Single Mutational Event Research Articles

Mutation accumulation in H. sapiens F508del CFTR countermands dN/dS type genomic analysis.

Understanding the mechanisms that underlie de novo mutations (DNMs) can be essential for interpreting human evolution, including aspects such as rapidly diverging genes, conservation of non-coding regulatory elements, and somatic DNA adaptation, among others. DNM accumulation in Homo sapiens is often limited to evaluation of human trios or quads across a single generation. Moreover, human SNPs in exons, pseudogenes, or other non-coding elements can be ancient and difficult to date, including polymorphisms attributable to founder effects and identity by descent. In this report, we describe multigenerational evolution of a human coding locus devoid of natural selection, and delineate patterns and principles by which DNMs have accumulated over the past few thousand years. We apply a data set comprising cystic fibrosis transmembrane conductance regulator (CFTR) alleles from 2,393 individuals homozygous for the F508del defect. Additional polymorphism on the F508del background diversified subsequent to a single mutational event during recent human history. Because F508del CFTR is without function, SNPs observed on this haplotype are effectively attributable to factors that govern accumulating de novo mutations. We show profound enhancement of transition, synonymous, and positionally repetitive polymorphisms, indicating appearance of DNMs in a manner evolutionarily designed to protect protein coding DNA against mutational attrition while promoting diversity.

Navigating Molecular Pathways: An Update on Drugs in Colorectal Cancer Treatment.

Colorectal cancer (CRC) is a multifaceted and heterogeneous ailment that affects the colon or rectum of the digestive system. It is the second most commonly occurring form of cancer and ranks third in terms of mortality rate. The progression of CRC does not occur due to a single mutational event; rather, it is the result of the sequential and cumulative accumulation of mutations in key driver genes of signaling pathways. The most significant signaling pathways, which have oncogenic potential due to their deregulation, include Wnt/β-catenin, Notch, TGF-β, EGFR/MAPK, and PI3K/AKT pathways. Numerous drug target therapies have been developed to treat CRC using small molecule inhibitors, antibodies, or peptides. Although drug-targeted therapy is effective in most cases, the development of resistance mechanisms in CRC has raised questions about their efficacy. To overcome this issue, a novel approach to drug repurposing has come to light, which utilizes already FDA-approved drugs to treat CRC. This approach has shown some promising experimental results, making it a crucial avenue of research in the treatment of CRC.

A new, all-encompassing aetiology of type 1 diabetes.

The aetiology of type 1 diabetes (T1D) is considered multifactorial with the contribution of the MHC on chromosome 6 being most important. Multiple factors also contribute to the aetiology of colorectal neoplasia, but the final event causing the change from normal mucosa to polyp and from polyp to cancer is due to a single somatic mutation event. Repeated formation of colorectal neoplasia within an at-risk population results in a predictable, tapering, exponential neoplasia distribution. Critical mutations driving colorectal neoplasia formation occur in mutation-prone DNA. These observations led to three hypotheses related to T1D. First, a single somatic mutation within the MHC of antigen presenting cells results in a change in phenotype from normal to T1D. Second, the distribution of additional autoimmune diseases (AAIDs) among persons with T1D adheres to a predictable, tapering, exponential distribution. And third, critical mutations driving development of T1D occur in mutation-prone DNA. To address the hypotheses in an orderly fashion, a new analytical method called genome-wide aetiology analysis (GWEA) consisting of nine steps is presented. All data required for GWEA of T1D are obtained from peer-reviewed publications or publicly available genome and proteome databases. Critical GWEA steps include AAID distribution among persons with T1D, analysis of at-risk HLA loci for mutation-prone DNA, determination of the role of non-MHC genes on GWAS, and verification of human data by cell culture or animal experiments. GWEA results show that distribution of AAID among persons with T1D adheres to a predictable, tapering, exponential distribution. A single, critical, somatic mutation within the epitope-binding groove of at-risk HLA loci alters HLA-insulin-peptide-T-cell-receptor (TCR) complex binding affinity and creates a new pathway that leads to loss of self-tolerance. The at-risk HLA loci, in particular binding pockets P1, P4 and P9, are encoded by mutation-prone DNA: GC-rich DNA sequence and somatic hypermutation hotspots. All other genes on GWAS can but do not have to amplify the new autoimmune pathway by facilitating DNA mutations, changing peptide binding affinity, reducing signal inhibition or augmenting signal intensity. Animal experiments agree with human studies. In conclusion, T1D is caused by a somatic mutation within the epitope-binding groove of an at-risk HLA gene that affects HLA-insulin-peptide-TCR complex binding affinity and initiates an autoimmune pathway. The nature of the peptide that binds to a mutated epitope-binding groove of an at-risk HLA gene determines the type of autoimmune disease that develops, that is, one at-risk HLA locus, multiple autoimmune diseases. Thus, T1D and AAIDs, and therefore common autoimmune diseases, share a similar somatic mutation-based aetiology.

