Identification Of Single Nucleotide Variants Research Articles

Modified Unit-Mediated Strand Displacement Reactions for Direct Detection of Single Nucleotide Variants in Active Double-Stranded DNA.

Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.

PAX5 Alterations in a Consecutive Childhood B-Cell Acute Lymphoblastic Leukemia Cohort Treated Using the ALL IC-BFM 2009 Protocol.

In this study, we aimed to identify patients within our B-ALL cohort with altered PAX5. Our objective was to use a comprehensive analysis approach to characterize the types of genetic changes, determine their origin (somatic/germline), and analyze the clinical outcomes associated with them. A consecutive cohort of 99 patients with B-ALL treated at the Children's Hospital of the UMC Ljubljana according to the ALL IC-BFM 2009 protocol was included in our study. We used RNA sequencing data for gene expression analysis, fusion gene detection and single nucleotide variant identification, multiplex-ligation dependent probe amplification for copy number variation assessment, and Sanger sequencing for germline variant detection. PAX5 was impacted in 33.3% of our patients, with the genetic alterations ranging from CNVs and rearrangements to SNVs. The most common were CNVs, which were found in more than a third of patients, followed by point mutations in 5.2%, and gene rearrangements in 4.1%. We identified eight patients with a PAX5-associated genetic subtype that were previously classified as "B-other", and they showed intermediate outcomes. We showed higher minimal residual disease values at the end of induction and poorer event-free survival in hyperdiploid cases carrying duplications in PAX5 compared to other hyperdiploid cases. We also report an interesting case of a patient with PAX5::FKBP15 and a pathogenic variant in PTPN11 who underwent an early relapse with a monocytic switch. In conclusion, this study provides valuable insights into the presence, frequency, and prognostic significance of diverse PAX5 alterations in B-ALL patients, highlighting the complexity of genetic factors and their impact on patient outcomes.

First investigation of RH gene polymorphism in patients with sickle cell disease and associated blood donors in Cameroon, Central Africa.

Although genetic polymorphism of the RH blood group system is well known in sub-Saharan Africa, national/regional specificities still remain to be described precisely. For the first time in Cameroon, Central Africa, and in order to better characterize the molecular basis driving RH phenotype variability, as well as to identify the main antigens that may be potentially responsible for alloimmunization, we sought 1) to study the RH genes in a cohort of 109 patients with sickle cell disease; 2) to study the same genes in the corresponding donors whose red blood cells (RBCs) were transfused to the patients (108 donors in 98 patients); 3) to predict RH phenotype on the basis of the molecular data and compare the results with serologic testing; and 4) to identify retrospectively patients at risk for alloimmunization. In order to generate an exhaustive dataset, the RH genes of all patient and donor samples were systematically investigated 1) by quantitative multiplex PCR of short fluorescent fragments (QMPSF) for characterization of RHD gene zygosity and potential structural variants (SVs), and 2) by Sanger sequencing for identification of single nucleotide variants (SNVs). Subsequent to molecular analysis, the genotypes and RH phenotype were deduced and predicted, respectively, from reference databases. In a total of 217 Cameroonian individuals, as many as 24 and up to 22 variant alleles were identified in the RHD and RHCE genes, respectively, in addition to the reference alleles. Interestingly, 65 patients with SCD (66.3%) were assumed to be exposed to one or more undesirable RH antigen(s) with varying degrees of clinical relevance. Beyond the comprehensive report of the nature and distribution of RH variant alleles in a subset of Cameroonian patients treated by transfusion therapy, this work highlights the need for an extensive review of current practice, including routine serologic typing procedures, preferably in the near future.

Empirical Bayes single nucleotide variant-calling for next-generation sequencing data

One of the fundamental computational problems in cancer genomics is the identification of single nucleotide variants (SNVs) from DNA sequencing data. Many statistical models and software implementations for SNV calling have been developed in the literature, yet, they still disagree widely on real datasets. Based on an empirical Bayesian approach, we introduce a local false discovery rate (LFDR) estimator for germline SNV calling. Our approach learns model parameters without prior information, and simultaneously accounts for information across all sites in the genomic regions of interest. We also propose another LFDR-based algorithm that reliably prioritizes a given list of mutations called by any other variant-calling algorithm. We use a suite of gold-standard cell line data to compare our LFDR approach against a collection of widely used, state of the art programs. We find that our LFDR approach approximately matches or exceeds the performance of all of these programs, despite some very large differences among them. Furthermore, when prioritizing other algorithms’ calls by our LFDR score, we find that by manipulating the type I-type II tradeoff we can select subsets of variant calls with minimal loss of sensitivity but dramatic increases in precision.

