Low-level Mutations Research Articles

Ultra-Low Level Somatic Mutations and Structural Variations in Focal Cortical Dysplasia Type II.

Brain somatic mutations in mTOR pathway genes are a major genetic etiology of focal cortical dysplasia type II (FCDII). Despite a greater ability to detect low-level somatic mutations in the brain by deep sequencing and analytics, about 40% of cases remain genetically unexplained. We included 2 independent cohorts consisting of 21 patients with mutation-negative FCDII without apparent mutations on conventional deep sequencing of bulk brain. To find ultra-low level somatic variants or structural variants, we isolated cells exhibiting phosphorylation of the S6 ribosomal protein (p-S6) in frozen brain tissues using fluorescence-activated cell sorting (FACS). We then performed deep whole-genome sequencing (WGS; >90×) in p-S6+ cells in a cohort of 11 patients with mutation-negative. Then, we simplified the method to whole-genome amplification and target gene sequencing of p-S6+ cells in independent cohort of 10 patients with mutation-negative followed by low-read depth WGS (10×). We found that 28.6% (6 of 21) of mutation-negative FCDII carries ultra-low level somatic mutations (less than 0.2% of variant allele frequency [VAF]) in mTOR pathway genes. Our method showed ~34 times increase of the average mutational burden in FACS mediated enrichment of p-S6+ cells (average VAF=5.84%) than in bulky brain tissues (average VAF=0.17%). We found that 19% (4 of 21) carried germline structural variations in GATOR1 complex undetectable in whole exome or targeted gene sequencing. Our method facilitates the detection of ultra-low level somatic mutations, in specifically p-S6+ cells, and germline structural variations and increases the genetic diagnostic rate up to ~80% for the entire FCDII cohort. ANN NEUROL 2023;93:1082-1093.

Comparative transcriptomics and genomics from continuous axenic media growth identifies Coxiella burnetii intracellular survival strategies.

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a 'reverse evolution' approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (Sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic, and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.

Enzymatic Methods for Mutation Detection in Cancer Samples and Liquid Biopsies.

Low-level tumor somatic DNA mutations in tissue and liquid biopsies obtained from cancer patients can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up, or early cancer detection. Unless detected, such low-frequency DNA alterations can misinform patient management decisions or become missed opportunities for personalized medicine. Next-generation sequencing technologies and digital-PCR can resolve low-level mutations but require access to specialized instrumentation, time, and resources. Enzymatic-based approaches to detection of low-level mutations provide a simple, straightforward, and affordable alternative to enrich and detect such alterations and is broadly available to low-resource laboratory settings. This review summarizes the traditional uses of enzymatic mutation detection and describes the latest exciting developments, potential, and applications with specific reference to the field of liquid biopsy in cancer.

Laser microdissection for the analysis of molecular heterogeneity in colorectal cancer

Oncogenic driver mutations in RAS/RAF oncogenes are frequent in colorectal cancer (CRC). The presence of different subclones within asingle tumor can lead to treatment failure in anti-EGFR/epidermal growth factor receptor-directed antibody therapies. The identification of different subclones and their mutational profiles within asingle tumor and the identification of morphologically distinct tumor areas might help to unravel novel aspects of tumor biology and therapy resistance. The aim of this study was to identify intratumoral heterogeneity in CRC by using laser microdissection (LMD) in comparison to the routinely used method. We hereby applied LMD to identify and investigate tumor heterogeneity in CRC. We established LMD and purified DNA from several morphologically distinct tumor areas (n = 13) in CRCs from 2patients and compared the results from routine testing to our newly established LMD approach. LMD enabled the comparative analysis of small tumor areas by cutting histologically selected elements under microscopic control using alaser beam. In some cases, potential low-level mutations (PLLM) could not be detected using the routine method since they were masked by high-level mutations (HLM). The application of LMD enabled the identification of concomitant PLLM in NRAS and BRAF genes in the identical patient sample. LMD improved spatial resolution in the molecular analysis of CRC tumor tissue compared to routine methods. Our results confirmed the presence of molecular heterogeneity in CRC. This should be kept in mind when interpreting sequencing results, since low frequency mutations can have an impact on the effectiveness of targeted therapy.