Effect of MAPK activation via mutations in NRAS, KRAS and BRAF on clinical outcome in newly diagnosed multiple myeloma.

Until now, next generation sequencing (NGS) data has not been incorporated into any prognostic stratification of multiple myeloma (MM) and no therapeutic considerations are based upon it. In this work, we correlated NGS data with (1) therapy response and survival parameters in newly diagnosed multiple myeloma, treated by VRd * and (2) MM disease stage: newly diagnosed multiple myeloma (ndMM) versus relapsed and/or refractory (relapsed/refractory multiple myeloma). We analyzed 126 patients, with ndMM and relapsed refractory multiple myeloma (rrMM), treated at the University Hospital of Bern (Inselspital). Next generation sequencing was performed on bone marrow, as part of routine diagnostics. The NGS panel comprised eight genes CCND1, DIS3, EGR1, FAM46C (TENT5C), FGFR3, PRDM1, TP53, TRAF3 and seven hotspots in BRAF, IDH1, IDH2, IRF4, KRAS, NRAS. The primary endpoint was complete remission (CR) after VRd in ndMM, in correlation with mutational profile. Mutational load was generally higher in rrMM, with more frequently mutated TP53: 11/87 (13%) in ndMM versus 9/11 (81%) in rrMM (OR 0.0857, p=0.0007). In ndMM, treated by VRd, mutations in MAPK-pathway members (NRAS, KRAS or BRAF) were associated with reduced probability of CR (21/38, 55%), as compared with wild type NRAS, KRAS or BRAF (34/40, 85%; OR 0.2225, p=0.006). NRAS c.181C>A (p.Q61K) as a single mutation event showed a trend to reduced probability of achieving CR (OR 0.0912, p=0.0247). Activation of MAPK pathway via mutated NRAS, KRAS and BRAF genes seems to have a negative impact on outcome in ndMM patients receiving VRd therapy. VRd* - bortezomib (Velcade®), lenalidomide (Revlimid®) and dexamethasone.

A founder event causing a dominant childhood epilepsy survives 800 years through weak selective pressure

Genetic epilepsy with febrile seizures plus (GEFS+) is an autosomal dominant familial epilepsy syndrome characterized by distinctive phenotypic heterogeneity within families. The SCN1B c.363C>G (p.Cys121Trp) variant has been identified in independent, multi-generational families with GEFS+. Although the variant is present in population databases (at very low frequency), there is strong clinical, genetic, and functional evidence to support pathogenicity. Recurrent variants may be due to a founder event in which the variant has been inherited from a common ancestor. Here, we report evidence of a single founder event giving rise to the SCN1B c.363C>G variant in 14 independent families with epilepsy. A common haplotype was observed in all families, and the age of the most recent common ancestor was estimated to be approximately 800 years ago. Analysis of UK Biobank whole-exome-sequencing data identified 74 individuals with the same variant. All individuals carried haplotypes matching the epilepsy-affected families, suggesting all instances of the variant derive from a single mutational event. This unusual finding of a variant causing an autosomal dominant, early-onset disease in an outbred population that has persisted over many generations can be attributed to the relatively mild phenotype in most carriers and incomplete penetrance. Founder events are well established in autosomal recessive and late-onset disorders but are rarely observed in early-onset, autosomal dominant diseases. These findings suggest variants present in the population at low frequencies should be considered potentially pathogenic in mild phenotypes with incomplete penetrance and may be more important contributors to the genetic landscape than previously thought.