Targeted haplotyping in pharmacogenomics using Oxford Nanopore Technologies' adaptive sampling.

Pharmacogenomics (PGx) studies the impact of interindividual genomic variation on drug response, allowing the opportunity to tailor the dosing regimen for each patient. Current targeted PGx testing platforms are mainly based on microarray, polymerase chain reaction, or short-read sequencing. Despite demonstrating great value for the identification of single nucleotide variants (SNVs) and insertion/deletions (INDELs), these assays do not permit identification of large structural variants, nor do they allow unambiguous haplotype phasing for star-allele assignment. Here, we used Oxford Nanopore Technologies' adaptive sampling to enrich a panel of 1,036 genes with well-documented PGx relevance extracted from the Pharmacogenomics Knowledge Base (PharmGKB). By evaluating concordance with existing truth sets, we demonstrate accurate variant and star-allele calling for five Genome in a Bottle reference samples. We show that up to three samples can be multiplexed on one PromethION flow cell without a significant drop in variant calling performance, resulting in 99.35% and 99.84% recall and precision for the targeted variants, respectively. This work advances the use of nanopore sequencing in clinical PGx settings.

CRISPR-based genome editing in disease treatment.

CRISPR-based genome editing tools used for genetic disease treatment mainly include Cas nucleases, base editors (BEs), and prime editors (PEs). High-throughput functional screenings enable accurate dissection of complex proteins, and identification of single nucleotide variants (SNVs) within the same gene, including those that confer sensitivity or resistance to drugs. Efficient delivery of genome editing machinery to target cells or tissues is crucial for successful genome modification either in or outside the body. Viral vectors such as lentivirus (LV), adenovirus (Adv), and adeno-associated virus (AAV) offer complementary advantages, and non-viral delivery provides the advantage of transient editing activity.

Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

Abstract 6: Applying Next-Generation Sequencing to Improve Pediatric Cancer Diagnosis in Mexico

Abstract Purpose: In Mexico, cancer is the second leading cause of mortality among children between 4 to 15 years old. The overall cure rates remain below 50%, compared to >90% in the United States. This discrepancy is in part explained by the limited access to methods for accurate diagnosis and identification of appropriate therapies in Mexico. Only 48% of large hospitals across Mexico report having the capacity to perform some of the complex testing necessary for subclassification of pediatric cancers. Pan-cancer next generation sequencing (NGS) panels are not available in Mexico. We developed a strategy to perform clinical NGS in pediatric cancer patients in Mexico for identification of single nucleotide variants, copy number changes, and fusion events that are useful for risk stratification and adjusted therapy. Methods: We established collaborative agreements between UCSF and the National Institute for Genomic Medicine (INMEGEN) in Mexico City. We developed a pediatric cancer focused panel (IMOP) encompassing 200 genes and created a cloud-based bioinformatic and analysis pipeline for remote data interpretation. We established concordance by comparing the sequencing results of clinical cases obtained with the validated UCSF500 panel and the IMOP panel. Results: The results of 8 clinical samples previously sequenced using the clinically validated UCSF500 panel were compared to results obtained using the IMOP panel. Clinically relevant SNVs and fusions were successfully detected by the IMOP panel at similar variant allelic frequencies. The IMOP panel was shipped to INMEGEN where two samples with prior sequencing results were successfully sequenced. Re-sequencing of the 8 clinical samples to establish concordance at INMEGEN is pending. Conclusion: Our ongoing pilot proof-of-concept study shows feasibility of a strategy to implement clinical NGS testing in a middle-income country. We hypothesize that implementation of centralized NGS for pediatric cancers at INMEGEN will improve diagnostic accuracy. Moving forward, we plan to complete the clinical validation of the IMOP panel for routine sequencing of pediatric cancer patients. We will use the RE-AIM implementation science framework to assess the effectiveness of the assay in refining clinical diagnosis and treatment planning for pediatric cancer cases in Mexico. Citation Format: Roberto Ruiz-Cordero, Ela Alcantara, Ana Quintanar-Alfaro, Carlos A Espinoza-Mendez, Katherine Van Loon, Lindsay Breithaupt, Jorge Melendez Zajgla, Alejandro Sweet-Cordero. Applying Next-Generation Sequencing to Improve Pediatric Cancer Diagnosis in Mexico [abstract]. In: Proceedings of the 11th Annual Symposium on Global Cancer Research; Closing the Research-to-Implementation Gap; 2023 Apr 4-6. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(6_Suppl):Abstract nr 6.

