Single Base Pair Substitutions Research Articles

Prediction of Deleterious Single Amino Acid Polymorphisms with a Consensus Holdout Sampler.

Single Amino Acid Polymorphisms (SAPs) or nonsynonymous Single Nucleotide Variants (nsSNVs) are the most common genetic variations. They result from missense mutations where a single base pair substitution changes the genetic code in such a way that the triplet of bases (codon) at a given position is coding a different amino acid. Since genetic mutations sometimes cause genetic diseases, it is important to comprehend and foresee which variations are harmful and which ones are neutral (not causing changes in the phenotype). This can be posed as a classification problem. Computational methods using machine intelligence are gradually replacing repetitive and exceedingly overpriced mutagenic tests. By and large, uneven quality, deficiencies, and irregularities of nsSNVs datasets debase the convenience of artificial intelligence-based methods. Subsequently, strong and more exact approaches are needed to address these problems. In the present work paper, we show a consensus classifier built on the holdout sampler, which appears strong and precise and outflanks all other popular methods. We produced 100 holdouts to test the structures and diverse classification variables of diverse classifiers during the training phase. The finest performing holdouts were chosen to develop a consensus classifier and tested using a k-fold (1 ≤ k ≤5) cross-validation method. We also examined which protein properties have the biggest impact on the precise prediction of the effects of nsSNVs. Our Consensus Holdout Sampler outflanks other popular algorithms, and gives excellent results, highly accurate with low standard deviation. The advantage of our method emerges from using a tree of holdouts, where diverse LM/AI-based programs are sampled in diverse ways.

Kataegis in clinical and molecular subgroups of primary breast cancer

Kataegis is a hypermutation phenomenon characterized by localized clusters of single base pair substitution (SBS) reported in multiple cancer types. Despite a high frequency in breast cancer, large-scale analyses of kataegis patterns and associations with clinicopathological and molecular variables in established breast cancer subgroups are lacking. Therefore, WGS profiled primary breast cancers (n = 791) with associated clinical and molecular data layers, like RNA-sequencing data, were analyzed for kataegis frequency, recurrence, and associations with genomic contexts and functional elements, transcriptional patterns, driver alterations, homologous recombination deficiency (HRD), and prognosis in tumor subgroups defined by ER, PR, and HER2/ERBB2 status. Kataegis frequency was highest in the HER2-positive(p) subgroups, including both ER-negative(n)/positive(p) tumors (ERnHER2p/ERpHER2p). In TNBC, kataegis was neither associated with PAM50 nor TNBC mRNA subtypes nor with distant relapse in chemotherapy-treated patients. In ERpHER2n tumors, kataegis was associated with aggressive characteristics, including PR-negativity, molecular Luminal B subtype, higher mutational burden, higher grade, and expression of proliferation-associated genes. Recurrent kataegis loci frequently targeted regions commonly amplified in ER-positive tumors, while few recurrent loci were observed in TNBC. SBSs in kataegis loci appeared enriched in regions of open chromatin. Kataegis status was not associated with HRD in any subgroup or with distinct transcriptional patterns in unsupervised or supervised analysis. In summary, kataegis is a common hypermutation phenomenon in established breast cancer subgroups, particularly in HER2p subgroups, coinciding with an aggressive tumor phenotype in ERpHER2n disease. In TNBC, the molecular implications and associations of kataegis are less clear, including its prognostic value.