PUMILIO competes with AUF1 to control DICER1 RNA levels and miRNA processing.

DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3′UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3′UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.

Experimental evolution of the megaplasmid pMPPla107 in Pseudomonas stutzeri enables identification of genes contributing to sensitivity to an inhibitory agent.

Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the Pseudomonas syringae megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of Pseudomonas stutzeri populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY. By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

APOBEC3-induced mutation of the hepatitis virus B DNA genome occurs during its viral RNA reverse transcription into (−)-DNA

APOBEC3s are innate single-stranded DNA cytidine-to-uridine deaminases that catalyze mutations in both pathogen and human genomes with significant roles in human disease. However, how APOBEC3s mutate a single-stranded DNA that is available momentarily during DNA transcription or replication in vivo remains relatively unknown. In this study, utilizing hepatitis B virus (HBV) viral mutations, we evaluated the mutational characteristics of individual APOBEC3s with reference to the HBV replication process through HBV whole single-strand (−)-DNA genome mutation analyses. We found that APOBEC3s induced C-to-T mutations from the HBV reverse transcription start site continuing through the whole (−)-DNA transcript to the termination site with variable efficiency, in an order of A3B >> A3G > A3H-II or A3C. A3B had a 3-fold higher mutation efficiency than A3H-II or A3C with up to 65% of all HBV genomic cytidines being converted into uridines in a single mutation event, consistent with the A3B localized hypermutation signature in cancer, namely, kataegis. On the other hand, A3C expression led to a 3-fold higher number of mutation-positive HBV genome clones, although each individual clone had a lower number of C-to-T mutations. Like A3B, A3C preferred both 5′-TC and 5′-CC sequences, but to a lesser degree. The APOBEC3-induced HBV mutations were predominantly detected in the HBV rcDNA but were not detectable in other intermediates including HBV cccDNA and pgRNA by primer extension of their PCR amplification products. These data demonstrate that APOBEC3-induced HBV genome mutations occur predominantly when the HBV RNA genome was reversely transcribed into (−)-DNA in the viral capsid.

Associations between interaction effects of mutational events and efficient immunotherapy in squamous NSCLC: Results from 373 patients.

e21136 Background: Predictive biomarkers might not be similarly predictive in every single subpopulation, and they might be merely valid under certain circumstances, which is termed by “interaction effect”. Here, we proposed to investigate the interaction effect of mutational events on predicting efficacy of immune checkpoint inhibitors (ICIs), and to explore whether adding these interaction terms could optimize the prediction model based on genomic alterations. Methods: In total, 373 patients with squamous NSCLC were included. 191 patients with both PFS and OS data receiving monotherapy from the MSKCC, DFCI, SNCC, and POPLAR/OAK cohorts were included in the training set. 130 patients from the POPLAR/OAK cohort treated with docetaxel were included in the control set. Validation set-1 consists of 34 patients receiving monotherapy from the NCC and MSKCC cohorts, and validation set-2 comprises 18 patients treated with combination therapy from the MSKCC cohort. Risk models using single mutations (uni-model) and both single mutations and interactions (inter-model) were developed via multivariable Cox proportional hazards regression model. Results: Four single mutational events, including mutations of NOTCH1/2/3, LRP1B, RB1, and members in PI3K pathway, and two interactions ( TP53* NFE2L2 and TP53*HRR pathway) were identified as predictive biomarkers. The score of inter-model exhibited higher correlation with immunotherapeutic PFS in the training sets, compared to the uni-model ( Dxy: 0.196 vs. 0.175). Uni-score low was barely associated with the PFS (HR=0.32, 95% CI 0.07-1.50, P=0.15) and OS (HR=1.20, 95% CI 0.45-3.17, P=0.71) in the validation set-1, and the PFS in the validation set-2 (HR=0.96, 95% CI 0.31-2.95, P=0.94). Inter-score low showed better association with longer PFS (HR=0.17, 95% CI 0.03-0.84, P=0.029) and OS (HR=0.33, 95% CI 0.12-0.92, P=0.033) in the validation set-1, and longer PFS in the validation set-2 (HR=0.50, 95% CI 0.13-1.85, P=0.30). In addition, no association was observed between inter-score low and the PFS and OS in the control set, indicating that inter-score low might not be a prognostic biomarker, but a positive predictive biomarker to efficacious immunotherapy. Conclusions: Two interaction effects of mutational events on predicting efficacious immunotherapy in squamous NSCLC ( TP53* NFE2L2 and TP53*HRR pathway) were identified. Adding these interaction terms to the prediction model could optimize the predictive utility. Deeper understanding of these inter-relations might provide insights for basic research on anti-tumor immunity.

