Single Base Deletion Research Articles

Heredity and fine mapping of a yellow leaf and less tillering mutant yllt10 in rice

Rice (Oryza sativa L.) is a major food crop and increasing rice yield is the primary objective of rice research. Photosynthesis and nitrogen utilization efficiency directly affect the tiller number of rice, which affects the yield of rice. In this study, a stable yellow leaf and less tillering rice mutant yllt10 (yellow leaf and less tillering 10) was obtained by heavy-ion beam mutagenesis of rice variety 'Ke-fu-geng 7'. Compared with the wild type, yllt10 showed reduced chlorophyll content, decreased photosynthesis rate, and abnormal chloroplast structure. The genetic analysis indicated that the phenotype of yllt10 was controlled by a single recessive nuclear gene. Map-based cloning localized YLLT10 between two molecular markers J4 and J5 on chromosome 10. The sequencing of candidate genes within this interval revealed that YLLT10 was an allelic mutation of CAO1/PGL with a single base deletion in the first exon resulting in the frame shift mutation of CAO1/PGL, and YLLT10 was a new allelic variation of CAO1/PGL. The mutant yllt10 was insensitive to changes in nitrogen concentration when being incubated with different nitrogen concentrations. YLLT10 controls leaf color and tiller number and affects photosynthesis and yield of rice. The study of this gene provides a theoretical basis for molecular breeding of rice.

Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors.

Canine RNF170 Single Base Deletion in a Naturally Occurring Model for Human Neuroaxonal Dystrophy.

Neuroaxonal dystrophy (NAD) is a group of inherited neurodegenerative disorders characterized primarily by the presence of spheroids (swollen axons) throughout the central nervous system. In humans, NAD is heterogeneous, both clinically and genetically. NAD has also been described to naturally occur in large animal models, such as dogs. A newly recognized disorder in Miniature American Shepherd dogs (MAS), consisting of a slowly progressive neurodegenerative syndrome, was diagnosed as NAD via histopathology. To describe the clinical and pathological phenotype together with the identification of the underlying genetic cause. Clinical and postmortem evaluations, together with a genome-wide association study and autozygosity mapping approach, followed by whole-genome sequencing. Affected dogs were typically young adults and displayed an abnormal gait characterized by pelvic limb weakness and ataxia. The underlying genetic cause was identified as a 1-bp (base pair) deletion in RNF170 encoding ring finger protein 170, which perfectly segregates in an autosomal recessive pattern. This deletion is predicted to create a frameshift (XM_038559916.1:c.367delG) and early truncation of the RNF170 protein (XP_038415844.1:(p.Ala123Glnfs*11)). The age of this canine RNF170 variant was estimated at ~30 years, before the reproductive isolation of the MAS breed. RNF170 variants were previously identified in human patients with autosomal recessive spastic paraplegia-85 (SPG85); this clinical phenotype shows similarities to the dogs described herein. We therefore propose that this novel MAS NAD could serve as an excellent large animal model for equivalent human diseases, particularly since affected dogs demonstrate a relatively long lifespan, which represents an opportunity for therapeutic trials. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Multidisciplinary follow-up in a patient with Morgagni hernia leads to diagnosis of Marfan syndrome

Backgroundcongenital diaphragmatic hernia (CDH) is a birth defect occurring in isolated or syndromic (chromosomal or monogenic) conditions. The diaphragmatic defect can be the most common one: left-sided posterolateral, named Bochdalek hernia; or it can be an anterior-retrosternal defect, named Morgagni hernia. Marfan syndrome (MFS) is a rare autosomal dominant inherited condition that affects connective tissue, caused by mutations in fibrillin-1 gene on chromosome 15. To date various types of diaphragmatic defects (about 30 types) have been reported in association with MFS, but they are heterogeneous, including CDH and paraesophageal hernia.Case presentationWe describe the case of a child incidentally diagnosed with Morgagni hernia through a chest X-ray performed due to recurrent respiratory tract infections. Since the diagnosis of CDH, the patient underwent a clinical multidisciplinary follow-up leading to the diagnosis of MFS in accordance with revised Ghent Criteria: the child had typical clinical features and a novel heterozygous de novo single-base deletion in exon 26 of the FBN1 gene, identified by Whole-Exome Sequencing. MFS diagnosis permitted to look for cardiovascular complications and treat them, though asymptomatic, in order to prevent major cardiovascular life-threatening events.ConclusionOur case shows the importance of a long-term and multidisciplinary follow-up in all children with diagnosis of CDH.