Identification of single nucleotide variants in SLC26A9 gene in patients with cystic fibrosis (p.Phe508del homozygous) and its association to Orkambi® (Lumacaftor and Ivacaftor) response in vitro

BackgroundSince patients with cystic fibrosis with different Cystic Fibrosis Transmembrane Regulator (CFTR) genotypes present a wide response variability for modulator drugs such as Orkambi®, it is important to screen variants in candidate genes with an impact on precision and personalized medicine, such as Solute Carrier Family 26, member 9 (SLC26A9) gene. MethodsSanger sequencing for the exons and intron–exon boundary junctions of the SLC26A9 gene was employed in nine individuals with p.Phe508del homozygous genotype for the CFTR gene who were not under CFTR modulators therapy. The sequencing variants were evaluated by in silico prediction tools. The CFTR function was measured by cAMP–stimulated current (ΔIsc-eq-FSK) in polarized CFTR of human nasal epithelial cells cultured in micro–Ussing chambers with Orkambi®. ResultsWe found 24 intronic variants, three in the coding region (missense variants – rs74146719 and rs16856462 and synonymous – rs33943971), and three in the three prime untranslated region (3′ UTR) region in the SLC26A9 gene. Twenty variants were considered benign according to American College of Medical Genetics and Genomics guidelines, and ten were classified as uncertain significance. Although some variants had deleterious predictions or possible alterations in splicing, the majority of predictions were benign or neutral. When we analyzed the ΔIsc-eq-FSK response to Orkambi®, there were no significant differences within the genotypes and alleles for all 30 variants in the SLC26A9 gene. ConclusionsAmong the nine individuals with p.Phe508del homozygous genotype for the CFTR gene, no pathogenic SLC26A9 variants were found, and we did not detect associations from the 30 SLC26A9 variants and the response to the Orkambi® in vitro.

Impact of genetic structural variants in factor XI deficiency: identification, accurate characterization, and inferred mechanism by long-read sequencing

BackgroundCongenital factor XI (FXI) deficiency is a probably underestimated coagulopathy that confers antithrombotic protection. Characterization of genetic defects in F11 is mainly focused on the identification of single-nucleotide variants and small insertion/deletions because they represent up to 99% of the alterations accounting for factor deficiency, with only 3 gross gene defects of structural variants (SVs) having been described. ObjectivesTo identify and characterize the SVs affecting F11. MethodsThe study was performed in 93 unrelated subjects with FXI deficiency recruited in Spanish hospitals over a period of 25 years (1997-2022). F11 was analyzed by next-generation sequencing, multiplex ligand probe amplification, and long-read sequencing. ResultsOur study identified 30 different genetic variants. Interestingly, we found 3 SVs, all heterozygous: a complex duplication affecting exons 8 and 9, a tandem duplication of exon 14, and a large deletion affecting the whole gene. Nucleotide resolution obtained by long-read sequencing revealed Alu repetitive elements involved in all breakpoints. The large deletion was probably generated de novo in the paternal allele during gametogenesis, and despite affecting 30 additional genes, no syndromic features were described. ConclusionSVs may account for a high proportion of F11 genetic defects implicated in the molecular pathology of congenital FXI deficiency. These SVs, likely caused by a nonallelic homologous recombination involving repetitive elements, are heterogeneous in both type and length and may be de novo. These data support the inclusion of methods to detect SVs in this disorder, with long-read–based methods being the most appropriate because they detect all SVs and achieve adequate nucleotide resolution.

Quality metrics for enhanced performance of an NGS panel using single-vial amplification technology

AimsTargeted next-generation sequencing (NGS) panels, which identify genomic alterations, are the stronghold of molecular oncology laboratories. In spite of technological advances, the quantity and quality of DNA from formalin-fixed paraffin-embedded...