Recent Developments in Mutation Enrichment and Detection Technologies.

Presence of excess unaltered, wild-type DNA (wtDNA) providing information of little clinical value may often mask low-level mutations containing important diagnostic or therapeutic clues. This is a recurring hurdle in biotechnology and medicine, including cancer, prenatal diagnosis, infectious diseases, and organ transplantation. Mutation enrichment techniques that allow reduction of unwanted DNA to enable the detection of low-level mutations have emerged since the early 1990s. They are continuously being refined and updated with new technologies. The burgeoning interest in liquid biopsies for residual cancer monitoring, detection of resistance to therapy, and early cancer detection has driven an expanded interest in new and improved methodologies for practical and effective mutation enrichment and detection of low-level mutations of clinical relevance. Newly developed mutation enrichment technologies are described and grouped according to the main principle of operation, PCR-blocking technologies, enzymatic methods, and physicochemical approaches. Special emphasis is given to technologies enabling pre-PCR blockage of wtDNA to bypass PCR errors [nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO) and UV-mediated cross-linking minor allele enrichment (UVME)] or providing high multiplexity followed by next-generation sequencing [Minor allele enriched sequencing through recognition oligonucleotides (MAESTRO)]. This review summarizes technological developments in rare mutation enrichment over the last 12 years, complementing pre-2010 reviews on this topic. The expanding field of liquid biopsy calls for improved limits of detection (LOD) and highly parallel applications, along with the traditional requirements for accuracy, speed, and cost-effectiveness. The current technologies are reviewed with regards to these new requirements.

Analysis of low-level somatic mosaicism reveals stage and tissue-specific mutational features in human development.

Most somatic mutations that arise during normal development are present at low levels in single or multiple tissues depending on the developmental stage and affected organs. However, the effect of human developmental stages or mutations of different organs on the features of somatic mutations is still unclear. Here, we performed a systemic and comprehensive analysis of low-level somatic mutations using deep whole-exome sequencing (average read depth ~500×) of 498 multiple organ tissues with matched controls from 190 individuals. Our results showed that early clone-forming mutations shared between multiple organs were lower in number but showed higher allele frequencies than late clone-forming mutations [0.54 vs. 5.83 variants per individual; 6.17% vs. 1.5% variant allele frequency (VAF)] along with less nonsynonymous mutations and lower functional impacts. Additionally, early and late clone-forming mutations had unique mutational signatures that were distinct from mutations that originated from tumors. Compared with early clone-forming mutations that showed a clock-like signature across all organs or tissues studied, late clone-forming mutations showed organ, tissue, and cell-type specificity in the mutation counts, VAFs, and mutational signatures. In particular, analysis of brain somatic mutations showed a bimodal occurrence and temporal-lobe-specific signature. These findings provide new insights into the features of somatic mosaicism that are dependent on developmental stage and brain regions.

Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia.

Mutations of the nucleophosmin1 (NPM1) gene represent the most frequent molecular alteration in acute myelogenous leukemia (AML), especially in patients with AML who have a normal karyotype. These alterations have been shown to carry favorable prognostic significance in patients with AML. Several methods have been developed for detection of NPM1 gene mutations. However, their ability to detect low levels of mutations in a wild-type background is limited. In this study, the Enhance improved and complete enrichment Co-amplification at Lower Denaturation temperature Polymerase Chain Reaction (E-ice-COLD-PCR) assay combined with High Resolution Melting (HRM) analysis was developed and validated for highly specific and sensitive screening for NPM1 gene mutations. A total of 83 blood samples from patients with AML were collected, and their DNA was extracted. For mutational analysis, the E-ice-COLD-PCR assay for the detection of NPM1 gene mutations was developed. PCR products were analyzed by HRM analysis. All positive samples were confirmed by direct sequencing. This assay enabled detection specificity and sensitivity of NPM1 mutations in 9/83 patients with AML. Direct sequencing results were 100% concordant with this method. In addition, the limit of detection was 12.5% mutant in the final concentration of 5 ng genomic DNA. The E-ice-COLD-PCR assay with HRM analysis is a highly specific and sensitive screening method for enrichment of detecting NPM1 gene mutations. This method has both a short turn around time and easier interpretation compared to those of other methods.