Introductory course-based research experience for university sophomores: Using testicular-feminized mice to identify genes underlying brain sexual differentiation

Like many educators, we are striving to incorporate more active learning and inquiry-driven exercises into our STEM curricula. Course-based undergraduate research experiences (CUREs) have become more common in undergraduate laboratory courses in many STEM fields. Here we detail a new CURE developed in a sophomore-level undergraduate molecular neuroendocrinology course at California State University Long Beach. The course centered on the mouse model carrying testicular feminization mutation ( Tfm). This mutation, resulting from a single base deletion, produces a nonfunctional androgen receptor (AR) protein. Thus, Tfm mice are insensitive to androgen through ARs, providing an excellent model to probe the physiological role of ARs in the development of the brain and behavior. Since AR is the main mediator of androgen activity by acting as a hormonal transcription regulator, gene expression profiling has been measured in the brains of Tfm mice and normal controls to identify the potential AR-regulated genes as downstream molecular effectors. Our CURE took a similar approach, aiming to determine (1) if the selected genes were expressed differentially between the sexes and (2) if their expression could be altered by loss of AR in specific brain regions. Three broad goals of having students think, communicate, and perform like scientists were organized in the learning objectives for the CURE. All participating students (n=21) were pursuing a biology or related major, and none had any experience conducting independent laboratory research. Here students first used polymerase chain reaction (PCR) genotyping to screen the mice carrying the Tfm mutation and their sexes. They next conducted a literature search and review, through which they learned how to select databases and how to combine key words to create a productive search string for identifying the potential AR-regulated genes. Last, students performed RT-qPCR in the wild-type and Tfm mouse hypothalamus and measured sex- and AR-dependent gene expression. These modules cover not only key concepts in genetics, molecular biology, and neuroendocrinology, but also the development of methods and techniques, including DNA/RNA extraction, electrophoresis, and RT-PCR/qPCR. We designed the CURE for a semester timeline with a single three-hour laboratory section meeting once a week for 15 weeks. All modules were taught and conducted in a research laboratory containing all materials and equipment necessary for research. At the end, students constructed and presented posters on their results. To assess how well students achieved the learning goals, we administered pre- and post-course surveys. Students reported striking gains related to the learning goals, with a high degree of satisfaction. Students also perceived the improved understanding of the scientific backgrounds and methods in the CURE. This CURE course illustrates that authentic research experiences, which use complex molecular techniques, can be offered to sophomore students. The course can serve as a template to ignite students’ intellectual curiosity and improve their understanding of the scientific process by engaging them in novel research. This work was supported by the Keck Undergraduate Research Experiences (KURE) Incubator funding. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

Possible contribution of 8-hydroxydeoxyguanosine to gene mutations in the kidney DNA of gpt delta rats following potassium bromate treatment

