agents are administered to the patient, these tumor subclones may respond differently to the treatment and may eventually cause relapse. Identifying resistant subclones, and their genotypes, after each chemotherapy would yield valuable insights into how the tumor population reacted to the previously administered chemo-agent, and how the treatment plan should be adjusted based on the genetic features of surviving subclones. Similarly, understanding the relationships between the primary and metastatic tumor cell populations would provide knowledge about the mechanisms driving, and potentially preventing, metastasis. Our method (SubcloneSeeker) is able to reconstruct subclone structures jointly across all samples (temporal or spatial), using somatic variants jointly clustered with their Cell Prevalence (CP), to infer a unified evolutionary structure. When more than one structure is able to explain the observed somatic variants and their CP values, we can compute a consensus structure that captures the most important features (i.e., which subclones and the corresponding genomic aberrations are responsible for the resistance of a certain chemotherapy drug), as well as suggest a panel of variants to be profiled with single-cell assay in order to reduce computational ambiguity. We applied this method to several datasets, including a breast cancer patient who had received three regimens of chemotherapy and had tumor samples taken at 4 consecutive time points. Our analysis revealed that the tumor population reacted to these treatment in two distinct patterns: 1) although a significant reduction in tumor burden was observed, most of the major subclones survived and expanded again after treatment, indicating that the treatment was effective, but terminated prematurely; 2) a single subclone with extra mutations (e.g., FBXL19, a tumor suppressor by inhibiting E-cadherin downregulation) survived the treatment and became the founding clone for the tumor population post-treatment, and the extra mutations were fixed at very high CP (~100%), indicating that these extra mutations are most likely to be causative for chemoresistance. We also applied our method to breast cancer patient rapid-autopsy datasets with 28 tumor samples. Our method was able to map out subclone evolution across multiple metastatic sites and highlight variants unique to each evolutionary lineage. In conclusion, our method is able to elucidate the dynamics of tumorigenesis, drug response, and metastasis by reconstructing unified subclone evolutionary structures, allowing more informed decisions to be made in adjusting treatment plans in real time and potentially facilitating personalized cancer medicine. Citation Format: Gabor Marth, Yi Qiao, Dillon Lee, Xiaomeng Huang. Real-time spatial and temporal monitoring on tumor subclonal evolution [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 39.

A Mechanism Underlying Attenuation of Recombinant Influenza A Viruses Carrying Reporter Genes.

Influenza A viruses (IAV) carrying reporter genes provide a powerful tool to study viral infection and pathogenesis in vivo, however, incorporating a non-essential gene into the IAV genome often results in virus attenuation and genetic instability. Very few studies have systematically compared different reporter IAVs, and most optimization attempts seem to lack authentic directions. In this study, we evaluated the ratio of genome copies to the number of infectious unit of two reporter IAVs, PR8-NS1-Gluc and PR8-PB2-Gluc. As a result, PR8-NS1-Gluc and PR8-PB2-Gluc produced 41.4 and 3.8 genomes containing noninfectious particles respectively for every such particle produced by parental PR8 virus. RdRp assay demonstrated that modification of segment NS by inserting reporter genes can interfere with the replication competitive property of the corresponding vRNAs, and the balance of the 8 segments of the reporter IAVs were drastically impaired in infected cells. As a consequence, large amounts of NS-null noninfectious particles were produced during the PR8-NS1-Gluc packaging. In summary, we unravel a mechanism underlying attenuation of reporter IAVs, which suggests a new approach to restore infectivity and virulence by introducing extra mutations compensating for the impaired replication property of corresponding segments.

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene

Background:Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies.Methods:We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs.Results:By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12).Conclusions:mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