Mosaic GLUD1 Mutations Associated with Hyperinsulinism Hyperammonemia Syndrome

Introduction: The hyperinsulinemia-hyperammonemia syndrome (HIHA) is the second most common cause of congenital hyperinsulinism and is caused by activating heterozygous missense mutations in GLUD1. In the majority of HIHA cases, the GLUD1 mutation is found to be de novo. We have identified 3 patients in whom clinical evaluation was suggestive of HIHA but with negative mutation analysis in peripheral blood DNA for GLUD1 as well as other known HI genes. Methods: We performed next-generation sequencing (NGS) on peripheral blood DNA from two children with clinical features of HIHA in order to look for mosaic mutations in GLUD1. Pancreas tissue was also available in one of these cases for NGS. In addition, NGS was performed on peripheral blood DNA from a woman with a history of HI in infancy whose child had HIHA due to a presumed de novo GLUD1 mutation. Results: Mosaic GLUD1 mutations were identified in these 3 cases at percent mosaicism ranging from 2.7% to 10.4% in peripheral blood. In one case with pancreas tissue available, the mosaic GLUD1 mutation was present at 17.9% and 28.9% in different sections of the pancreas. Two unique GLUD1 mutations were identified in these cases, both of which have been previously reported (c.1493c>t/p.Ser445Leu and c.820c>t/p.Arg221Cys). Conclusion: The results suggest that low-level mosaic mutations in known HI genes may be the underlying molecular mechanism in some children with HI who have negative genetic testing in peripheral blood DNA.

A systematic pan-cancer analysis of the gasdermin (GSDM) family of genes and their correlation with prognosis, the tumor microenvironment, and drug sensitivity.

Background: Pyroptosis is a programmed cell death process mediated by the gasdermin (GSDM) protein. However, limited research has been conducted to comprehensively analyze the contribution of the GSDM family in a pan-cancer setting. Methods: We systematically evaluated the gene expression, genetic variations, and prognostic values of the GSDM family members. Furthermore, we investigated the association between the expression of GSDM genes and immune subtypes, the tumor microenvironment (TME), the stemness index, and cancer drug sensitivities by means of a pan-cancer analysis. Results: GSDM genes were highly upregulated in most of the tested cancers. Low-level mutation frequencies within GSDM genes were common across the examined types of cancer, and their expression levels were associated with prognosis, clinical characteristics, TME features, and stemness scores in several cancer types, particularly those of the urinary system. Importantly, we found that the expressions of GSDMB, GSDMC, and GSDMD were higher in kidney carcinomas, and specifically kidney renal clear cell carcinoma (KIRC); which adversely impacted the patient outcome. We showed that GSDMD was potentially the most useful biomarker for KIRC. The drug sensitivity analysis demonstrated that the expressions of GSDM genes were correlated with the sensitivity of tumor cells to treatment with chemotherapy drugs nelarabine, fluphenazine, dexrazoxane, bortezomib, midostaurin, and vincristine. Conclusion: GSDM genes were associated with tumor behaviors and may participate in carcinogenesis. The results of this study may therefore provide new directions for further investigating the role of GSDM genes as therapeutic targets in a pan-cancer setting.

Performance of ImproGene cfDNA blood collection tubes for mutation analysis in cancer patients

With the widely application of liquid biopsy and the development of detection technology, the standardization of pre-analysis procedures is necessary. For controlling pre-analysis variation of circulating tumor DNA (ctDNA) in blood samples, the blood collection tubes for ctDNA preservation particularly contribute a lot. The objective of this study was to investigate whether ImproGene® Cell Free DNA Tube (ImproGene tube) can be used in sample collection, preservation and NGS based mutation detection for ctDNA. We investigated hemolysis and cell free DNA (cfDNA) concentration of blood samples stored in ImproGene tubes and detected β-actin, LINE1 and exogenous gene level by qPCR. We compared cfDNA and RNA quantity between samples in ImproGene tube and Streck Cell-Free DNA BCT® (Streck tube). And 10 gene mutations and three fusion mutations analysis were compared by sequencing. When stored at room temperature within 7 days in ImproGene tubes, blood samples had no visible hemolysis and the cfDNA concentration, levels of β-actin, LINE1 and exogenous gene remained stable which means no genomic DNA release and cfDNA was protected. There was no significant difference in cfDNA and RNA quantity between ImproGene tubes and Streck tubes. Furthermore, based on this limited data set, ImproGene tubes showed increased detection rates of low-level mutations. Therefore, ImproGene Cell Free DNA Tubes may have promising applications in sample collection, preservation and NGS based mutation detection for ctDNA by its good preservation performance.

