Low-frequency Mutations Research Articles

Comparative Analysis of Within-Host Mutation Patterns and Diversity of Hepatitis C Virus Subtypes 1a, 1b, and 3a.

Understanding within-host evolution is critical for predicting viral evolutionary outcomes, yet such studies are currently lacking due to difficulty involving human subjects. Hepatitis C virus (HCV) is an RNA virus with high mutation rates. Its complex evolutionary dynamics and extensive genetic diversity are demonstrated in over 67 known subtypes. In this study, we analyzed within-host mutation frequency patterns of three HCV subtypes, using a large number of samples obtained from treatment-naïve participants by next-generation sequencing. We report that overall mutation frequency patterns are similar among subtypes, yet subtype 3a consistently had lower mutation frequencies and nucleotide diversity, while subtype 1a had the highest. We found that about 50% of genomic sites are highly conserved across subtypes, which are likely under strong purifying selection. We also compared within-host and between-host selective pressures, which revealed that Hyper Variable Region 1 within hosts was under positive selection, but was under slightly negative selection between hosts, which indicates that many mutations created within hosts are removed during the transmission bottleneck. Examining the natural prevalence of known resistance-associated variants showed their consistent existence in the treatment-naïve participants. These results provide insights into the differences and similarities among HCV subtypes that may be used to develop and improve HCV therapies.

A digital coding combination analysis for mutational genotyping using pyrosequencing.

In the present study, we developed a novel digital coding combination analysis (DCCA) to analyze the gene mutation based on the sample combination principle. The principle is that any numerically named sample is divided into two groups, any two samples are not grouped in the same two groups, and any sample can be tested within the detection limit. Therefore, we proposed a specific combination that N samples were divided into M groups. Then N samples were analyzed, which could obtain the mutation results of M mixed groups. If only two groups showed positive (mutant type) signals, the same sample number from two positive signal groups would be the positive sample, and the remaining samples were negative (wild type). If three groups or more exhibited positive results, the same sample number from three positive signal groups would be the positive sample. If some samples remained uncertain, individual samples could be analyzed on a small scale. In the present study, we used the two genotypes of a mutation site (A5301G) to verify whether it was a useful and promising method. The results showed that we could quantitatively detect mutations and demonstrate 100% consistent results against a panel of defined mixtures with the detection limit using pyrosequencing. This method was suitable, sensitive, and reproducible for screening and analyzing low-frequency mutation samples, which could reduce reagent consumption and cost by approximately 70-80% compared with conventional clinical methods.

Topological Data Analysis: A New Method to Identify Genetic Alterations in Cancer

Cancer is the largest health problem worldwide. A number of targeted therapies are currently employed for the treatment of different cancers. Determining the molecular mechanisms that are necessary for cancer development and progression is the most critical step in targeted therapies. Currently, many studies have identified a large number of frequently mutated cancer-associated genes using recurrence-based methods. However, only the cancer-associated mutations with a mutation frequency >15% can be identified by these methods. In other words, they cannot be used to identify driver genes that have low mutation frequency but play a major role in tumorigenesis and development. Thus, there is an urgent need for a method for identifying cancer-associated genes that are not based on recurrence. In a study, recently published in Nature Communications, research team led by Prof. Raúl Rabadán from the Columbia University successfully devised a novel topological data analysis approach to identify low-prevalence cancer-associated gene mutations using expression data from multiple cancers.

Distinct Somatic Alteration Features Identified by Gene Panel Sequencing in Korean Triple-Negative Breast Cancer with High Ki67 Expression.