Abstract 6127: Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals

Abstract Background: Approximately 50% of high-grade serous ovarian carcinomas (HGSOC) are homologous recombination deficient (HRD). HRD is associated with increased T-cell infiltration and improved survival. This study aimed to describe the distribution of HRD features and cytotoxic T-cell infiltration by gene expression subtype among Black HGSOC cases. Methods: We included Black HGSOC cases from the African American Cancer Epidemiology Study and the North Carolina Ovarian Cancer Study. We used RNASeq data to assign gene expression subtypes (n=160), and whole exome sequencing data from matched blood and tumor formalin-fixed paraffin-embedded specimens (n=206) to characterize HRD features. We defined HRD features as (1) germline variants and somatic mutations in HRD genes, and (2) HRD-associated signatures (SBS3 and ID6) that we obtained from de novo mutational signature analysis for single-base pair substitution, insertion, and deletion. We estimated the proportion of intratumoral cytotoxic T cells using multiplex immunofluorescence (n=125). Results: The subtype distribution in our study was 39% immunoreactive, 29% mesenchymal 27% proliferative, and 4% differentiated. Germline variants and somatic mutations in any HRD gene were observed in 27% and 23% of cases, respectively, and at least one of these was observed in 42% of cases. Pathogenic germline variants and somatic mutations were observed in 7% and 3% of cases, respectively. The prevalence of germline or somatic variants by gene was 16% for BRCA1, 14% for BRCA2, 11% for ATM, 5% for RAD51, and 2% each for BRIP1, PALB2, and BARD1. We observed a COSMIC HRD signature in 43% of cases, with 25% SBS3 only, 5% ID6 only, and 13% with both signatures. The prevalence of any HRD gene variant or signature was higher in immunoreactive cases (52% and 59%, respectively) than in mesenchymal (28% and 43%) or proliferative (44% and 28%) cases. Germline HRD variants were more common in the proliferative subtype (44%) compared with immunoreactive (25%) and mesenchymal (17%). In contrast, somatic HRD mutations were more common in the immunoreactive subtype (33%), compared with proliferative (16%) and mesenchymal (15%). This was due to a higher occurrence of BRCA1 somatic mutations in the immunoreactive subtype (25%) compared with proliferative (9%) and mesenchymal (8%). A higher proportion of cytotoxic T-cells (≥1% vs <1%) was also observed in the immunoreactive subtype (44%) compared with mesenchymal (31%) and proliferative (11%). Conclusion: We observed differences in the distribution of HRD features and cytotoxic T-cells by gene expression subtype. In particular, in the immunoreactive subtype, the most common gene expression subtype in Black HGSOC cases, we observed a higher occurrence of any HRD feature, somatic BRCA1 mutations, and cytotoxic T cell infiltration than in any of the other gene expression subtypes. Citation Format: Katherine Lawson-Michod, Mollie E. Barnard, Natalie Davidson, Lindsay J. Collin, Courtney Johnson, Lucas A. Salas, Casey Greene, Jeffrey R. Marks, Lauren Peres, Joellen M. Schildkraut, Jennifer A. Doherty. Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6127.

A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Role of KatG in Francisella tularensis Susceptibility to Resazurin.

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the risk of potential release of antibiotic-resistant F. tularensis strains, new therapeutics against F. tularensis must be developed. Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The mode of action of resazomycins is not understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The FTL_1504 gene was identified as being mutated in all of the Rz-resistant (Rzr) strains sequenced. FTL_1504 encodes for the catalase KatG which catalyzes the decomposition of hydrogen peroxide to water and oxygen. Moreover, KatG has also been shown to play an essential role in the activity of a different antibiotic, isonazid. Four different coding mutations were characterized in katG that included single base pair substitutions and a deletion with all resulting in the introduction of a premature stop codon. All Rzr mutants tested had reduced catalase activity compared to wild-type F. tularensis LVS with three of the four mutations resulting in loss of KatG expression. Based on this data, we hypothesize KatG plays a role in the susceptibility of F. tularensis to resazurin. To address this, we are generating a katG deletion mutant in F. tularensis LVS and measuring its Rz susceptibility via agar dilution assays. Investigation of the contribution of KatG may unveil a unique role for the oxidative stress response in the mode of action of this family of antibiotics

Abstract 3506: High-grade serous ovarian cancer somatic mutational signatures in Black and White individuals