Interaction effect between mutational events on predicting efficacious immunotherapy in nonsquamous NSCLC: Results from 1,083 patients.

e21100 Background: Despite that the biomarkers of single mutational events predicting efficacy of immune checkpoint inhibitors (ICIs) have been intensively studied in non-squamous NSCLC, the interaction effects between mutations on prediction were scarcely investigated due to the limited sample size. Deeper understanding of these inter-relations could optimize the prediction model and provide insights for basic research on anti-tumor immunity. Methods: In total, 1083 patients with non-squamous NSCLC were included. 288 patients with both PFS and OS data receiving monotherapy from the MSKCC, DFCI, and SNCC cohorts were included in the training set-1. 304 and 294 patients from the POPLAR/OAK cohort treated with atezolizumab or docetaxel were included in the training set-2 and the control set respectively. Validation set-1 and validation set-2 respectively consists of 104 patients receiving monotherapy with only OS data and 93 patients treated with combination therapy from the MSKCC cohort. Risk models using single mutations (uni-model) and both single mutations and interactions (inter-model) were developed via multivariable Cox proportional hazards regression model. Results: By cross-validation in training set-1/2, five single mutational events, including mutations of EGFR, STK11, PTPRD, members in PI3K pathway, and members in HRR pathway, and five interactions ( EGFR* PI3K pathway, TP53* KRAS, TP53* ERBB4, cell cycle pathway*HRR pathway, and PI3K pathway*chromatin remodeling pathway) were identified as predictive biomarkers. The score of inter-model exhibited higher correlation with immunotherapeutic PFS in the training sets, compared to the uni-model ( Dxy: 0.173 vs. 0.139). Uni-score low was moderately associated with longer OS in the validation set-1 (HR = 0.62, 95% CI 0.35-1.08, P = 0.092), and PFS in the validation set-2 (HR = 0.32, 95% CI 0.18-0.60, P < 0.001). Inter-score low showed better predictive utility in the validation set-1 (HR = 0.53, 95% CI 0.30-0.94, P = 0.030), and the validation set-2 (HR = 0.23, 95% CI 0.10-0.50, P < 0.001). On the contrary, inter-score low was associated with worse PFS and OS in the control set, indicating that inter-score low might be a negative prognostic biomarker, but positive predictive biomarker to efficacious immunotherapy. Conclusions: Our study suggests that the predictive biomarkers might not be similarly predictive in every single subpopulation, and they might be merely valid under certain circumstances, which is termed by “interaction effect”. Given this, the future researches probing into immunotherapeutic predictor should focus on the mutual influence between distinct biomarkers, for instance, adding an interaction term in the Cox proportional hazards regression model.

Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Cancer Cells in Myeloproliferative Neoplasms.

SummarySome cancers originate from a single mutation event in a single cell. Blood cancers known as myeloproliferative neoplasms (MPNs) are thought to originate when a driver mutation is acquired by a hematopoietic stem cell (HSC). However, when the mutation first occurs in individuals and how it affects the behavior of HSCs in their native context is not known. Here we quantified the effect of the JAK2-V617F mutation on the self-renewal and differentiation dynamics of HSCs in treatment-naive individuals with MPNs and reconstructed lineage histories of individual HSCs using somatic mutation patterns. We found that JAK2-V617F mutations occurred in a single HSC several decades before MPN diagnosis—at age 9 ± 2 years in a 34-year-old individual and at age 19 ± 3 years in a 63-year-old individual—and found that mutant HSCs have a selective advantage in both individuals. These results highlight the potential of harnessing somatic mutations to reconstruct cancer lineages.