This study aimed to clarify the genetic difference between Korean triple-negative breast cancer (TNBC) and other breast cancer (BC) subtypes. TNBC was defined as the absence of hormonal receptors and human epidermal growth factor receptor 2 (HER2) amplification. DNA panel of the Ion Torrent Oncomine Comprehensive Assay (OCA) v3 was performed to identify somatic alteration in 48 specimens. In a total of 102 alterations (37 nonsense, 35 missense, 8 frameshift and 22 amplifications), 30 nucleotide alterations (24 nonsense, 1 missense, and 5 frameshift) were newly identified. The eight most commonly altered genes were PIK3CA, TP53, ERBB2, BRCA2, FANCD2, AKT1, BRCA1, and FANCA. TNBC had significantly lower mutation frequency in PIK3CA (TNBC vs. hormone receptor-positive and HER2-negative BC [HRPBC], p = 0.009), but higher mutation frequency in TP53 (TNBC vs. HRPBC, p = 0.036; TNBC vs. hormone receptor-positive and HER2- positive BC [HHPBC], p = 0.004). TNBC showed frequently higher Ki-67 expression than any positive BC (p = 0.004) due to HRPBC (p < 0.001). TNBC with high Ki-67/unmutated PIK3CA/mutated TP53 appears at a younger age (52.2 ± 7.6 years), compared to other subtypes (63.7 ± 11.0 years). TNBC with high Ki-67/unmutated PIK3CA/mutated TP53 may be related to relatively early onset BCThese findings demonstrate the genomic heterogeneity between TNBC and other BC subtypes and could present a new approach for molecular targeted therapy in TNBC patients.

Predominance of HBV Genotype B and HDV Genotype 1 in Vietnamese Patients with Chronic Hepatitis.

Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; n = 115), liver cirrhosis (LC; n = 21), and hepatocellular carcinoma (HCC; n = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the “a” determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (n = 204) and 7% (n = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; n = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; n = 134), followed by HBV-C (31%; n = 64), HBV-D, and HBV-G (3%; n = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; n = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (n = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections.

A simple method to estimate the in-house limit of detection for genetic mutations with low allele frequencies in whole-exome sequencing analysis by next-generation sequencing

BackgroundNext-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency.ResultsAllele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%.ConclusionsFor properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

Stratum corneum markers of innate and T helper cell-related immunity and their relation to the disease severity in Croatian patients with atopic dermatitis.

Atopic dermatitis (AD) presents with the wide spectrum of clinical phenotypes within and between various populations. Recent study showed low frequency of filaggrin loss-of-function (FLG LOF) mutations in Croatian AD patients. At present, there are no data on biomarkers of immune response in Croatian AD patients that might be useful in the selection and monitoring of novel immune therapies. To investigate levels of cytokines of various signature in the stratum corneum (SC) collected from lesional and non-lesional skin of AD patients and healthy controls and to evaluate their relationship with the severity of disease and skin barrier function. SC samples were collected from 100 adult patients with moderate-to-severe AD and 50 healthy controls. The levels of 21 cytokines were measured by multiplex immunoassay. We conducted machine learning analysis to assess whether a small number of cytokine measurements can discriminate between healthy controls and AD patients and can predict AD severity (SCORAD). The SC levels of thirteen cytokines representing innate immunity, Th-1, Th-2 and Th-17/22 immune response showed significant differences between healthy and AD skin. Our analysis demonstrated that as few as three cytokines measured in lesional skin can discriminate healthy controls and AD with an accuracy of 99% and that the predictive models for SCORAD did not achieve a high accuracy. Cytokine levels were highly correlated with the levels of filaggrin degradation products and skin barrier function. Stratum corneum analysis revealed aberrant levels of cytokines representing innate immunity, Th-1-, Th-2- and Th-17/22-mediated immune response in Croatian AD patients. Increased Th-2 cytokines and their strong association with natural moisturizing factor (NMF) can explain low NMF levels despite of low frequency of FLG LOF mutations in Croatian population. Predictive models for SCORAD identified cytokines associated with SCORAD but warrants further investigation.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations

BackgroundMosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation.MethodsTo overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples.ResultsThe Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions.ConclusionsMIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq) is a new ultra-sensitive UMI-based NGS library preparation technology for use with cfDNA and cfRNA

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20–25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

Accurate, Efficient and User-Friendly Mutation Calling and Sample Identification for TILLING Experiments.

