High-confidence Mutations Research Articles

Haploinsufficiency of Syngap1 in striatal indirect pathway neurons alters motor and goal-directed behaviors in mice.

SYNGAP1 is a high-confidence autism spectrum disorder (ASD) risk gene and mutations in SYNGAP1 lead to a neurodevelopmental disorder (NDD) that presents with epilepsy, ASD, motor developmental delay, and intellectual disability. SYNGAP1 codes for Ras/Rap GTP-ase activating protein SynGAP (SynGAP). In mice, SynGAP is located in the postsynaptic density of glutamatergic synapses and regulates glutamate receptor trafficking in an activity-dependent manner. In addition to forebrain glutamatergic neurons, Syngap1 is highly expressed in the striatum, although the functions of SynGAP in the striatum have not been extensively studied. Here we show that Syngap1 is expressed in both direct and indirect pathway striatal projection neurons (dSPNs and iSPNs) in mice of both sexes. In a mouse model of Syngap1 haploinsufficiency, dendritic spine density, morphology, and intrinsic excitability are altered primarily in iSPNs, but not dSPNs. At the behavioral level, SynGAP reduction alters striatal-dependent motor learning and goal-directed behavior. Several behavioral phenotypes are reproduced by iSPN-specific Syngap1 reduction and, in turn, prevented by iSPN-specific Syngap1 rescue. These results establish the importance of SynGAP to striatal neuron function and pinpoint the indirect pathway as a key circuit in the neurobiology of SYNGAP1-related NDD.Significance statement SYNGAP1 mutations cause a neurodevelopmental disorder presenting with intellectual disability, motor problems, epilepsy, autism spectrum disorder, and a constellation of other behavioral and psychiatric conditions. SynGAP protein is highly expressed in the striatum but its functions in this brain region have not yet been explored. This study shows that loss of one copy of the Syngap1 gene from striatal indirect, but not direct, pathway neurons alters synaptic properties, cellular excitability, motor behaviors, and goal-directed responding in mice. This work provides a new perspective on the functions of SynGAP and suggests that altered activity in striatal circuits may be an important driver of the motor and learning alterations in people with SYNGAP1 disorder.

A fully sequenced collection of homozygous EMS mutants for forward and reverse genetic screens in Arabidopsis thaliana.

Genetic screens are powerful tools for biological research and are one of the reasons for the success of the thale cress Arabidopsis thaliana as a research model. Here, we describe the whole-genome sequencing of 871 Arabidopsis lines from the Homozygous EMS Mutant (HEM) collection as a novel resource for forward and reverse genetics. With an average 576 high-confidence mutations per HEM line, over three independent mutations altering protein sequences are found on average per gene in the collection. Pilot reverse genetics experiments on reproductive, developmental, immune and physiological traits confirmed the efficacy of the tool for identifying both null, knockdown and gain-of-function alleles. The possibility of conducting subtle repeated phenotyping and the immediate availability of the mutations will empower forward genetic approaches. The sequence resource is searchable with the ATHEM web interface (https://lipm-browsers.toulouse.inra.fr/pub/ATHEM/), and the biological material is distributed by the Versailles Arabidopsis Stock Center.

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data.

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials-isoniazid, rifampin, ethambutol, and pyrazinamide-for MTBC isolates tested between the years 2018-2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks.

Specific Mycobacterium tuberculosis Strain Circulating in Prison Revealed by Cost-Effective Amplicon Sequencing.

This scientific study focuses on tuberculosis (TB) within prison settings, where persons deprived of liberty (PDL) face significantly higher rates of the disease compared to the general population. The research employs the low-cost amplicon sequencing of Mycobacterium tuberculosis strains, aiming first to identify specific lineages and also to detect mutations associated with drug resistance. The method involves multiplex amplification, DNA extraction, and sequencing, providing valuable insights into TB dynamics and resistance-mutation profiles within the prison system at an affordable cost. The study identifies a characteristic lineage (X) circulating among PDLs in the penitentiary system in Uruguay, absent in the general population, and notes its prevalence at prison entry. No high-confidence mutations associated with drug resistance were found. The findings underscore the importance of molecular epidemiology in TB control, emphasizing the potential for intra-prison transmissions and the need for broader studies to understand strain dynamics.