A Natural Mutational Event Uncovers a Life History Trade-Off via Hormonal Pleiotropy

Environmental signals often control central life history decisions, including the choice between reproduction and somatic maintenance. Such adaptive developmental plasticity occurs in the nematode Caenorhabditis elegans, where environmental cues govern whether larvae will develop directly into reproducing adults or arrest their development to become stress-resistant dauer larvae. Here, we identified a natural variant underlying enhanced sensitivity to dauer-inducing cues in C.elegans: a 92-bp deletion in the cis-regulatory region of the gene eak-3. This deletion reduces synthesis or activity of the steroid hormone dafachronic acid (DA), thereby increasing environmental sensitivity for dauer induction. Consistent with known pleiotropic roles of DA, this eak-3 variant significantly slows down reproductive growth. We experimentally show that, although the eak-3 deletion can provide a fitness advantage through facilitated dauer production in stressful environments, this allele becomes rapidly outcompeted in favorable environments. The identified eak-3 variant therefore reveals a trade-off in how hormonal responses influence both the pace of developmental timing and the way in which environmental sensitivity controls adaptive plasticity. Together, our results show how a single mutational event altering hormonal signaling can lead to the emergence of a complex life history trade-off.

Descendant distributions for the impact of mutant contagion on networks

Contagion, broadly construed, refers to anything that can spread infectiously from peer to peer. Examples include communicable diseases, rumors, misinformation, ideas, innovations, bank failures, and electrical blackouts. Sometimes, as in the 1918 Spanish flu epidemic, a contagion mutates at some point as it spreads through a network. Here, using a simple susceptible-infected (SI) model of contagion, we explore the downstream impact of a single mutation event. Assuming that this mutation occurs at a random node in the contact network, we calculate the distribution of the number of "descendants," $d$, downstream from the initial "Patient Zero" mutant. We find that the tail of the distribution decays as $d^{-2}$ for complete graphs, random graphs, small-world networks, networks with block-like structure, and other infinite-dimensional networks. This prediction agrees with the observed statistics of memes propagating and mutating on Facebook, and is expected to hold for other effectively infinite-dimensional networks, such as the global human contact network. In a wider context, our approach suggests a possible starting point for a mesoscopic theory of contagion. Such a theory would focus on the paths traced by a spreading contagion, thereby furnishing an intermediate level of description between that of individual nodes and the total infected population. We anticipate that contagion pathways will hold valuable lessons, given their role as the conduits through which single mutations, innovations, or failures can sweep through a network as a whole.

The Fabry disease-causing mutation, GLA IVS4+919G>A, originated in Mainland China more than 800 years ago.

The Fabry disease-causing mutation, the GLA IVS4+919G>A (designated GLA IVS4), is very prevalent in patients with hypertrophic cardiomyopathy in Taiwan. This X-linked mutation has also been found in patients in Kyushu, Japan and Southeast Asia. To investigate the age and the possible ancestral origin of this mutation, a total of 33 male patients with the GLA IVS4+919G>A mutation, born in Taiwan, Japan, Singapore, Malaysia, Vietnam, and the Fujian and Guangdong provinces of China, were studied. Peripheral bloods were collected, and the Ilumina Infinium CoreExome-24 microarray was used for dense genotyping. A mutation-carrying haplotype was discovered which was shared by all 33 patients. This haplotype does not exist in 15 healthy persons without the mutation. Rather, a wide diversity of haplotypes was found in the vicinity of the mutation site, supporting the existence of a single founder of the GLA IVS4 mutation. The age of the founder mutation was estimated by the lengths of the mutation-carrying haplotypes based on the linkage-disequilibrium decay theory. The first, second, and third quartile of the age estimates are 800.7, 922.6, and 1068.4 years, respectively. We concluded that the GLA IVS4+919G>A mutation originated from a single mutational event that occurred in a Chinese chromosome more than 800 years ago.

Misannotation of multiple-nucleotide variants risks misdiagnosis.