Validation of a Next-Generation Sequencing Panel for AML Routine Diagnosis

Numerous recurrently mutated genes have been described in the last years for acute myeloid leukemia (AML). Nevertheless, only a few of them (NPM1, CEBPA, FLT3), and usually individually analyzed, have been systematically incorporated into clinical laboratories to stratify prognosis and guide risk-adapted therapy. So far, technology made impossible their simultaneous study, however, development of high throughput techniques as "next generation sequencing" (NGS) allows a parallel assay. Recently, our group designed a NGS panel (custom panel) interrogating the complete coding sequence of 87 genes potentially involved in AML. Interactome analysis revealed that 13 genes were highly implicated in AML pathogenesis. Although NGS has been broadly utilized in the investigational scope, its application to the routine diagnosis is still far from become a standard. The aim of this study is to validate the clinical applicability to routine laboratories of a hotspot NGS panel that includes these 13 genes and other potentially actionable targets.We included 62 "de novo" AML patients in which we tested the Ion Ampliseq AML community panel (Life Technologies) (IAAC panel) in the Ion PGM/Proton platforms. This panel includes hotspots of ASXL1 (exon 12), BRAF (V600E), CBL (exons 8-9), FLT3 (codons 676, 830-850), IDH1 (exon 4), IDH2 (exon 4), JAK 2 (exon 14), KIT (exons 8, 10, 11 and 17), KRAS (exons 2-4), NRAS (exons 2-4), PTPN11 (exons 3,7,8,13), RUNX1 (exons 3-8) y WT1 (exons 7 and 9), and the entire coding sequence of CEBPA, DNMT3A, GATA, TET2 and TP53. The design does not include the FLT3-ITD region. This panel requires only a total of 40 ng of DNA, far less than the custom panel (250 ng), which is very convenient in a clinical laboratory, where the input sample can be limited.The hotspot NGS panel detected 153 variants in 62 patients (2.47 mutations/patient). Mean read depth and uniformity were 1580 and 94.89%, respectively. The IAAC panel detected 25 NPM1 mutations, 20 TET2 and DNMT3A mutations, 14 in CEBPA, 12 in TP53, 11 in RUNX1, 8 in FLT3, 7 in IDH2, 6 in ASXL1, GATA2 and NRAS, 5 in PTPN11 and KRAS, 3 in WT1, 2 in CBL and 1 mutation in IDH1, KIT, and BRAF. Only 4 patients (6%) remained wild-type for these genes after the analysis with the IAAC panel.These samples had previously been analyzed by conventional molecular biology techniques (CMBT) for FLT3-D835, NPM1-T288, DNMT3A-R882 and CEBPA. The IAAC panel found 100% of these previously known mutations plus 5 extra mutations that were negative by CMBT. These mutations were reconfirmed by Sanger sequencing. Therefore, the IAAC panel detects more mutations than CMBT.To further assess the suitability of the hotspot panel, we selected a subset of 25 patients that had been also analyzed by the custom panel (Sure Desing Tool (Agilent) and an Illumina platform). This allowed us to analyze the complete coding sequence of the genes included in the IAAC panel and thus look for mutations outside the hotspots. A total of 53 variants were found with the custom panel. Fifty of these variants (95%) were also detected with the IAAC panel. The remaining 3 variants (5%) were located outside the hotspot regions. Additionally, the IAAC panel detected 13 variants in the overlapping regions that were not found with the custom panel. This could be explained because when focusing on recurrent regions of particular genes, it is possible to increase the mean read depth and therefore reach higher sensibility, which can be achieved with the IAAC panel but not with the custom panel.In conclusion, the Ion Ampliseq AML community panel detects mutations currently analyzed in most of clinical laboratories with validated prognostic relevance (NPM1 and CEBPA) in one assay, with low sample input requirement and with 100% sensibility compared with CMBT. In addition, this panel is able to find out alterations in these genes that are lost by CMBT. Moreover, other mutations with probable diagnostic or prognostic value and/or potential therapeutic targets are also studied and identified with high sensibility. Only a few changes are excluded of the covered regions. Therefore, IAAC panel is useful for routine diagnosis; however, detection of FLT3 internal tandem duplications is not possible, which limits its clinical utility. DisclosuresNo relevant conflicts of interest to declare.