Abstract Causes of racial disparities in ovarian cancer survival are likely multifactorial, including socio-environmental, structural, and biological factors. Mutational signatures reflect endogenous and exogenous exposures, which may differ by race and ethnicity. This study aims to characterize mutational signatures and their associated etiologies among Black high-grade serous ovarian cancer (HGSC) cases, and to compare the occurrence and distribution of signatures with White TCGA cases. Mutational profiling with whole exome sequencing in matched blood and tumor formalin-fixed paraffin-embedded specimens was performed in 271 HGSC cases identifying as Black or African American and participating in the African American Cancer Epidemiology Study (AACES) or the North Carolina Ovarian Cancer Study (NCOCS). After filtering, we identified 28,601 high-confidence coding mutations in 256 samples that passed quality control assessments. The median number of mutations per sample was 67, similar to that in TCGA at 66. Mutation frequencies of the major genes identified in TCGA were nearly identical in Black AACES/NCOCS cases; mutations in TP53 were present in 96% and 89%, respectively, and mutation frequencies in BRCA1/2, CSMD3, NF1, CDK12, FAT3, GABRA6, and RB1 differed by only 0-2%. We performed mutational signature analysis using SigProfiler and refitting to the Catalogue of Somatic Mutations in Cancer (COSMIC) previously published single base-pair substitution (SBS) signatures reflecting mutational processes resulting from known endogenous and exogenous exposures. SBS signatures are defined by 96 mutation features including the single nucleotide variant class (e.g., C>A, C>G, C>T, T>A, T>C, and T>G) and the identity of the 5’/3’ flanking bases. We observed much higher frequencies of the mismatch repair deficiency, homologous recombination deficiency, ultraviolet light, and treatment exposure signatures in Black AACES/NCOCS cases (26%, 22%, 9%, and 8%, respectively) than in White TCGA cases (3%, 10%, 3%, and 2%, respectively). The frequencies of the remaining signatures with known etiologies (clock-like, reactive oxygen species, base excision repair, mutagen exposure, DNA polymerase epsilon, and tobacco signatures) differed by less than 5% between Black and White cases. Many mismatch repair deficiency signatures identified in Black AACES/NCOCS cases are dominated by C>T or T>C mutations and may represent an artifact from formalin fixation. While we observed that the somatic mutation frequencies in major genes associated with HGSC were similar in Black and White cases, the frequencies of some known mutational signatures were considerably higher in Black HGSC. In particular, despite similar frequencies of BRCA1/2 mutations, the homologous recombination deficiency signature was considerably more common in Black than White HGSC. Citation Format: Katherine A. Lawson-Michod, David Nix, Lindsay Collin, Natalie Davidson, Ariel Hippen, Chad Huff, Aaron Atkinson, Lucas A. Salas, Lauren Peres, Casey Greene, Joellen Schildkraut, Jeffrey Marks, Jennifer A. Doherty. High-grade serous ovarian cancer somatic mutational signatures in Black and White individuals [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 3506.