Abstract PO5-03-12: Genomic differences of primary triple negative breast cancer in patients younger than 45 years vs. patients older than 45 years of age

Abstract Background: Triple negative breast cancer (TNBC) is often associated with an aggressive clinical course, especially in younger women. We previously demonstrated that there are differences in the genomic spectrum of metastatic TNBC in patients who are ≤45 years and those who are >45 years of age (Vidula et al, SABCS, 2020). Here we explore genomic differences of primary TNBC in patients ≤45 years and patients >45 years to understand variations in the overall number of non-silent mutations and proportion of patients with specific gene mutations. Methods: We obtained filtered mutation calls generated by the Multi-Center Mutation Calling in Multiple Cancers (MC3) working group (Ellrott et al, Cell Syst, 2018), from whole exome sequencing data of 9079 The Cancer Genome Atlas (TCGA) samples across 33 cancer types that had been used for the comprehensive characterization of cancer driver genes and mutations (Bailey et al, Cell, 2018). Receptor subtype and age at diagnosis of 1247 TCGA breast cancer samples were obtained from the UCSC Xena browser (Goldman et al, Nat Biotechnol, 2020). These datasets were combined to identify 119 patients with primary TNBC with harmonized high confidence mutation calls for analysis. We compared the overall number of non-silent mutations between patients ≤45 years vs. >45 years using the Wilcoxon rank sum test. We also compared the proportion of patients with non-silent mutations in genes mutated at >10% frequency between age groups using the Fisher Exact test. Results: Altogether, 25 patients ≤45 years and 94 patients >45 years were identified with primary TNBC. No significant difference in the number of non-silent mutations among patients ≤45 years vs. >45 years was seen (median [range], ≤45 years: 65 [5-228] vs. >45 years: 74 [2-742], Wilcoxon rank sum p=0.57). In total, 35 unique genes were mutated at >10% frequency in either patients ≤45 years (30 genes) or >45 years (7 genes); notably, TP53 mutations were commonly seen in both age groups (≤45 years: 76%; >45 years: 79%, p=0.79) and TTN mutations were also frequently mutated in both cohorts (≤45 years: 20%; >45 years: 27%, p=0.61). Among the 35 genes identified, 12 genes were mutated at significantly different frequencies among patients ≤45 years vs. those >45 years, as depicted in Table 1. Conclusions: Relatively high frequencies of TP53 and TTN mutations were seen in both patients ≤45 years and those >45 years with primary TNBC. However, many genes associated with varying cellular functions including motility, signaling receptors, and growth were more frequently mutated in patients ≤45 years. These results suggest that there are genomic differences of primary TNBC in patients ≤45 years vs. those >45 years. Further research is needed to validate these findings in a larger cohort of primary TNBC since genomic differences may affect tumor biology and clinical behavior. Table 1. Significant Gene Mutation Differences in Primary TNBC Based on Age. Citation Format: Neelima Vidula, Leif Ellisen, Aditya Bardia, Christina Yau. Genomic differences of primary triple negative breast cancer in patients younger than 45 years vs. patients older than 45 years of age [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-03-12.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

High Neoantigen Load Confers a Major Benefit from Autologous Stem Cell Transplantation Compared to Consolidation in Newly Diagnosed Multiple Myeloma