Development of an Ultra-Deep Sequencing Method on Ion Torrent PGM to Detect Mutations in a Panel of Genes Relevant to Disease Progression and Drug Resistance in Chronic Lymphocytic Leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western world. The disease is characterised by a great variability in clinical course and response to treatment. Acquired somatic mutations in genes related to multiple signal pathways have recently emerged as important factors contributing to this heterogeneity. These mutations accumulate with disease progression or development of therapy resistance. However, with conventional methods they are very difficult to be detected in a single test, particularly when the size of the affected cell population is small. Therefore, there is a clinical need for a rapid, reliable, affordable and highly sensitive sequencing method to monitor these mutations from an early stage of this disease.Herein we developed an ultra-deep next generation sequencing approach based on Ion Torrent PGM to sequence a total of 246 exons of 15 genes including TP53, ATM, BIRC3, NOTCH1, SF3B1, MYD88, LRP1B, SAMHD1, FBXW7, POT1, HISTIH1E, XPO1, CHD2, PCLO and ZFPM2. These genes were selected because mutations in them have been reported to involve at least 5% of CLL patients and associate with poor outcome of CLL. Serial CLL samples were collected at multiple time points from diagnosis to disease progression/drug resistance from a cohort of 33 patients. Initially, target DNA in samples taken at advanced disease stages, without or with mutations in TP53 as detected and quantified by FASAY assay and Sanger sequencing, was enriched and amplified using Agilent HaloPlex probes, with 4604 amplicons for a sequenceable size of 135.91 Kbp of each sample. On each Ion 318 Chip (1 GB output), barcoded DNA enrichment preparations from 4 patients were sequenced by the PGM. For confirmation, the same patient samples were tested repeatedly in an independent experiment starting from DNA preparation. The sequence data were aligned to a human reference genome (Hg19); variants were called by the Torrent Variant Caller (v4.0-6) and visualised with the IGV. Results showed that the target coverage was 99.999% and an average coverage depth 3941x. Importantly, all of the known TP53 mutations were repeatedly detected at expected frequencies. In addition, extra mutations at lower levels (5% - 20% alleles) in multiple genes, including TP53 and SF3B1, were detected.In a sensitivity test of this method, DNA samples from 5 cases, each with a known % of mutation in TP53measured by FASAY assay and Sanger sequencing, were pooled together and then mixed with a wild-type DNA sample to serially dilute these mutations to 20% - 0.2% alleles before target enrichment. With an average coverage depth of 1843x (range: 1610 - 2187) for these 5 target locations, each mutation was readily detected at 20%, 5% and 1%, with the average quality scores being 1497.3, 61.2 and 29.4, respectively. However, only 3 out the 5 mutations could be detected at the level of 0.2%.Taken together, we have successfully developed a sensitive next generation sequencing method for detecting mutations in a CLL gene panel. This allows us to monitor multiple mutations affecting as low as 1% of alleles in CLL samples throughout the disease course and possibly discover clinically useful biomarker(s) for CLL progression and resistance to therapy. DisclosuresNo relevant conflicts of interest to declare.

Linkage analysis and whole-exome sequencing exclude extra mutations responsible for the parkinsonian phenotype of spinocerebellar ataxia-2

CAG expansion within the exon 1 of ataxin-2 (ATXN2) gene responsible for spinocerebellar ataxia-2 (SCA2) has been reported to cause pure parkinsonism and other neurodegenerative disorders. However, it remains unclear whether CAG expansion is the only cause for SCA2 and its clinical alternatives, and whether extra mutations exist to modify the phenotypic diversity. To address this, we have conducted fine genetic mapping and exome sequencing for a large Chinese SCA2 pedigree predominantly manifesting parkinsonism (called SCA2-P). In addition, we compared the CAG expansions between the SCA2-P and 16 SCA2 families presenting as pure ataxia (SCA2-A). As a result, CAG repeat expansions, ranging from 37 to 40 copies, were detected among 10 affected and 8 nonsymptomatic members of the SCA2-P family. The CAG repeats in the diseased alleles were interrupted by CAA in the 3′-end. In contrast, CAG expansion ranging from 36 to 54 without CAA interruption was detected in all probands of the SCA2-A families. Genetic mapping located the SCA2-P pedigree on 12q24.21, which spans the ATXN2 gene. Exome sequencing for 3 patients and 1 normal member revealed no extra mutations in this family. In addition, by genotyping single-nucleotide polymorphisms around SCA2 locus, we have excluded the existence of haplotypes predisposing different patterns of CAG expansion. These results demonstrate that the ATXN2 CAG expansion is the sole causative mutation responsible for SCA2-P, and that genetic modifiers may not be the major cause of the phenotypic diversity of SCA2.

Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Free‐energy computations identify the mutations required to confer trans‐sialidase activity into Trypanosoma rangeli sialidase

Trypanosoma rangeli's sialidase (TrSA) and Trypanosoma cruzi's trans-sialidase (TcTS) are members of the glycoside hydrolase family 33 (GH-33). They share 70% of sequence identity and their crystallographic Cα RMSD is 0.59 Å. Despite these similarities they catalyze different reactions. TcTS transfers sialic acid between glycoconjugates while TrSA can only cleave sialic acid from sialyl-glyconjugates. Significant effort has been invested into unraveling the differences between TrSA and TcTS, and into conferring TrSA with trans-sialidase activity through appropriate point mutations. Recently, we calculated the free-energy change for the formation of the covalent intermediate (CI) in TcTS and performed an energy decomposition analysis of that process. In this article we present a similar study for the formation of the CI in TrSA, as well as in a quintuple mutant (TrSA5mut), which has faint trans-sialidase activity. The comparison of these new results with those previously obtained for TcTS allowed identifying five extra mutations to be introduced in TrSA5mut that should create a mutant (TrSA10mut ) with high trans-sialidase activity.

Monitoring of therapy in patients with chronic hepatitis B virus

The aims of this study were to evaluate therapy with lamivudine (LAM) and adefovir dipivoxil (ADV) monotherapy in chronic hepatitis B virus (HBV)-infected patients with frequent measurements of DNA levels, to characterize HBV genotypes, and to determine the emergence of nucleos(t)ide analogue mutants before and during the therapy by direct-sequencing the reverse transcriptase region and by INNO-LiPA HBV DR v3. A total of 15 chronic HBV patients were analysed: 11 were treated with ADV and four were treated with LAM. Viral genotype was determined, showing the presence of genotype D (73%) in 11 patients and genotype A (27%) in four patients. In the viral response to treatment, three patients developed substitutions at rtM204I associated with LAM resistance and one of these patients presented rtM204V/I plus rtL180M mutation. In contrast, of the 11 patients treated with ADV, three patients developed mutations (rtN236T; rtA181V; rtA181V plus rtN236T). With regard to this case, the same results were observed by INNO-LiPA HBV DR v3 and direct sequencing, but by direct sequencing we detected an extra mutation rtQ215S that was present in two patients: one patient who was on treatment with LAM had an rtQ215S mutation in addition to an rtM204I, and the second patient treated with ADV had rtA181V. Direct sequence analysis is an essential tool to optimize therapeutic management of HBV chronic infection in clinical practice to choose the appropriate nucleos(t)ide analogues and to detect extra mutations that are not included in the commercial kit.

Characterization of sulphonamide-resistant Escherichia coli using comparison of sul2 gene sequences and multilocus sequence typing

The sul2 gene encodes sulphonamide resistance (Sul(R)) and is commonly found in Escherichia coli from different hosts. We typed E. coli isolates by multilocus sequence typing (MLST) and compared the results to sequence variation of sul2, in order to investigate the relation to host origin of pathogenic and commensal E. coli strains and to investigate whether transfer of sul2 into different genomic lineages has happened multiple times. Sixty-eight E. coli isolated in Denmark and Norway from different hosts and years were MLST typed and sul2 PCR products were sequenced and compared. PFGE was performed in a subset of isolates. All isolates were divided into 45 different sequence types (STs), with clonal complexes CC10, CC23, CC168, CC350 and CC69 being the most frequent. The sul2 gene from the majority of E. coli strains had only two point mutations, at positions 159 and 197, leading to a synonymous and a non-synonymous change, respectively. Five strains had extra single mutations. All poultry, poultry meat, and Danish human blood isolates had the same sul2 ST and some of these strains clustered under the same MLST STs, indicating that they shared habitats. Most PFGE profiles clustered according to source, but some included different sources. Sul(R) E. coli from different animals, food, human faeces and infections did not cluster according to their origin, suggesting that these habitats share E. coli and sul2 gene types. However, while pig isolates on one occasion clustered with urinary tract infection isolates, poultry isolates seemed more related to isolates from bloodstream infections in humans. Presence of mainly two types of the sul2 gene in both human and animal isolates, irrespective of date and geography, and the presence of both types in the same clonal lineages, suggest horizontal transfer of sul2.

Addressing Cumulative Selection

Addressing Cumulative Selection