Dynamics of the most common pathogenic mtDNA variant m.3243A > G demonstrate frequency-dependency in blood and positive selection in the germline.

The A-to-G point mutation at position 3243 in the human mitochondrial genome (m.3243A > G) is the most common pathogenic mtDNA variant responsible for disease in humans. It is widely accepted that m.3243A > G levels decrease in blood with age, and an age correction representing ~ 2% annual decline is often applied to account for this change in mutation level. Here we report that recent data indicate that the dynamics of m.3243A > G are more complex and depend on the mutation level in blood in a bi-phasic way. Consequently, the traditional 2% correction, which is adequate 'on average', creates opposite predictive biases at high and low mutation levels. Unbiased age correction is needed to circumvent these drawbacks of the standard model. We propose to eliminate both biases by using an approach where age correction depends on mutation level in a biphasic way to account for the dynamics of m.3243A > G in blood. The utility of this approach was further tested in estimating germline selection of m.3243A > G. The biphasic approach permitted us to uncover patterns consistent with the possibility of positive selection for m.3243A > G. Germline selection of m.3243A > G shows an 'arching' profile by which selection is positive at intermediate mutant fractions and declines at high and low mutant fractions. We conclude that use of this biphasic approach will greatly improve the accuracy of modelling changes in mtDNA mutation frequencies in the germline and in somatic cells during aging.

Localized islet nuclear enlargement hyperinsulinism (LINE-HI) due to ABCC8 and GCK mosaic mutations.

Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in children. In addition to typical focal or diffuse HI, some cases with diazoxide-unresponsive congenital HI have atypical pancreatic histology termed Localized Islet Nuclear Enlargement (LINE) or mosaic HI, characterized by histologic features similar to diffuse HI, but confined to only a region of pancreas. Our objective was to characterize the phenotype and genotype of children with LINE-HI. The phenotype and genotype features of 12 children with pancreatic histology consistent with LINE-HI were examined. We compiled clinical features of 12 children with LINE-HI and performed next-generation sequencing on specimens of pancreas from eight of these children to look for mosaic mutations in genes known to be associated with diazoxide-unresponsive HI (ABCC8, KCNJ11, and GCK). Children with LINE-HI had lower birth weights and later ages of presentation compared to children with typical focal or diffuse HI. Partial pancreatectomy in LINE-HI cases resulted in euglycemia in 75% of cases; no cases have developed diabetes. Low-level mosaic mutations were identified in the pancreas of six cases with LINE-HI (three in ABCC8, three in GCK). Expression studies confirmed that all novel mutations were pathogenic. These results indicate that post-zygotic low-level mosaic mutations of known HI genes are responsible for some cases of LINE-HI that lack an identifiable germ-line mutation and that partial pancreatectomy may be curative for these cases.

Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications.

Simple SummaryLiquid biopsies provide a non-invasive approach to tracing tumor-derived biomarkers in blood, with broad applications in medicine. During the past decade, circulating free DNA (cfDNA) has been turned into an informative resource for cancer management. Mutation-enrichment methods enhance the detection of tumor-derived, low-level mutations in blood. These methods increase the frequency of low-level mutations insofar as they become detectable via routine diagnostic techniques. Enriching mutations prior to PCR (pre-PCR) offers distinctive advantages. Applying the mutation-enrichment process directly to genomic DNA or cfDNA circumvents PCR errors and provides enriched mutation-containing products that different technologies can detect without any required changes in their protocols. In this review, we discuss the recent developments in pre-PCR enrichment methods from the perspective of their applications in liquid biopsies.Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies.

Combining cell-free RNA (cfRNA) with cell-free total nucleic acid (cfTNA) as a new paradigm for liquid biopsy.