Phylogeny, molecular evolution, and dating of divergences in Lagerstroemia using plastome sequences

Lagerstroemia L. (Lythraceae) is a widely distributed genus of trees and shrubs native to tropical and subtropical environments from Southeast Asia to Australia, with numerous species highly valued as ornamentals. Although the plastomes of many species in this genus have been sequenced, the rates of functional gene evolution and their effect on phylogenetic analyses have not been thoroughly examined. We compared three plastome sequence matrices to elucidate how differences in these datasets affected phylogenetic analyses. Robust phylogenetic relationships for Lagerstroemia species were reconstructed based on different plastome sequence partitions and multiple phylogenetic methods. Identification of single-nucleotide variants within different genes also provides basic data on the patterns of functional gene evolution in Lagerstroemia and may provide insights into how those mutations affect protein structure and potentially drive divergence via cytonuclear incompatibility. These results as well as analyses of non-synonymous and synonymous mutations, indicate that heterotachic modes of evolution are present in functional plastome genes and should be accounted for in the analyses of molecular evolution. In addition, divergence events within the Lagerstroemia were dated for the first time. Several of the divergence estimates corresponded to well-known Earth history events, such as the reduction in global temperatures at the Eocene/Oligocene boundary. Our analyses conducted in Lagerstroemia here dissects the various patterns in the divergence of Lagerstroemia and may provide a useful guide to help plant breeders, as well as the necessity of using plastomic data and as possible as to combine evidence from morphological characteristics to investigate the complicated interspecies relationship and the evolutionary dynamics of species.

Abstract 3992: Modular ddPCR assay design for highly sensitive single nucleotide variant detection

Abstract Introduction: Single-nucleotide variants (SNVs) are key drivers of both oncogenesis and emergent resistance to targeted therapies. Although high-throughput Identification of SNVs can be achieved with Next Generation Sequencing (NGS) technologies, the cost is significant, and proportional to the desired sensitivity of variant detection. Conversely, while only small numbers of SNVs can be queried in an individual quantitative PCR (qPCR) or droplet digital PCR (ddPCR) reaction, a fully optimized qPCR or ddPCR assay can detect SNVs at very low frequencies for a fraction of the cost of sequencing. Herein, we present a generalizable approach to PCR assay design, using a combination of mutation-sensitive induced mismatch primers, wild-type suppressors, and TaqMan probes, that permits detection of SNVs below 0.1%. Method: Known oncogenic hotspot regions were selected for development of mutation-detecting PCR assays. For each hotspot, a mutation-specific-mismatch primer, suppressor, and probe (MSMPSP) cassette was designed consisting of 1) a mutation-bearing forward primer with engineered mismatches, 2) a wild-type, extension-locked blocker oligo, 3) a fluorescent TaqMan probe, and 4) a downstream reverse primer. Designed mutation assays were evaluated using plasmid control sequences, remnant clinical specimens, and healthy donor peripheral blood samples spiked with known quantities of mutation-bearing plasmid or cell lines and processed on the BioRad AutoDG/QX200 droplet digital PCR system. Results: Variant detection below 0.1% was achieved for all tested mutations, including those residing in homopolymeric and GC-rich regions, from as low as 100 ng DNA input, in a single day. At higher variant frequencies accessible to multiple platforms, results with MSMPSP-based ddPCR assays exhibited excellent concordance with NGS and qPCR results and yielded linear signal decline down to 0.01% variant allele frequency. Overall assay sensitivity was proportional to DNA input, and improved detection limits were achieved by increased DNA input and combined well analysis. Conclusion: Utilizing the MSMPSP approach, coupled with ddPCR, variant detection for targeted mutations below 0.1% is achievable in diverse genomic contexts. Furthermore, simple design principles and flexible probe-binding sites permit rapid development of new assays, allowing critical mutations to be tracked as they are discovered. While multiplexing capabilities are reduced compared with NGS, short time-to-results, coupled with low cost-per-sample for MSMPSP ddPCR, permits faster treatment decisions at reduced cost to the healthcare system. Citation Format: Sarah Heine, Ashley Beams, Nathan Riccitelli, Shabnam Tangri. Modular ddPCR assay design for highly sensitive single nucleotide variant detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3992.