MP33-19 MOLECULAR CHARACTERISTICS AND ACTIONABLE TARGETS OF TESTICULAR GERM CELL TUMORS IN REAL-WORLD CONDITIONS

You have accessJournal of UrologyCME1 Apr 2023MP33-19 MOLECULAR CHARACTERISTICS AND ACTIONABLE TARGETS OF TESTICULAR GERM CELL TUMORS IN REAL-WORLD CONDITIONS Rafael Morales-Grimany, Cesar Delgado, Fady Baky, Armon Amini, Thomas Gerald, Rohit R Badia, Jacob Taylor, Luke L Wang, Juan Javier-Desloges, Vitaly Margulis, Solomon L. Woldu, Amir Salmasi, Fred Millard, Rana R. Mckay, and Aditya Bagrodia Rafael Morales-GrimanyRafael Morales-Grimany More articles by this author , Cesar DelgadoCesar Delgado More articles by this author , Fady BakyFady Baky More articles by this author , Armon AminiArmon Amini More articles by this author , Thomas GeraldThomas Gerald More articles by this author , Rohit R BadiaRohit R Badia More articles by this author , Jacob TaylorJacob Taylor More articles by this author , Luke L WangLuke L Wang More articles by this author , Juan Javier-DeslogesJuan Javier-Desloges More articles by this author , Vitaly MargulisVitaly Margulis More articles by this author , Solomon L. WolduSolomon L. Woldu More articles by this author , Amir SalmasiAmir Salmasi More articles by this author , Fred MillardFred Millard More articles by this author , Rana R. MckayRana R. Mckay More articles by this author , and Aditya BagrodiaAditya Bagrodia More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003266.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Molecular features of testicular germ cell tumors (GCT) in various clinical states (pre- vs post-chemotherapy, localized versus metastatic) may inform treatment options for patients with recurrence after definitive therapy. In his study, we describe molecular features and potential therapeutic targets in a cohort of patients with testicular GCT. METHODS: We retrospectively examined clinicopathologic and next-generation sequencing (NGS) data from 27 patients with GCT. Tumors were sequenced using the TempusxT solid tumor assay, which includes DNA sequencing of 595-648 genes at 500x coverage and RNA sequencing for all human coding genes. Tumor mutational burden (TMB) was measured for all tumors and PD-L1 levels were assessed qualitatively by 22C3 pharmDx immunohistochemistry assay in 8 patients. All genetic variants detected were quantified and analyzed to identify potentially actionable targets. RESULTS: We identified 13 (48%) stage I GCT, 11 (41%) stage II, and 3 (11%) stage III. There were 7 seminomas and 20 nonseminomas. 12 tumor specimen resections were obtained from orchiectomy, and 15 from retroperitoneal lymph node dissection (RPLND), of which, 8 were chemotherapy-naïve and 7 were post-chemotherapy. Chemo-naïve RPLND histology showed a combination of teratoma, seminoma, and mixed GCT, while post-chemo histology revealed 6 teratomas and 9 benign pathologies. The median TMB for the cohort was 0.75 mutations/megabase. Somatic mutations were identified in 55% of patients and most commonly within: KRAS (25.9%), KIT (11.1%), and PIK3CB (7.4%). PD-L1 expression was observed in 75% of the tumors measured (60% positivity at stage I and 100% positivity at stage III). Microsatellite stability was stable in 18 tumors tested. DNA alterations- [single base pair substitutions, insertions, and deletions]- in KRAS (GTPase) proto-oncogenes were detected in 7 tumors and tyrosine kinase receptor gene variants (KIT, P1K3CB) were found at similar frequencies across disease stages. Whole transcriptome NGS RNA expression assays were performed on 21 untreated specimens revealing overexpression of MTOR (33%), MAPK1(14%), and MET (8.0%). Actionable targets with FDA-approved therapies in other organ tissues were detected in 11 patients (40.7%). Incidental germline mutations, including MSH6, RB1, and MSH2, were identified in 9 patients though all were variants of unknown significance. CONCLUSIONS: In our study, a significant proportion of patients had potentially actionable molecular targets across the disease spectrum. The identified genetic alterations provide a genomic landscape for risk stratification, future therapies, and molecularly informed treatment paradigms for GCT patients. Source of Funding: Cancer Prevention and Research Institute © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e458 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rafael Morales-Grimany More articles by this author Cesar Delgado More articles by this author Fady Baky More articles by this author Armon Amini More articles by this author Thomas Gerald More articles by this author Rohit R Badia More articles by this author Jacob Taylor More articles by this author Luke L Wang More articles by this author Juan Javier-Desloges More articles by this author Vitaly Margulis More articles by this author Solomon L. Woldu More articles by this author Amir Salmasi More articles by this author Fred Millard More articles by this author Rana R. Mckay More articles by this author Aditya Bagrodia More articles by this author Expand All Advertisement PDF downloadLoading ...