3048 Background: Expressed RNA can capture mutations, gene fusions, and biomarker profiles. In principle, each abnormal cell has one copy of mutated gene, but numerous copies of mutated RNA. Cell-free RNA (cfRNA) is not used due to the assumption that it is degraded. Next Generation Sequencing (NGS) by design is particularly adaptable for fragmented DNA and RNA. We developed an approach to isolate cell-free total nucleic acid (cfTNA) and cell-free RNA (cfRNA) from peripheral blood. Using targeted sequencing, we explored the potential of this approach to detect mutations, fusion mRNA, and copy number variation (CNV) in solid tumors and hematologic neoplasms. Methods: Peripheral blood cfTNA and cfRNA were extracted from B-cell lymphoid neoplasms (#105), T-cell neoplasms (#16), Myeloid neoplasms (#73), solid tumors (#44), and Normal individuals (#51), and sequenced using a targeted panel of 1459 genes. Results: Numbers of mutations detected in solid tumors and hematologic neoplasms were significantly (P > 0.0001) higher in cfRNA (No. = 1229) than in cfTNA (No. = 1004). Overall variant allele frequency (VAF) was significantly higher in cfRNA than in cfTNA (P < 0.0001). However, numerous mutations detected by RNA were not detected by cfTNA and vice versa. In general, nonsense mutations were more likely to be detected by cfTNA than by cfRNA and at higher VAF. Low-level mutations (VAF < 10%) were more likely to be detected by cfRNA than by cfTNA. For example, 136 mutations in TP53 gene were detected using cfRNA and only 70 mutations were detected in cfTNA. KRAS mutations were also higher in cfRNA (#33) as compared with cfTNA (#21). In contrast, when most of the mutations were nonsense, as in ASXL1 gene, more mutations were detected by cfTNA (24 vs 23). When mutations were detected in both cfRNA and cfTNA, mutation load (level of mutant copies) was overall slightly higher in cfTNA (P = 0.06), likely due to higher degradation of RNA, but varied significantly dependent on the type of mutated gene and type of mutation. cfRNA was reliable in detecting fusion transcripts in solid tumors and in hematologic neoplasms (SLC34A2-ROS1, DDX5-BCL6, ETV6-RUNX1, RUNX1T1-RUNX1, PML-RARA, RUNX1-ZFPM2, DEK-NUP214, EP300-ZNF384) irrespective of the breakpoint or partner gene. The cfTNA detected various CNVs expected by cytogenetic analysis when tumor fraction was adequate (VAF > 10%). Conclusions: This data demonstrates that using cfRNA and cfTNA provides complementary comprehensive information for evaluating mutations, fusion genes, and CNV. This approach increased sensitivity and reliability of liquid biopsy. Furthermore, the cfRNA provides critical information on relative expression of various genes that can be used as biomarkers in characterizing the neoplastic process (see ASCO abstract, Liquid Biopsy Based on Cell-Free RNA and Biomarkers profiling of hematologic and solid tumors).

Identification of Mutations Associated With Macozinone-Resistant in Mycobacterium Tuberculosis.

Novel anti-tuberculosis drug macozinone (MCZ) is identified as a drug candidate and is currently under clinical development for the treatment of tuberculosis. However, the mutations conferring resistance to MCZ remain inadequately characterized. In this study we investigated resistant mutations to the MCZ through de novo resistance selection in vitro. This was accomplished by passing the Mycobacterium tuberculosis H37Rv on solid agar plates, which were infused with the antibiotic. Through whole genome sequencing and targeted PCR genomic resistance conferring SNPs were catalogued, and the resistant phenotype was analyzed by MABA test. Of MCZ-resistant clones obtained in vitro the mutation of C387S in dprE1 is only observed in high-level resistant clones (MIC90 > 500ng/mL) indicating that C387S mutation is directly related to high-level MCZ-resistance. In addition, high-level resistance to MCZ can occur in clone grew on agar plates infused with low concentration MCZ, which means low concentration MCZ induction can produce high-level drug-resistance and it is easy to produce high-level MCZ-resistance. We also found two new mutations (G61A and G248A) in dprE1 for the first time. The MIC90 of other clones except the clones carrying the C387S mutation was at the same level (20ng/mL > MIC90 > 2ng/mL). Of low-level resistant clones other gene mutations involved in drug efflux or membrane permeability were found (pepQ, Rv0678, arsC, etc.), with highest mutation frequency in Rv0678 (50/64, 78.12%). It suggests that there may be new mechanisms independent of dprE1 mutations.