Autologous stem cell transplant (ASCT) is the current standard of care for eligible patients with multiple myeloma (MM). The UK national trial CARDAMON investigated whether, following carfilzomib, cyclophosphamide, and dexamethasone (KCD) induction, ASCT was of benefit compared with further cycles of KCD as consolidation. All patients received carfilzomib maintenance. KCD consolidation did not show non-inferiority, but the difference was marginal, and we previously concluded “that further studies are warranted to explore deferred autologous HSCT in some subgroups.” Whilst some of the benefit of ASCT derives from the myeloablative effect of melphalan, there is evidence that immune reprogramming has an important role. It has also been reported that neoantigen burden has prognostic significance in MM, with patients with a high neoantigen load having worse outcomes. Taken together, we hypothesized that patients with higher neoantigen load would gain most benefit from ASCT, due to immune reprogramming. We therefore developed a pipeline to predict neoantigen load in myeloma patients ( Fig. 1A) and explored whether this had prognostic significance in the context of consolidation or ASCT in patients enrolled in CARDAMON. We collected paired tumour and germline whole exome sequencing and RNA-Seq data. We called nonsynonymous and frame-shift mutations using MuTECT2 and calculated RNA-Seq expression using Salmon. We used ARCAS-HLA for HLA genotyping. We translated high-confidence mutations within a sliding window and predicted HLA allele binding using NETMHCpan-4.0, predicting binding when the IC50 was less than 200nM, per the standard definition of a strong binding peptide. We removed any neoantigens that were not expressed, using the RNA-Seq data. Neoantigen load for an individual patient was taken as the number of predicted expressed neoantigens. We chose a neoantigen cut-off based on mutation rates distinguishing immunologically “cold” or “hot” tumours. Consequently, patients with <= 130 neoantigens were defined as low antigen load, and those with >130 were defined as high neoantigen load. We observed a total of 15,162 high affinity neoantigens across 58 patients, with a median of 200 neoantigens per patient (range 34 - 653). Forty-six neoantigens were shared across patients, mostly NRAS, KRAS, and BRAF. We first compared patients by randomization group. For patients who received consolidation, there was no significant difference in progression-free survival (PFS) between high and low antigen loads (p=0.32). For patients in the ASCT arm, a high antigen load was associated with a longer PFS (p=0.013). This was surprising, as high antigen load has previously been shown to be a poor prognostic factor, although our numbers were small and confirmation in independent data is ongoing. We then considered patients split by antigen load. For those with low antigen load, we saw no difference in PFS between those who received ASCT and those who received consolidation. However, for patients with a high antigen load, there was a marked improvement in PFS for those undergoing ASCT ( Fig. 1B Upper; median PFS not reached vs. 25 months; hazard ratio 0.50, 95% confidence interval 0.20-0.65; p=0.0039). This difference translated to overall survival ( Fig. 1B Lower; median OS not reached in either group; hazard ratio 0.12, 95% confidence interval 0.014-0.50; p=0.028). The marked benefit of ASCT over consolidation in this subgroup analysis is in contrast to the marginal difference between the two therapies for allcomers in the CARDAMON trial. Whilst upfront ASCT remains the treatment of choice for eligible patients, the risk/benefit ratio may be less clear in the more frail and in the context of deep response to induction. In summary: We have developed a bioinformatic pipeline to predict neoantigen burden in newly diagnosed myeloma, which suggests higher neoantigen load is associated with a major benefit from ASCT. This approach provides novel insight into the biology of ASCT and could be used to stratify patient need for ASCT in the future, although the immunological mechanisms implicated in these findings remain to be identified. Neoantigen subtype effect (frameshift vs SNV) and validation in a larger cohort is ongoing.

Direct detection of drug-resistant Mycobacterium tuberculosis using targeted next generation sequencing.

Mycobacterium tuberculosis complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens. A multiplex PCR was designed to amplify a group of thirteen full-length genes and promoter regions with mutations known to be involved in resistance to first- and second-line MTBC drugs. Long-read amplicon libraries were sequenced with Oxford Nanopore Technologies platforms and high-confidence resistance mutations were identified in real-time using an in-house developed bioinformatics pipeline. Sensitivity, specificity, reproducibility, and accuracy of the tNGS assay was assessed as part of a clinical validation study. In total, tNGS was performed on 72 primary specimens and 55 MTBC-positive cultures and results were compared to clinical whole genome sequencing (WGS) performed on paired patient cultures. Complete or partial susceptibility profiles were generated from 82% of smear positive primary specimens and the resistance mutations identified by tNGS were 100% concordant with WGS. In addition to performing tNGS on primary clinical samples, this assay can be used to sequence MTBC cultures mixed with other mycobacterial species that would not yield WGS results. The assay can be effectively implemented in a clinical/diagnostic laboratory with a two to three day turnaround time and, even if batched weekly, tNGS results are available on average 15 days earlier than culture-derived WGS results. This study demonstrates that tNGS can reliably predict MTBC drug resistance directly from clinical specimens or cultures and provide critical information in a timely manner for the appropriate treatment of patients with DR tuberculosis.