Identification of multiple single-nucleotide variants for clinical evaluation of Helicobacter pylori drug resistance

Helicobacter pylori infections are threats to public health due to their high infection rate and drug resistance. Identification of single-nucleotide variants (SNVs) in H. pylori is crucial for both diagnosis and therapy. Yet the clinical testing of resistant H. pylori mutants is still facing some challenges, such as the selectivity is not good enough for SNVs in abundant wild-type DNA, the lack of clinical validation and the economical burden on patients. Herein, an X-shaped DNA probe with a toehold initiator was designed, which could specifically hybridize with certain genotype DNA due to the thermodynamically driven reaction. A competitive reaction was developed to amplify the thermodynamic difference between wild-type DNA and SNVs, diminishing the interference of wild-type DNA. By this means, multiple SNVs in H. pylori were successfully identified and two SNVs related to clarithromycin resistance are chosen as model targets. A paper strip was fabricated for visual, fast screening of SNVs. Furthermore, the approach was validated using clinical samples, and a point-of-care (POCT) testing diagnosis was executed on saliva samples, demonstrating its potential for the prevention and cure of H. pylori infections.

Abstract 2214: The genomic landscape of lung cancer in post-menopausal never-smoker women

Abstract Lung cancer is the deadliest form of cancer worldwide. Cigarette smoking is the primary risk factor for developing lung cancer, fueling global tobacco cessation programs. In the United States, it is projected that by 2065 there will be a 63% reduction in smoking-associated lung cancer burden. However, even with this decline, lung cancer will still account for a significant proportion of cancer-associate mortality, suggesting that factors beyond smoking contribute to lung tumorigenesis. Lung cancer in never smokers is a distinct sub-group that needs specific exploration. It is the 7th highest cause of cancer-deaths worldwide, with an increasing trend in its incidence. Thus far, the majority of tumor genome-wide sequencing efforts in lung cancer have been focused on patient cohorts with a smoking history. We know that LCNS is distinct from lung adenocarcinoma found in smokers at the genetic level, however large gaps in our understanding of this population persist. Through a unique collaboration with the Women's Health Initiative (WHI) we now have access to a large repository of tumor tissue from LCNS patients. We have performed exome-sequencing on this cohort and here we describe novel mutations, structural variants and copy number changes in this cohort. Whole-exome sequencing of 75 tumor-normal pairs was performed. Mutations were identified for the entire cohort using different computational methods. For the identification of single nucleotide variants a consensus dual-caller and filtering methodology was employed. Variants were then analyzed to identify genes with significant mutations using MutSig2CV. Structural variant analysis was performed using SvABA and copy number analysis was performed using TITAN. Finally, we perform correlative analysis of mutations with the metadata curated by the WHI and highlight key features of this cohort. Our mutational analysis reveals showed that there is an enrichment of known oncogenes including KRAS, EGFR and TP53. Additionally, we identified mutations in STK11. Here we describe novel genes and mutations previously unknown to occur in LCNS cohort and describe their potential functional implications. We also report novel structural variants and copy number alterations. Additionally we performed mutational signature analysis and identified novel signatures specific to this cohort. Mutational analysis of our LCNS cohort reveals that this cohort in general has a lower tumor mutational burden compared to their control cohort with a history of smoking. Additionally, we find that this cohort has mutations that have been previously reported in studies in LCNS patients. However, the existence of novel mutations and structural alterations may offer a unique opportunity for future target identification and therapeutic intervention. Citation Format: Sitapriya Moorthi, Amy Paguirigan, Garnet Anderson, Gavin Ha, Alice Berger. The genomic landscape of lung cancer in post-menopausal never-smoker women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2214.

A new approach to decode DNA methylome and genomic variants simultaneously from double strand bisulfite sequencing.