Postmortem Diagnosis of the Proteus Syndrome by Next Generation Sequencing of Affected Brain Tissue.

We report a case of a somatic overgrowth syndrome diagnosed at forensic autopsy with the aid of next generation sequencing as Proteus syndrome. Somatic overgrowth syndromes result from spontaneous somatic mutations that arise early in development and display a mosaic pattern of expression in patient tissues. Due to the temporal and anatomic heterogeneity of these syndromes, phenotypes vary widely, resulting in clinical overlap. Furthermore, the variable ratio of mutated to nonmutated cells in patient tissue can result in low-level mutations that could be missed using Sanger sequencing. Due to these factors, recent literature points to next generation sequencing (NGS) as an adjunct to diagnosis of these rare entities. A male in his fourth decade of life presented to our forensic autopsy service with physical features suggestive of a somatic overgrowth syndrome. Due to the paucity of clinical information accompanying the individual, a definitive diagnosis based on physical characteristics, alone, was not possible. Next generation sequencing of affected formalin-fixed and paraffin-embedded brain tissue confirmed the presence of the variant in AKT1 (c.49G>A, p.Glu17Lys, in 14.13% of reads) found in Proteus syndrome. To our knowledge, this is the first report of the mosaic variant of AKT1 detected in brain tissue and the first reported case of a postmortem diagnosis of Proteus syndrome with the aid of NGS. We conclude that NGS can be used as an adjunctive method to support a specific diagnosis among the somatic overgrowth syndromes postmortem in the absence of sufficient clinical history.

Trends in rifampicin and isoniazid resistance in patients with presumptive TB.

BACKGROUND: Early diagnosis of drug-resistant TB (DR-TB) is crucial in preventing the spread of the disease in the community. Introduction of upfront decentralised drug susceptibility testing to district-level as part of universal drug susceptibility testing (UDST) policy increased the feasibility of rapid and early testing for drug resistance closer to the patient and has resulted in reduced circumstances for transmission. The introduction of the first-line line-probe assay (FL-LPA), GenoType® MTBDRplus v2, has had an extensive impact on the management of multidrug-resistant TB (MDR-TB) in India.MATERIALS and METHODS: Sputum samples of patients with presumptive TB and DR-TB from selected districts of Tamil Nadu received through National TB Elimination Programme (NTEP) were subjected to FL-LPA as per programme guidelines. In this study, we present trends in genotypic resistance to isoniazid (INH) and rifampicin (RIF) during the 4 years (2016-2019) among these patients. Band patterns were analysed as per the updated GLI (Global Laboratory Initiative) LPA interpretation and reporting guidelines.RESULTS: A total of 26,349 samples were received during the study period. Smear-positive samples (n = 20231) were directly subjected to FL-LPA; smear-negative samples were cultured in liquid media and M. tuberculosis-positive cultures were tested using FL-LPA. A total of 18,441 were MTB-positive on FL-LPA. INH monoresistance, RIF monoresistance and MDR-TB was observed in respectively 8.7%, 1.1% and 3.3% of the samples. There was a decreasing trend in all types of resistance observed particularly after 2017 (P < 0.001). MDR-TB showed a steady decrease from 5.6% to 1.8%. S531L (19.5%) and S315T (61.1%) were the most common mutations identified in the rpoB and katG genes, respectively. The percentage of inhA-c-15t promoter mutation, indicating low-level INH resistance, showed a consistent increase (P < 0.001).CONCLUSION: The impact of the UDST policy on the NTEP may have led to this decreasing trend in RIF and INH resistance observed in the study period. The increase in low-level INH resistance mutation inhA-c-15t may be associated with ethionamide/prothionamide resistance, and this should be taken into account when designing DR-TB regimen.

Biallelic Variants in ENDOG Associated with Mitochondrial Myopathy and Multiple mtDNA Deletions.