Whole-Genome Sequence-Based Characterization of Pre-XDR M. tuberculosis Clinical Isolates Collected in Kazakhstan.

Kazakhstan has a high burden of multidrug-resistant tuberculosis in the Central Asian region. This study aimed to perform genomic characterization of Mycobacterium tuberculosis strains obtained from Kazakhstani patients with pre-extensively drug-resistant tuberculosis diagnosed in Kazakhstan. Whole-genome sequencing was performed on 10 pre-extensively drug-resistant M. tuberculosis strains from different regions of Kazakhstan. All strains had high-confidence resistance mutations according to the resistance grading system previously established by the World Health Organization. The genome analysis was performed using TB-Profiler, Mykrobe, CASTB, and ResFinder. Valuable information for understanding the genetic diversity of tuberculosis in Kazakhstan can also be obtained from whole-genome sequencing. The results from the Phenotypic Drug Susceptibility Testing (DST) of bacterial strains were found to be consistent with the drug resistance information obtained from genomic data that characterized all isolates as pre-XDR. This information can help in developing targeted prevention and control strategies based on the local epidemiology of tuberculosis. Furthermore, the data obtained from whole-genome sequencing can help in tracing the transmission pathways of tuberculosis and facilitating early detection of outbreaks. The results from whole-genome sequencing of tuberculosis clinical samples in Kazakhstan provide important insights into the drug resistance patterns and genetic diversity of tuberculosis in the country. These results can contribute to the improvement of tuberculosis control and management programs in Kazakhstan.

Abstract 211: The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling

Abstract Whole genome sequencing (WGS) is widely used for cancer diagnostic and therapeutic applications in clinical practice and clinical trials. Currently, the Truseq PCR-free library preparation methods are routinely used in genomics laboratories. However, microgram amounts of DNA input is a limitation due to limited materials often from clinically-derived specimens. Here, we evaluated a novel low input (100-300 ng) PCR-free tagmentation (TAG) based library preparation for WGS. Replicates of TAG- and ligation-based (Illumina TruSeq) sequencing libraries were prepared from 3 pairs of breast cancer-derived tumors (HCC1195, HCC1143 and HCC1187) and matched B-lymphocyte-derived normal (HCC1195BL, HCC1143BL and HCC1187BL) cell lines. Libraries were sequenced on Illumina Novaseq 6000 platforms to target 30x and 90x mean coverage for normal and tumor samples respectively. Raw sequencing data were aligned to the hg38 by Isaac Aligner before variant analysis by Strelka2. Technical sequencing metrics demonstrated high similarity between TAG- and ligation-based workflows, including passing filter reads, Q30%, aligned reads, mean coverage. Germline variants of HCC1395BL showed greater than 98% precision and sensitivity using ligation-based variants as a reference. For somatic mutation calling from TAG-based libraries, HCC1395, HCC1143 and HCC1187, 88%, 85% and 86% precision and 81%, 70%, 68% sensitivity was observed, respectively, compared to the ligation-based method. Furthermore, using the SEQC2 Consortium high-confidence mutation set, TAG- and ligation- based variant calls of HCC1395 had 90% and 93% precision and 74% and 73% sensitivity, respectively with this high confidence reference. Further investigation showed that a high fraction of false negative calls was associated with low variant allele frequency (<10%). Compared to an available reference dataset for benchmarking somatic calling pipelines from New York Genome Center, TAG- and ligation-based variant calls of HCC1143 yielded 85% and 83% precision and 63% and 63% sensitivity, respectively; TAG- and ligation-based variant calls of HCC1187 resulted in 91% and 92% precision and 81% and 79% sensitivity, respectively. In summary, the low input TAG-based WGS protocol produced highly reproducible variant calls with highly concordant somatic variant calls compared to the commonly-used PCR-free ligation-based methods and to reference callset. Further evaluation is warranted to broaden clinical application from minimal starting material.The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Liqun Jiang, Xijun Zhang, Camille Alba, Gauthaman Sukumar, Elizabeth A. Rice, Matthew D. Wilkerson, Clifton L. Dalgard. The performance characteristic of the low input tagmentation-based whole genome sequencing in high quality somatic variant calling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 211.