Genetic and epigenetic contributions to various diseases and biological processes have been well-recognized. However, simultaneous identification of single-nucleotide variants (SNVs) and DNA methylation levels from traditional bisulfite sequencing data is still challenging. Here, we develop double strand bisulfite sequencing (DSBS) for genome-wide accurate identification of SNVs and DNA methylation simultaneously at a single-base resolution by using one dataset. Locking Watson and Crick strand together by hairpin adapter followed by bisulfite treatment and massive parallel sequencing, DSBS simultaneously sequences the bisulfite-converted Watson and Crick strand in one paired-end read, eliminating the strand bias of bisulfite sequencing data. Mutual correction of read1 and read2 can estimate the amplification and sequencing errors, and enables our developed computational pipeline, DSBS Analyzer (https://github.com/tianguolangzi/DSBS), to accurately identify SNV and DNA methylation. Additionally, using DSBS, we provide a genome-wide hemimethylation landscape in the human cells, and reveal that the density of DNA hemimethylation sites in promoter region and CpG island is lower than that in other genomic regions. The cost-effective new approach, which decodes DNA methylome and genomic variants simultaneously, will facilitate more comprehensive studies on numerous diseases and biological processes driven by both genetic and epigenetic variations.

Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes

BackgroundATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane-anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans.MethodsTo expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles, we performed exome sequencing for identification of single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3a Gal4 knockin-null allele was generated using CRISPR-Cas9 genome editing technology to aid the interpretation of variants.ResultsWe report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. The variants included four missense variants inherited in trans to loss-of-function alleles (p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val)), a homozygous missense variant p.(Arg327Pro), and a heterozygous non-frameshift indel p.(Lys568del). Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, Arg327Pro, and Lys568del are severe loss-of-function alleles leading to early developmental lethality. Further, we showed that Phe50Leu, Gly236Val, and Arg327Pro cause neurogenesis defects. On the contrary, Leu77Val and Arg170Trp are partial loss-of-function alleles that cause progressive locomotion defects and whose expression leads to an increase in autophagy and mitophagy in adult muscles.ConclusionOur findings expand the allelic spectrum of ATAD3A variants and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma.

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

Improvement of Molecular Diagnosis Using Domain-Level Single-Nucleotide Variants by Eliminating Unexpected Secondary Structures.

Identification of single-nucleotide variants (SNVs) is of great significance in molecular diagnosis. The problem that should not be ignored in the identification process is that the unexpected secondary structure of the target nucleic acid may greatly affect the detection accuracy. Herein, we proposed a conditional domain-level SNV diagnosis strategy, in which the subsequent SNV detection can only be carried out after eliminating the unexpected secondary structure of target DNA. Specifically, the target DNA is assembled into a rigid double strand, which makes folding the target DNA difficult and the unexpected secondary structure is eliminated. Based on this double-stranded structure, specially designed probes are used to detect double-stranded properties and report abundant domain-level oligonucleotide information to improve the effective information in the detection results and complete domain-level SNV diagnosis. If the unexpected secondary structure is not eliminated, the detector will first detect it and feed back to us, ensuring the accuracy of the subsequent detection results. With the occurrence (or not) of SNV and the change of the SNV site, in the proof-of-concept experiment, we successfully identified the four homologous sequences to be tested related to BRAF gene.

Identification of single nucleotide variants in the Moroccan population by whole-genome sequencing

BackgroundLarge-scale human sequencing projects have described around a hundred-million single nucleotide variants (SNVs). These studies have predominately involved individuals with European ancestry despite the fact that genetic diversity is expected to be highest in Africa where Homo sapiens evolved and has maintained a large population for the longest time. The African Genome Variation Project examined several African populations but these were all located south of the Sahara. Morocco is on the northwest coast of Africa and mostly lies north of the Sahara, which makes it very attractive for studying genetic diversity. The ancestry of present-day Moroccans is unknown and may be substantially different from Africans found South of the Sahara desert, Recent genomic data of Taforalt individuals in Eastern Morocco revealed 15,000-year-old modern humans and suggested that North African individuals may be genetically distinct from previously studied African populations.ResultsWe present SNVs discovered by whole genome sequencing (WGS) of three Moroccans. From a total of 5.9 million SNVs detected, over 200,000 were not identified by 1000G and were not in the extensive gnomAD database. We summarise the SNVs by genomic position, type of sequence gene context and effect on proteins encoded by the sequence. Analysis of the overall genomic information of the Moroccan individuals to individuals from 1000G supports the Moroccan population being distinct from both sub-Saharan African and European populations.ConclusionsWe conclude that Moroccan samples are genetically distinct and lie in the middle of the previously observed cline between populations of European and African ancestry. WGS of Moroccan individuals can identify a large number of novel SNVs and aid in functional characterisation of the genome.