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetics method in plant functional genomics and breeding to identify mutagenized individuals with improved behavior for a trait of interest. Pooled high throughput sequencing (HTS) of the targeted genes allows efficient identification and sample assignment of variants within genes of interest in hundreds of individuals. Although TILLING has been used successfully in different crops and even applied to natural populations, one of the main issues for a successful TILLING experiment is that most currently available bioinformatics tools for variant detection are not designed to identify mutations with low frequencies in pooled samples or to perform sample identification from variants identified in overlapping pools. Our research group maintains the Next Generation Sequencing Experience Platform (NGSEP), an open source solution for analysis of HTS data. In this manuscript, we present three novel components within NGSEP to facilitate the design and analysis of TILLING experiments: a pooled variants detector, a sample identifier from variants detected in overlapping pools and a simulator of TILLING experiments. A new implementation of the NGSEP calling model for variant detection allows accurate detection of low frequency mutations within pools. The samples identifier implements the process to triangulate the mutations called within overlapping pools in order to assign mutations to single individuals whenever possible. Finally, we developed a complete simulator of TILLING experiments to enable benchmarking of different tools and to facilitate the design of experimental alternatives varying the number of pools and individuals per pool. Simulation experiments based on genes from the common bean genome indicate that NGSEP provides similar accuracy and better efficiency than other tools to perform pooled variants detection. To the best of our knowledge, NGSEP is currently the only tool that generates individual assignments of the mutations discovered from the pooled data. We expect that this development will be of great use for different groups implementing TILLING as an alternative for plant breeding and even to research groups performing pooled sequencing for other applications.

Low Frequency of Integrase Inhibitor Resistance Mutations Among Therapy-Naïve HIV Patients in Southeast China.

BackgroundWith the widespread use of integrase strand transfer inhibitors (INSTIs) in the clinical setting, transmission of INSTIs-resistance mutations may increase. Data regarding transmitted drug resistance mutations (TDRM) to INSTIs in Chinese HIV patients are limited. The aim of this study was to summarize the INSTIs TDRM, including the frequency of protease inhibitors (PIs) and reverse transcriptase (RT) inhibitors (RTIs) mutations in treatment-naïve patients in Southeast China.MethodsHIV-1 positive patients were retrospectively selected between April 2018 and October 2020 from the Mengchao Hepatobiliary Hospital of Fujian Medical University, the largest designated HIV/AIDS care hospital in Southeast China. Individuals who were antiretroviral therapy-naïve and received antiretroviral drug resistance testing at baseline were included. Clinical data including demographic data, CD4 counts, HIV-RNA loads, and drug resistance mutations were collected.ResultsA total of 147 patients were enrolled. INSTIs TDRM was rare, with only one primary integrase mutation E138K observed in one sample and one secondary mutation E157Q detected in another sample. The overall prevalence of INSTIs TDRM was 1.36%. A substantial proportion of patients harbored common INSTIs-associated polymorphic variants. Two samples harbored the T215S, M184V and K70E mutations related to nucleoside RTIs (NRTIs). Twelve patients carried nonnucleoside RTIs (NNRTIs)-resistance mutations. Two individuals harbored PIs-resistance mutations: Q58E in one patient and M46I, I54V, V82A, L10F, and Q58E mutations in another patient. The total TDRM rate for RTIs and PIs was 10.20% (15/147), but only 0.68% (1/147) was according to the WHO recommendations on TDRM.ConclusionThe rate of INSTIs TDRM was low among therapy-naïve HIV patients in Southeast China. INSTIs as a first-line regimen are suitable for untreated HIV-1 patients in Southeast China. But special attention must be still paid to INSTIs TDRM in clinical practice.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

BackgroundPatients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease.ConclusionsHemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Plant Mitochondria are a Riddle Wrapped in a Mystery Inside an Enigma.

A fundamental paradox motivates the study of plant mitochondrial genomics: the mutation rate is very low (lower than in the nucleus) but the rearrangement rate is high. A landmark paper published in Journal of Molecular Evolution in 1988 established these facts and revealed the paradox. Jeffrey Palmer and Laura Herbon did a prodigious amount of work in the pre-genome sequencing era to identify both the high frequency of rearrangements between closely related species, and the low frequency of mutations, observations that have now been confirmed many times by sequencing. This paper was also the first to use molecular data on rearrangements as a phylogenetic trait to build a parsimonious tree. The work was a technical tour-de-force, its findings are still at the heart of plant mitochondrial genomics, and the underlying molecular mechanisms that produce this paradox are still not completely understood.

Prognostic Implications of Pan-Cancer CMTM6 Expression and Its Relationship with the Immune Microenvironment.

CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) reportedly stabilizes programmed death-ligand 1 (PD-L1) and enhances the efficacy of immunotherapy. However, correlations between CMTM6 expression and the immune microenvironment and its prognostic value remain unknown in a variety of tumors. CMTM6 expression data were obtained from The Cancer Genome Atlas (TCGA) for 33 cancer types classified into high and low expression subgroups according to the median CMTM6 expression value. Pan-cancer analysis of CMTM6 protein expression in 20 tumor types was performed using a cohort from the Human Protein Atlas (HPA). PD-L1 protein expression data were obtained from The Cancer Proteome Atlas (TCPA) for 32 cancer types. Frequencies of CMTM6 copy number alterations and mutations were analyzed using cBioPortal. MANTIS was employed to estimate microsatellite instability in the TCGA cohort. CIBERSORT and the ESTIMATE algorithm were applied to estimate the relative fractions of infiltrating immune cell types and immune scores, respectively. Kaplan–Meier survival curve analysis was performed to assess the pan-cancer prognostic value of CMTM6.CMTM6 is heterogeneously expressed in diverse cancers. Further, the results revealed low CMTM6 mutation frequencies in multiple cancers. Among them, CMTM6 mutation frequency was the highest in uterine cancer. Additionally, CMTM6 expression was related to PD-L1 protein expression in breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, glioblastoma multiforme (GBM), head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma, sarcoma (SARC), stomach adenocarcinoma, and uterine carcinosarcoma. Increased CMTM6 expression may be associated with increased infiltration of neutrophils in some types of cancer. Finally, pan-cancer analysis indicated that CMTM6 expression was closely related to overall survival in adrenocortical carcinoma, GBM, acute myeloid leukemia, liver hepatocellular carcinoma, mesothelioma, SARC, thymoma, and uveal melanoma. Taken together, these findings highlight that CMTM6 plays an important role in the tumor immune microenvironment, and CMTM6 has been identified to have prognostic value in some types of cancers. Thus, CMTM6 is a potential target for cancer immunotherapy and effective prognostic biomarker.

V-pipe: a computational pipeline for assessing viral genetic diversity from high-throughput data.

MotivationHigh-throughput sequencing technologies are used increasingly not only in viral genomics research but also in clinical surveillance and diagnostics. These technologies facilitate the assessment of the genetic diversity in intra-host virus populations, which affects transmission, virulence and pathogenesis of viral infections. However, there are two major challenges in analysing viral diversity. First, amplification and sequencing errors confound the identification of true biological variants, and second, the large data volumes represent computational limitations.ResultsTo support viral high-throughput sequencing studies, we developed V-pipe, a bioinformatics pipeline combining various state-of-the-art statistical models and computational tools for automated end-to-end analyses of raw sequencing reads. V-pipe supports quality control, read mapping and alignment, low-frequency mutation calling, and inference of viral haplotypes. For generating high-quality read alignments, we developed a novel method, called ngshmmalign, based on profile hidden Markov models and tailored to small and highly diverse viral genomes. V-pipe also includes benchmarking functionality providing a standardized environment for comparative evaluations of different pipeline configurations. We demonstrate this capability by assessing the impact of three different read aligners (Bowtie 2, BWA MEM, ngshmmalign) and two different variant callers (LoFreq, ShoRAH) on the performance of calling single-nucleotide variants in intra-host virus populations. V-pipe supports various pipeline configurations and is implemented in a modular fashion to facilitate adaptations to the continuously changing technology landscape.Availabilityand implementationV-pipe is freely available at https://github.com/cbg-ethz/V-pipe.Supplementary information Supplementary data are available at Bioinformatics online.

Detection of Low-Frequency KRAS Mutations in cfDNA From EGFR-Mutated NSCLC Patients After First-Line EGFR Tyrosine Kinase Inhibitors.

BackgroundMolecular profiling of advanced EGFR mutated NSCLC has recently demonstrated the co-existence of multiple genetic alterations. Specifically, co-existing KRAS-mutations in EGFR NSCLCs have been described, despite their prevalence at progression and their role in the response to EGFR tyrosine kinase inhibitors (TKIs) remain marginally explored. Aim of our study was to investigate the prevalence of co-existing KRAS mutations at the time of progressive disease and explore their impact on clinical outcome.Materials and MethodsWe retrospectively analyzed by digital droplet PCR prevalence of KRAS co-mutations in 106 plasma samples of EGFR mutated NSCLC patients, in progressive disease after EGFR TKI treatment as first-line therapy.Results KRAS co-mutations (codon 12 and 13) were identified in 3 patients (2.8% of analyzed samples), with low allelic frequency (<0.2%), and had a negative impact on clinical outcome to first-line EGFR TKI.ConclusionDetection of KRAS mutations in cell-free DNA of EGFR mutant NSCLC patients at progression after first or second generation EGFR TKI is a rare event. Due to their low abundance, the negative impact of KRAS mutations on the response to EGFR TKI remains to be confirmed in larger studies.

Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice

DNA methylation regulates gene transcription and is involved in various physiological processes in mammals, including development and hematopoiesis. It is catalyzed by DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b. For Dnmt3b, its effects on transcription can result from its own DNA methylase activity, the recruitment of other Dnmts to mediate methylation, or transcription repression in a methylation-independent manner. Low-frequency mutations in human DNMT3B are found in hematologic malignancies including cutaneous T-cell lymphomas, hairy cell leukemia, and diffuse large B-cell lymphomas. Moreover, Dnmt3b is a tumor suppressor in oncogene-driven lymphoid and myeloid malignancies in mice. However, it is poorly understood how the different Dnmt3b activities contribute to these outcomes. We modulated Dnmt3b activity in vivo by generating Dnmt3b+/− mice expressing one wild-type allele as well as Dnmt3b+/CI and Dnmt3bCI/CI mice where one or both alleles express catalytically inactive Dnmt3bCI. We show that 43% of Dnmt3b+/− mice developed T-cell lymphomas, chronic lymphocytic leukemia, and myeloproliferation over 18 months, thus resembling phenotypes previously observed in Dnmt3a+/− mice, possibly through regulation of shared target genes. Interestingly, Dnmt3b+/CI and Dnmt3bCI/CI mice survived postnatal development and were affected by B-cell rather than T-cell malignancies with decreased penetrance. Genome-wide hypomethylation, increased expression of oncogenes such as Jdp2, STAT1, and Trip13, and p53 downregulation were major events contributing to Dnmt3b+/− lymphoma development. We conclude that Dnmt3b catalytic activity is critical to prevent B-cell transformation in vivo, whereas accessory and methylation-independent repressive functions are important to prevent T-cell transformation.

Frequency of BMP15 and GDF9 mutations increasing litter size and their phenotypic effects in Olkuska sheep population

Abstract Two mutations affecting the ovulation rate and litter size are segregating in Olkuska sheep population, FecXO in the BMP15 gene, and the G7 site mutation in GDF9 gene. Homozygous carriers of both mutations are hyperprolific, contrary to the sterility observed in homozygous carriers of most other BMP15 and GDF9 mutations. The objective of this study was to assess frequency and phenotypic effects of both mutations. Blood samples were obtained from 740 individuals, 111 rams and 629 ewes, out of which 91 rams and 561 ewes were successfully genotyped for the BMP15 and GDF9 loci. The reproductive performance included a number of lambs born/born alive and a number of lambs reared until 60 days of age, and for a subset of ewe ovulation rates. The study proved a high frequency of the FecXO mutation in two flocks that have been selected for many years for increased litter size (0.7–1.0 in breeding ewes and rams respectively), and a moderate frequency in another 19 private flocks (0.4–0.5). The frequency of the GDF9 mutation was low, with only 50 sheep out of 312 genotyped being carriers of the GDF9/G7 mutation, including three homozygous carriers. The FecXO mutation in the BMP15 had a significant effect on both litter size and the ovulation rate. The single copy in heterozygous carriers increased litter size by 0.255 (0.063), while the effect of two copies in homozygous genotypes was +0.874 (0.081) lambs born. Due to the low frequency of the GDF9 mutation, it can only be preliminarily concluded that litter size has been increased in double carriers of both the BM15 and GDF9 mutation, which may suggest their additive interaction. The positive effect of both mutations supports their direct use in selection programmes.