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GIGYF1 disruption associates with autism and impaired IGF-1R signaling.

Autism spectrum disorder (ASD) represents a group of neurodevelopmental phenotypes with a strong genetic component. An excess of likely gene-disruptive (LGD) mutations in GIGYF1 was implicated in ASD. Here, we report that GIGYF1 is the second-most mutated gene among known ASD high–confidence risk genes. We investigated the inheritance of 46 GIGYF1 LGD variants, including the highly recurrent mutation c.333del:p.L111Rfs*234. Inherited GIGYF1 heterozygous LGD variants were 1.8 times more common than de novo mutations. Among individuals with ASD, cognitive impairments were less likely in those with GIGYF1 LGD variants relative to those with other high-confidence gene mutations. Using a Gigyf1 conditional KO mouse model, we showed that haploinsufficiency in the developing brain led to social impairments without significant cognitive impairments. In contrast, homozygous mice showed more severe social disability as well as cognitive impairments. Gigyf1 deficiency in mice led to a reduction in the number of upper-layer cortical neurons, accompanied by a decrease in proliferation and increase in differentiation of neural progenitor cells. We showed that GIGYF1 regulated the recycling of IGF-1R to the cell surface. KO of GIGYF1 led to a decreased level of IGF-1R on the cell surface, disrupting the IGF-1R/ERK signaling pathway. In summary, our findings show that GIGYF1 is a regulator of IGF-1R recycling. Haploinsufficiency of GIGYF1 was associated with autistic behavior, likely through interference with IGF-1R/ERK signaling pathway.

Genetic determinants of lung cancer: Understanding the oncogenic potential of somatic missense mutations

BackgroundTreatment of lung cancer is getting more personalized nowadays and medical practitioners are moving away from conventional histology-driven empirical treatments, platinum-based chemotherapy, and other invasive surgical resections and have started adopting alternate therapies in which therapeutic targets are patient's molecular oncogenic drivers. AimThe aim of the current study is to extract meaningful information from the online somatic mutation data (retrieved from cBioPortal) of 16 most significantly mutated oncogenes in non-small-cell lung cancer (NSCLC), namely EGFR, NRAS, KRAS, HER2 (ERBB2), RET, MET, ROS1, FGFR1, BRAF, AKT1, MEK1 (MAP2K1), PIK3CA, PTEN, DDR2, LKB1 (STK11) and ALK, for improving our understanding of the pathobiology of the lung cancer that can aid decision-making on critical clinical and therapeutic considerations. MethodsUsing an integrated approach comprising 4 steps, the oncogenic potential of 661 missense non-synonymous single nucleotide polymorphisms (nsSNPs) in 16 genes was ascertained using 2059 NSCLC (1575 lung adenocarcinomas, 484 lung squamous cell carcinomas) patients' online mutation data. The steps used comprise sequence/structure homology-based prediction, scoring of conservation of mutated residues and positions, prediction of resulting molecular and functional consequences using machine-learning and structure-guided approach. ResultsOut of a total of 661 nsSNPs analyzed, a set of 29 nsSNPs has been identified as conserved high confidence mutations in 10 of 16 genes relevant to the under study. Out of 29 conserved high confidence nsSNPs, 4 nsSNPs (EGFR N1094Y, BRAF M620I, DDR2 R307L, ALK P1350T) have been found to be putative novel rare genetic markers for NSCLC. ConclusionsThe current study, the first of its kind, has provided a list of deleterious non-synonymous somatic mutations in a selected pool of oncogenes that can be considered as a promising target for future drug design and therapy for patients with lung adenocarcinomas and squamous cell carcinomas.