Scarless DNA Recombineering.

The method described here allows editing of the bacterial genome without leaving any secondary changes (scars) behind. This method uses a tripartite selectable and counterselectable cassette comprising an antibiotic-resistance gene (cat or kan) and the tetR repressor gene linked to a Ptet promoter-ccdB toxin gene fusion. In the absence of induction, the tetR gene product represses the Ptet promoter, preventing ccdB expression. The cassette is first inserted at the target site by selecting for chloramphenicol or kanamycin resistance. It is subsequently replaced by the sequence of interest by selecting for growth in the presence of anhydrotetracycline (AHTc), which inactivates the TetR repressor thereby causing CcdB-induced lethality. Unlike other CcdB-based counterselection schemes, which require specifically designed λ-Red delivery plasmids, the system described here uses the popular plasmid pKD46 as the source of λ-Red functions. This protocol allows a wide variety of modifications, including the intragenic insertion of fluorescent or epitope tags, gene replacements, deletions, and single base-pair substitutions, to be made. In addition, the procedure can be used to place the inducible Ptet promoter at a chosen position in the bacterial chromosome.

Molecular features and actionable targets for testicular germ cell tumors in a real-world setting.

430 Background: Molecular features of testicular germ cell tumors (GCT) in various clinical states (pre- vs post-chemotherapy, localized versus metastatic) may inform treatment options for patients with recurrence after definitive therapy. In his study, we describe molecular features and potential therapeutic targets in a cohort of patients with testicular GCT. Methods: We retrospectively examined clinicopathologic and next-generation sequencing (NGS) data from 27 patients with GCT. Tumors were sequenced using the Tempus|xT solid tumor assay, which includes DNA sequencing of 595-648 genes at 500x coverage and RNA sequencing for all human coding genes. Tumor mutational burden (TMB) was measured for all tumors and PD-L1 levels were assessed qualitatively by 22C3 pharmDx immunohistochemistry assay in 8 patients. All genetic variants detected were quantified and analyzed to identify potentially actionable targets. Results: We identified 13 (48%) stage I GCT, 11 (41%) stage II, and 3 (11%) stage III. There were 7 seminomas and 20 nonseminomas. 12 tumor specimen resections were obtained from orchiectomy, and 15 from retroperitoneal lymph node dissection (RPLND), of which, 8 were chemotherapy-naïve and 7 were post-chemotherapy. Chemo-naïve RPLND histology showed a combination of teratoma, seminoma, and mixed GCT, while post-chemo histology revealed 6 teratomas and 9 benign pathologies. The median TMB for the cohort was 0.75 mutations/megabase. Somatic mutations were identified in 55% of patients and most commonly within: KRAS (25.9%), KIT (11.1%), and PIK3CB (7.4%). PD-L1 expression was observed in 75% of the tumors measured (60% positivity at stage I and 100% positivity at stage III). Microsatellite stability was stable in 18 tumors tested. DNA alterations- [single base pair substitutions, insertions, and deletions]- in KRAS (GTPase) proto-oncogenes were detected in 7 tumors and tyrosine kinase receptor gene variants (KIT, P1K3CB) were found at similar frequencies across disease stages. Whole transcriptome NGS RNA expression assays were performed on 21 untreated specimens revealing overexpression of MTOR (33%), MAPK1(14%), and MET (8.0%). Actionable targets with FDA-approved therapies in other organ tissues were detected in 11 patients (40.7%). Incidental germline mutations, including MSH6, RB1, and MSH2, were identified in 9 patients though all were variants of unknown significance. Conclusions: In our study, a significant proportion of patients had potentially actionable molecular targets across the disease spectrum. The identified genetic alterations provide a genomic landscape for risk stratification, future therapies, and molecularly informed treatment paradigms for GCT patients.

A Review on Advanced CRISPR-Based Genome-Editing Tools: Base Editing and Prime Editing.