Endonuclease G (ENDOG) is a nuclear-encoded mitochondrial-localized nuclease. Although its precise biological function remains unclear, its proximity to mitochondrial DNA (mtDNA) makes it an excellent candidate to participate in mtDNA replication, metabolism and maintenance. Indeed, several roles for ENDOG have been hypothesized, including maturation of RNA primers during mtDNA replication, splicing of polycistronic transcripts and mtDNA repair. To date, ENDOG has been deemed as a determinant of cardiac hypertrophy, but no pathogenic variants or genetically defined patients linked to this gene have been described. Here, we report biallelic ENDOG variants identified by NGS in a patient with progressive external ophthalmoplegia, mitochondrial myopathy and multiple mtDNA deletions in muscle. The absence of the ENDOG protein in the patient’s muscle and fibroblasts indicates that the identified variants are pathogenic. The presence of multiple mtDNA deletions supports the role of ENDOG in mtDNA maintenance; moreover, the patient’s clinical presentation is very similar to mitochondrial diseases caused by mutations in other genes involved in mtDNA homeostasis. Although the patient’s fibroblasts did not present multiple mtDNA deletions or delay in the replication process, interestingly, we detected an accumulation of low-level heteroplasmy mtDNA point mutations compared with age-matched controls. This may indicate a possible role of ENDOG in mtDNA replication or repair. Our report provides evidence of the association of ENDOG variants with mitochondrial myopathy.

Diffuse astrocytoma with mosaic IDH1-R132H-mutant immuno-phenotype and low subclonal allele frequency.

Molecular alterations found in gliomas are now considered entity-defining features. The World Health Organization (WHO) classification system currently classifies the vast majority of gliomas utilizing an integrated genotype-phenotype approach. We present a case of diffuse astrocytoma with a mosaic isocitrate dehydrogenase (IDH)1-R132H-mutant immunophenotype and low subclonal allele frequency. A 35-year-old patient with a history of IDH1-R132H mutated diffuse astrocytoma in 20014 presented to the hospital again in 2019. MRI examination showed a non-enhancing abnormal signal in the periphery of her previous surgical cavity. Histopathological examination revealed that the tumor was hypercellular and without high grade histopathological features. The neoplastic cells were immunohistologically positive for GFAP, Olig2, and ATRX. However, only some scattered tumor cells were positive for IDH1-R132H. Cytogenetic studies revealed a lack of chromosomal 1p/19q co-deletion. Further next-generation sequencing (NGS) demonstrated a low-level IDH1-R132H mutation and allele frequency. Based on these findings, the diagnosis of diffuse astrocytoma with mosaic IDH1- R132H-mutant immunophenotype and low subclonal allele frequency (WHO grade II) was generated. This case indicates that gliomas may have heterogeneous molecular profile and the intra-tumoral molecular heterogeneity highlights the need to further characterize the molecular profile for glioma classification and clinical management.

Detecting rifampin and isoniazid resistance in Mycobacterium tuberculosis direct from patient sputum using an automated integrated system

While there has been progress in detection of drug resistant tuberculosis globally, WHO estimates only about half of the patients with bacteriologically confirmed tuberculosis were tested for rifampicin resistance over the past two years. To close this drug resistance diagnostic gap, an expansion of testing for rifampicin and isoniazid resistance is critically needed. The Akonni Biosystem Integrated System combines DNA extraction and a Lab-on-a-Film assembly (LFA) to perform rapid probe and PCR-based detection of resistance associated mutations to first-line anti-tuberculosis drugs. Using raw sputum samples from 25 tuberculosis patients at risk for drug resistance, we conducted a proof-of-concept study of the Integrated System with an MDR-TB assay. Performance of the Integrated System was compared to liquid Mycobacteria Growth Indicator Tube (MGIT) culture reference phenotypes using 2012 WHO endorsed critical concentrations for rifampicin and isoniazid. The overall percent agreement for rifampicin and isoniazid was 91.7% and 100% respectively, with agreement for rifampicin increasing to 95.7% after low-level resistance mutations in rpoB were excluded. The Integrated System, combining DNA extraction and LFA amplification, is a promising new tool for detection of both rifampicin and isoniazid using liquefied raw sputum.