PD12-10 URINE COMPREHENSIVE GENOMIC PROFILING OF UROTHELIAL CARCINOMA PATIENTS UNDERGOING INTRAVESICAL THERAPY ENABLES DETECTION OF ACTIONABLE DNA MUTATIONS ASSOCIATED WITH DRUG RESPONSE AND RISK STRATIFICATION

You have accessJournal of UrologyCME1 May 2022PD12-10 URINE COMPREHENSIVE GENOMIC PROFILING OF UROTHELIAL CARCINOMA PATIENTS UNDERGOING INTRAVESICAL THERAPY ENABLES DETECTION OF ACTIONABLE DNA MUTATIONS ASSOCIATED WITH DRUG RESPONSE AND RISK STRATIFICATION Goran Rac, Shalin Desai, Hiten D. Patel, Chirag Doshi, Ryan Dornbier, Vincent Caruso, Peter Lentz, Brian C. Mazzarella, Kevin G. Phillips, Trevor Levin, Alan J. Wolfe, and Gopal N. Gupta Goran RacGoran Rac More articles by this author , Shalin DesaiShalin Desai More articles by this author , Hiten D. PatelHiten D. Patel More articles by this author , Chirag DoshiChirag Doshi More articles by this author , Ryan DornbierRyan Dornbier More articles by this author , Vincent CarusoVincent Caruso More articles by this author , Peter LentzPeter Lentz More articles by this author , Brian C. MazzarellaBrian C. Mazzarella More articles by this author , Kevin G. PhillipsKevin G. Phillips More articles by this author , Trevor LevinTrevor Levin More articles by this author , Alan J. WolfeAlan J. Wolfe More articles by this author , and Gopal N. GuptaGopal N. Gupta More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002538.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prediction of intravesical therapy (IVT) response and risk stratification in patients with urothelial carcinoma (UC) remains a challenge. Previous attempts using biomarkers have not gained broad adoption. Urinary comprehensive genomic profiling (uCGP) has significant potential to aid in both quantitative assessment of therapeutic response and risk stratification. METHODS: uCGP was performed on 26 prospectively enrolled patients undergoing IVT induction (BCG=88%, Gemcitabine=12%). Samples were collected after visually complete TURBT immediately prior to IVT instillation. Urine DNA was sequenced and profiled across 60 genes using the CLIA-validated UroAmplitude test (Convergent Genomics). Recurrence predictions were performed using a previously validated algorithm. Subjects were grouped into high, intermediate, and low risk of recurrence. RESULTS: Stage distribution was Tis (15%), Ta (23%), T1 (62%). Mean follow-up after IVT was 22 months. Among samples analyzed, 25% contained no detectable mutations while 75% contained ≥1 high confidence mutation. The high and intermediate risk classification identified 100% of pathologically confirmed recurrence. Among low recurrence risk classifications, no clinical recurrence was identified within follow-up (100% NPV). TP53 mutations were strongly correlated with post-treatment recurrence OR=28, 95%CI (1.99, 394.42), p=0.038. Two subjects had detected aneuploidy post-treatment and had T1 recurrence, supporting earlier observations of aneuploidy correlating with invasive cancer. CONCLUSIONS: uCGP is a quantitative diagnostic with potential to identify therapeutic response. Low risk patients have low mutation burden prior to treatment, intermediate risk have notable disease burden pre-treatment with notable reduction after IVT, and high risk do not respond to treatment. These data suggest that uCGP performed before and after IVT has significant potential to stratify recurrence risk in non-muscle invasive UC patients. Additional studies are underway to further support the generalizability of these findings. Source of Funding: Support for this research is provided by the NCI through a SBIR Grant, 5R44CA200174, provided to Convergent Genomics © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e199 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Goran Rac More articles by this author Shalin Desai More articles by this author Hiten D. Patel More articles by this author Chirag Doshi More articles by this author Ryan Dornbier More articles by this author Vincent Caruso More articles by this author Peter Lentz More articles by this author Brian C. Mazzarella More articles by this author Kevin G. Phillips More articles by this author Trevor Levin More articles by this author Alan J. Wolfe More articles by this author Gopal N. Gupta More articles by this author Expand All Advertisement PDF DownloadLoading ...