In the field of medicine, it is axiomatic that the need of a precise gene-editing tool is critical to employ therapeutic approaches toward pathogenic mutations, occurring in human genome. Today we know that most of genetic defects are caused by single-base pair substitutions in genomic DNA. The ability to make practically any targeted substitutions of DNA sequences at specified regions in the human genome gives us the chance to employ gene therapy in most known diseases associated with genetic variants. In this regard, CRISPR/Cas9 applications is becoming more and more popular along with the significant advancements of life sciences, by employing this technology in genome-editing and high-throughput screenings. Several CRISPR/Cas-based mammalian cell gene-editing techniques have been developed during the last decade, including nucleases, base editors, and prime editors, all of which have the exact mechanism at first glance. However, they address a subset of known pathogenic sequence mutations using different methods. First, we highlight the development of CRISPR-based gene-editing tools. Then we describe their functions and summarize the conducted research studies, which are increasing the reliability of these strategies to better efficiencies for prospective gene therapies in the near future. Lastly, we compare the capabilities of all these platforms together besides their probable limitations.

A Simple, Robust, and Cost-effective Method for Genotyping Small-scale Mutations.

Background and objectives:Genotyping is an important tool for studying gene functions in animals or detecting genetic variants in humans. Various methods using low to high concentrations of agarose or polyacrylamide gel electrophoresis have been developed for genotyping. These methods rely on the detection of large-size differences (20–2,000 bp) of targeted PCR products between a wild-type gene and a mutant gene. Endonuclease digestion was introduced to identify heterozygous mutations, but it was not possible to differentiate the wild-type from the homozygous mutants with the same or similar size. This study thus developed a novel, simple, and reliable test for genotyping animals or cells following genetic modifications.Methods:We developed an improved and simple method that used 2% agarose gel electrophoresis following T7E1 or Surveyor endonuclease digestion to firstly separate the heterozygous mutations from the wild-type or homozygous mutations. By adding a wild-type PCR product to a potentially homozygous product, which would form heteroduplexes, we could then separate the wild-type from a homozygous mutation with a nearly identical size or only a single base pair substitution without Sanger sequencing.Results:We verified this method in genotyping zebrafish mutants with a 2-8-bp deletion or insertion and mouse mutants with a 1- or 8-bp substitution. The wild-type, heterozygous, and homozygous mutations ranged 1–8 bp were clearly differentiated on agarose gel. Sanger sequencing also confirmed our genotyping results.Conclusions:This novel and improved genotyping method may have a broad application in many clinical and research laboratories for rapid and economical genotyping of patients and animals with a small area deletion or single base pair substitution, particularly in the era of gene editing or in those with naturally occurring mutations.

HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing

Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.

Genotype-phenotype correlations for the type 1 lissencephaly of eight Tunisian children

Background: Lissencephaly represents a rare subgroup of genetically distinct neurological disorders of neuronal migration characterized by a paucity or absence of cerebral gyration. The most common form of lissencephaly has been isolated and referred to as classic or type 1 lissencephaly. It is frequently related to abnormalities within LIS1 or DCX genes, with abnormalities ranging from single base pair substitutions to contiguous gene deletions. Methods: In this study, we report, for the first time, a clinical and genetic characterization of eight unrelated Tunisian children presenting type 1 lissencephaly. We screened LIS1 and DCX abnormalities thanks to a combination of molecular cytogenetic methods and next generation sequencing. Results: One deletion of DCX and three deletions of LIS1 were observed. One of these deletions was inherited from a maternal reciprocal translocation and estimated to approximately 2,9 Mb length. In addition, a 26 Kb LIS1 deletion was detected and refined between exon 3 up to exon 11 by target capture and sequencing. The last LIS1 rearrangement was a mutation (c.779T>A, p.V260E). Finally, two novel DCX mutations were found out (c.910G>C, p.G304R/c.436T>C, p.F146L). Conclusions: Our data confirm the individuality and originality of type 1 lissencephaly on both the phenotypic and the genetic levels. Furthermore, our data confirm again that LIS1 and DCX are the most genes associated with type 1 lissencephaly and spotlight the usefulness of developing approaches and methods for detecting a large number of known causative gene mutations.