Expanded Low Allele Frequency RAS and BRAF V600E Testing in Metastatic Colorectal Cancer as Predictive Biomarkers for Cetuximab in the Randomized CO.17 Trial

Expanded RAS/BRAF mutations have not been assessed as predictive for single-agent cetuximab in metastatic colorectal cancer (mCRC), and low mutant allele frequency (MAF) mutations are of unclear significance. We aimed to establish cetuximab efficacy in optimally selected patients using highly sensitive beads, emulsion, amplification, and magnetics (BEAMing) analysis, capable of detecting alterations below standard clinical assays. CO.17 trial compared cetuximab versus best supportive care (BSC) in RAS/BRAF-unselected mCRC. We performed RAS/BRAF analysis on microdissected tissue of 242 patients in CO.17 trial using BEAMing for KRAS/NRAS (codons 12/13/59/61/117/146) and BRAF V600E. Patients without BEAMing but with previous Sanger sequencing-detected mutations were included. KRAS, NRAS, and BRAF mutations were present in 53%, 4%, and 3% of tumors, respectively. Cetuximab improved overall survival [OS; HR, 0.51; 95% confidence interval (CI), 0.32-0.81; P = 0.004] and progression-free survival (PFS; HR, 0.25; 95% CI, 0.15-0.41; P < 0.0001) compared with BSC in RAS/BRAF wild-type patients. Cetuximab did not improve OS/PFS for KRAS-, NRAS-, or BRAF-mutated tumors, and tests of interaction confirmed expanded KRAS (P = 0.0002) and NRAS (P = 0.006) as predictive, while BRAF mutations were not (P = 0.089). BEAMing identified 14% more tumors as RAS mutant than Sanger sequencing, and cetuximab lacked activity in these patients. Mutations at MAF < 5% were noted in 6 of 242 patients (2%). One patient with a KRAS A59T mutation (MAF = 2%) responded to cetuximab. More NRAS than KRAS mutations were low MAF (OR, 20.50; 95% CI, 3.88-96.85; P = 0.0038). We establish single-agent cetuximab efficacy in optimally selected patients and show that subclonal RAS/BRAF alterations are uncommon and remain of indeterminate significance.

Analysis of Serial Multidrug-Resistant Tuberculosis Strains Causing Treatment Failure and Within-Host Evolution by Whole-Genome Sequencing.

The cure rate of multidrug-resistant tuberculosis (MDR-TB) is relatively low in China. The reasons for the treatment failure and within-host evolution during treatment have not been sufficiently studied. All MDR-TB patients receiving standard treatment from January 2014 to September 2016 at a designated TB Hospital in Zhejiang Province were retrospectively included and grouped according to their known treatment outcome. Clinical information was collected. Baseline strains of all patients and serial strains of treatment-failure patients were revived. Drug susceptibility tests (DSTs) of 14 drugs and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were performed. The genetic distance and within-host evolution were investigated based on SNPs. In total, 20 treatment failure patients and 74 patients who succeeded in treatment were included. The number of effective drugs for patients who failed treatment was no more than three. Eighteen (90.0%) treatment-failure patients were characterized by a continuous infection of the primary strain, of which 14 patients (77.8%) developed phenotypic or genotypic acquired drug resistance under ineffective treatment. Acquired resistance to amikacin and moxifloxacin (2.0 mg/ml) was detected most frequently, in 5 and 4 patients, respectively. The insufficient number of effective drugs in the combined treatment regimen was the main reason for MDR-TB treatment failure. The study emphasizes the importance of DST for second-line drugs when implementing the second-line drug regimen in MDR-TB patients. For patients with risk factors for MDR-TB, DST of second-line antituberculosis drugs should be performed at initiation of treatment. Second-line drugs should be selected based on the results of DST to avoid acquired resistance. WGS detects low-frequency resistance mutations and heterogeneous resistance with high sensitivity, which is of great significance for guiding clinical treatment and preventing acquired resistance.IMPORTANCE Few studies have focused on the reasons for the low cure rate of multidrug-resistant tuberculosis in China and within-host evolution during treatment, which is of great significance for improving clinical treatment regimens. Acquired resistance events were common during the ineffective treatment, among which resistance to amikacin and high-level moxifloxacin were the most common. The main reason for the treatment failure of MDR-TB patients was insufficient effective drugs, which may lead to higher levels of drug resistance in MDR-TB strains. Therefore, the study emphasizes the importance of DST in the development of second-line treatment regimen when there is a risk of MDR. By performing whole-genome sequencing of serial strains from patients with treatment failure, we found that WGS can detect low-frequency resistance mutations and heterogeneous resistance with high sensitivity. It is thus recommended to conduct drug susceptibility tests at the beginning of treatment and repeat the DST when the sputum bacteria remain positive.