Deciphering Isoniazid Drug Resistance Mechanisms on Dimeric Mycobacterium tuberculosis KatG via Post-molecular Dynamics Analyses Including Combined Dynamic Residue Network Metrics.

Resistance mutations in Mycobacterium tuberculosis (Mtb) catalase peroxidase protein (KatG), an essential enzyme in isoniazid (INH) activation, reduce the sensitivity of Mtb to first-line drugs, hence presenting challenges in tuberculosis (TB) management. Thus, understanding the mutational imposed resistance mechanisms remains of utmost importance in the quest to reduce the TB burden. Herein, effects of 11 high confidence mutations in the KatG structure and residue network communication patterns were determined using extensive computational approaches. Combined traditional post-molecular dynamics analysis and comparative essential dynamics revealed that the mutant proteins have significant loop flexibility around the heme binding pocket and enhanced asymmetric protomer behavior with respect to wild-type (WT) protein. Heme contact analysis between WT and mutant proteins identified a reduction to no contact between heme and residue His270, a covalent bond vital for the heme-enabled KatG catalytic activity. Betweenness centrality calculations showed large hub ensembles with new hubs especially around the binding cavity and expanded to the dimerization domain via interface in the mutant systems, providing possible compensatory allosteric communication paths for the active site as a result of the mutations which may destabilize the heme binding pocket and the loops in its vicinity. Additionally, an interesting observation came from Eigencentrality hubs, most of which are located in the C-terminal domain, indicating relevance of the domain in the protease functionality. Overall, our results provide insight toward the mechanisms involved in KatG-INH resistance in addition to identifying key regions in the enzyme functionality, which can be used for future drug design.

Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations.

The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.

A short plus long-amplicon based sequencing approach improves genomic coverage and variant detection in the SARS-CoV-2 genome.

High viral transmission in the COVID-19 pandemic has enabled SARS-CoV-2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma

Bone marrow derived stromal cells from myelodysplastic syndromes are altered but not clonally mutated in vivo

The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients.

Genetic analysis of sinonasal undifferentiated carcinoma discovers recurrent SWI/SNF alterations and a novel PGAP3-SRPK1 fusion gene

BackgroundSinonasal Undifferentiated Carcinoma (SNUC) is a rare and aggressive skull base tumor with poor survival and limited treatment options. To date, targeted sequencing studies have identified IDH2 and SMARCB1 as potential driver alterations, but the molecular alterations found in SMARCB1 wild type tumors are unknown.MethodsWe evaluated survival outcomes in a cohort of 46 SNUC patients treated at an NCI designated cancer center and identify clinical and disease variables associated with survival on Kaplan-Meier and Cox multivariate survival analysis. We performed exome sequencing to characterize a series of SNUC tumors (n = 5) and cell line (MDA8788–6) to identify high confidence mutations, copy number alterations, microsatellite instability, and fusions. Knockdown studies using siRNA were utilized for validation of a novel PGAP3-SRPK1 gene fusion.ResultsOverall survival analysis revealed no significant difference in outcomes between patients treated with surgery +/− CRT and CRT alone. Tobacco use was the only significant predictor of survival. We also confirmed previously published findings on IDH and SMARC family mutations and identified novel recurrent aberrations in the JAK/STAT and PI3K pathways. We also validated a novel PGAP3-SRPK1 gene fusion in the SNUC cell line, and show that knockdown of the fusion is negatively associated with EGFR, E2F and MYC signaling.ConclusionCollectively, these data demonstrate recurrent alterations in the SWI/SNF family as well as IDH, JAK/STAT, and PI3K pathways and discover a novel fusion gene (PGAP3-SRPK1). These data aim to improve understanding of possible driver mutations and guide future therapeutic strategies for this disease.