Editing gene families by CRISPR/Cas9: accelerating the isolation of multiple transgene-free null mutant combinations with much reduced labor-intensive analysis.

Editing gene families by CRISPR/Cas9: accelerating the isolation of multiple transgene-free null mutant combinations with much reduced labor-intensive analysis.

O-090 Correcting a PLCζ mutation in the human germ line to overcome hereditary infertility

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G>C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p > 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (>18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

CRISPR/Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6.

The number of seeds per pod (NSPP) is one of the critical components that affect soybean yield (Liu et al., 2020). Ln, which encodes GmJAGGED1 (GmJAG1), is a major locus regulating NSPP and leaflet shape in soybean (Fang et al., 2013; Jeong et al., 2012). A single base-pair substitution of Ln locus from guanine (G) to cytosine (C) led to an amino acid change in the conserved EAR motif of GmJAG1, resulting in the loss-of-function of GmJAG1 and ln (Fang et al., 2013; Jeong et al., 2012). ln can significantly increase NSPP (Fang et al., 2013) and has been widely used in soybean breeding.

Genotype-phenotype studies in a large cohort of Brazilian patients with Hunter syndrome.

Mucopolysaccharidosis type II (MPS II) is an X-linked inherited disease caused by pathogenic variants in the IDS gene, leading to deficiency of the lysosomal enzyme iduronate-2-sulfatase and consequent widespread storage of glycosaminoglycans, leading to several clinical consequences, with progressive manifestations which most times includes cognitive decline. MPS II has wide allelic and clinical heterogeneity and a complex genotype-phenotype correlation. We evaluated data from 501 Brazilian patients diagnosed with MPS II from 1982 to 2020. We genotyped 280 of these patients (55.9%), which were assigned to 206 different families. Point mutations were present in 70% of our patients, being missense variants the most frequent. We correlated the IDS pathogenic variants identified with the phenotype (neuronophatic or non-neuronopathic). Except for two half-brothers, there was no discordance in the genotype-phenotype correlation among family members, nor among MPS II patients from different families with the same single base-pair substitution variant. Mothers were carriers in 82.0% of the cases. This comprehensive study of the molecular profile of the MPS II cases in Brazil sheds light on the genotype-phenotype correlation and helps the better understanding of the disease and the prediction of its clinical course, enabling the provision of a more refined genetic counseling to the affected families.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

SummaryFree asparagine is the precursor for acrylamide, which forms during the baking, toasting and high‐temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4‐gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

Prediction of Deleterious Protein Mutations

Human Single Amino Acid Polymorphisms (SAPs) or Single Amino Acid Variants (SAVs) represent the most frequent type of genetic variation among the population. They originate from non-synonymous single nucleotide variations (missense variants) where a single base pair substitution alters the genetic code in such a way that it produces a different amino acid at a given position. Since mutations are commonly associated with the development of various genetic diseases, it is of utmost importance to understand and predict which variations are deleterious and which are neutral. Computational tools based on machine learning are becoming promising alternatives to tedious and highly costly mutagenic experiments. Generally, varying quality, incompleteness and inconsistencies of SAVs datasets degrade the usefulness of machine learning approaches. Consequently, robust and more accurate approaches are essential to address these issues. In this paper, we present the application of a consensus classifier based on the holdout sampling, which shows robust and accurate results, outperforming currently available tools. We generated 100 holdouts to sample different classifiers’ architectures and different classification variables during the training stage. The best performing holdouts were selected to construct a consensus classifier and tested by blindly utilizing a K5-fold cross-validation approach. We also performed an analysis of the best protein attributes for predicting the effects of SAVs by calculating their discriminatory power.