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses that would previously have been made by standard Sanger (dideoxy) sequencing. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. This risk will be increased as confirmatory Sanger sequencing of Single Nucleotide variants (SNVs) ceases to be standard practice. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

Misannotation of multiple-nucleotide variants risks misdiagnosis

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

Misannotation of multiple-nucleotide variants risks misdiagnosis

Multiple Nucleotide Variants (MNVs) are miscalled by the most widely utilised next generation sequencing analysis (NGS) pipelines, presenting the potential for missing diagnoses that would previously have been made by standard Sanger (dideoxy) sequencing. These variants, which should be treated as a single insertion-deletion mutation event, are commonly called as separate single nucleotide variants. This can result in misannotation, incorrect amino acid predictions and potentially false positive and false negative diagnostic results. This risk will be increased as confirmatory Sanger sequencing of Single Nucleotide variants (SNVs) ceases to be standard practice. Using simulated data and re-analysis of sequencing data from a diagnostic targeted gene panel, we demonstrate that the widely adopted pipeline, GATK best practices, results in miscalling of MNVs and that alternative tools can call these variants correctly. The adoption of calling methods that annotate MNVs correctly would present a solution for individual laboratories, however GATK best practices are the basis for important public resources such as the gnomAD database. We suggest integrating a solution into these guidelines would be the optimal approach.

Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmosomal Variants Are Rarely De Novo.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with pathogenic/likely pathogenic (P/LP) variants in genes encoding the cardiac desmosomal proteins. Origin of these variants, including de novo mutation rate and extent of founder versus recurrent variants has implications for variant adjudication and clinical care, yet this has never been systematically investigated. We identified arrhythmogenic right ventricular cardiomyopathy probands who met 2010 Task Force Criteria and had undergone genotyping that included sequencing of the desmosomal genes (PKP2, DSP, DSG2, DSC2, and JUP) from 3 arrhythmogenic right ventricular cardiomyopathy registries in America and Europe. We classified the desmosomal variants, defined the contribution of unique versus nonunique (ie, not family-specific) P/LP variants, and identified the frequency and characteristics of de novo variants. Next, we haplotyped nonunique variants to determine how often they likely represent a single mutation event in a common ancestor (implied by shared haplotypes) versus multiple mutation events at the same genetic location. Of 501 arrhythmogenic right ventricular cardiomyopathy probands, 322 (64.3%) carried 327 desmosomal P/LP variants. Most variants (n=247, 75.6%, in 245 patients) were identified in more than one proband and, therefore, considered nonunique. For 212/327 variants (64.8%) genetic cascade screening was performed extensively enough to identify the parental origin of the P/LP variant. Only 3 variants were de novo, 2 of which were whole gene deletions. For 24 nonunique P/LP PKP2 variants, haplotyping was conducted in 183 available families. For all 24 variants, multiple seemingly unrelated families sharing identical haplotypes were identified, suggesting that these variants originate from common founders. Most desmosomal P/LP variants are inherited, nonunique, and originate from ancient founders. Two of 3 de novo variants were large deletions. These observations inform genetic testing, cascade screening, and variant adjudication.

Gastrointestinal Stromal Tumor With Multiple Primary Tyrosine Kinase Mutations-Clinicopathologic and Molecular Characterization.

A unique cohort of chemo-naive gastrointestinal stromal tumors (GISTs) with double-primary tyrosine kinase mutations was characterized particularly to determine whether coexistent mutations represent a single mutational event. Up to 2013, 4 UK centers reported 9 GISTs with 2 primary tyrosine kinase mutations. In each of 8 cases validated by next generation sequencing, both mutations were present in the same allele of the same exon (KIT exon 11 or 17, or PDGFRA exon 18). One case showed the second mutation only on some of the mutant alleles. Seven cases showed both mutations in all the reads, but in 2 cases, additional variants were found only in some reads. Clinicopathologic features of the 8 cases were similar to GISTs with single-primary mutations. When GIST genotyping rarely uncovers multiple tyrosine kinase variants in an exon, they occur in the same allele but are likely to represent separate mutational events and lack clinical significance.