8-Hydroxydeoxyguanosine (8-OHdG) is well known not only as an effective biomarker of oxidative stress but also as a mutagenic DNA modification. Incorporation of dAMP at the opposite site of 8-OHdG induces G>T or A>C transversions. However, in vivo analyses of gene mutations caused by potassium bromate (KBrO3), which can induce 8-OHdG at carcinogenic target sites, showed that G>T was prominent in the small intestines of mice, but not in the kidneys of rats. Because KBrO3 was a much clearer carcinogen in the kidneys of rats, detailed analyses of gene mutations in the kidney DNA of rats treated with KBrO3 could improve our understanding of oxidative stress-mediated carcinogenesis. In the current study, site-specific reporter gene mutation assays were performed in the kidneys of gpt delta rats treated with KBrO3. Groups of 5 gpt delta rats were treated with KBrO3 at concentrations of 0, 125, 250, or 500 ppm in the drinking water for 9 weeks. At necropsy, the kidneys were macroscopically divided into the cortex and medulla. 8-OHdG levels in DNA extracted from the cortex were dramatically elevated at concentrations of 250 ppm and higher compared with those from the medulla. Cortex-specific increases in mutant frequencies in gpt and red/gam genes were found at 500 ppm. Mutation spectrum and sequence analyses of their mutants demonstrated significant elevations in A>T transversions in the gpt gene and single base deletions at guanine or adenine in the gpt or red/gam genes. While A>T transversions and single base deletions of adenine may result from the oxidized modification of adenine, the contribution of 8-OHdG to gene mutations was limited despite possible participation of the 8-OHdG repair process in guanine deletion.

Novel Soybean Variety Lacking Raffinose Synthase 2 Activity.

Variation in the raffinose family oligosaccharide (RFO) content in soybean is advantageous for livestock farming and health science. In this study, a soybean variety (GmJMC172) with a significantly low stachyose content in its seeds was identified in the NARO Genebank core collection. The results of the single-nucleotide polymorphism (SNP) analysis suggested that this phenomenon was related to a single-base deletion, inducing a frameshift mutation in raffinose synthase 2 (RS2), rather than the polymorphisms in the RS3, RS4, and stachyose synthase (STS) sequences. Differences in the enzymatic properties between the native RS2 and truncated RS2 were examined by using a three-dimensional model predicted using Alphafold2. In addition to revealing the missing active pocket in truncated RS2, the modeled structure explained the catalytic role of W331* and suggested a sufficient space to bind both sucrose and raffinose in the ligand-binding pocket. The soybean line, with seeds available from the NARO Genebank, could serve as breeding materials for manipulating the RFO content.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

SAT079 A Novel Variant In The Carboxyl Ester Lipase (CEL) Gene Causing MODY

Abstract Disclosure: C.R. Medavarapu: None. M. McNutt: None. Introduction: Maturity-onset diabetes of the young (MODY) is a group of autosomal dominantly inherited disorders of non-autoimmune diabetes mellitus. Here, we report a patient initially diagnosed with type 2 DM and later found to have carboxyl ester lipase (CEL)-MODY. Case: A 51 year-old female was diagnosed with DM at the age of 44 and initially categorized as having type 2 DM based on normal GAD-65 antibodies. The patient’s DM was well controlled on semaglutide. The patient, however, reported chronic diarrhea/steatorrhea and intermittent dull abdominal pain which was not associated with semaglutide. The patient continued to have GI symptoms despite stopping the semaglutide. Abdominal imaging was unremarkable with a normal-appearing pancreas. Interestingly, the patient's daughter had similar gastric inflammatory symptoms and multiple members of the family have early-onset DM. Due to this symptom complex and similar presentation in the family, an inflammatory pathologic syndrome was suspected. A monogenetic auto-inflammatory syndrome was also considered in differential and the whole exome was analyzed. Genetic testing identified a novel, likely pathogenic heterozygous variant in CEL(NM_001807.5): c.1875del. Pathogenic variants in CEL are associated with MODY, type VIII. Discussion: Single-base deletions in the CEL gene can cause exocrine and endocrine pancreatic dysfunction. Our patient presented with a novel c.1875del variant in CEL that has not been reported in the literature as causative of disease nor as a variant in the general population. This variant lies in the variable number of tandem repeats (VNTR)-containing exon 11 of CEL. Two other single nucleotide deletions in this exon were described in the original report of CEL-MODY. The c.1875del variant results in the deletion of one nucleotide at position c.1875 of the CEL gene, causing a frameshift in the protein reading frame. Loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay is expected. The c.1875del CEL variant is classified as likely pathogenic, and it correlated with our patient's clinical and biochemical findings. Typically, patients with CEL-MODY develop pancreatic exocrine dysfunction in early childhood and later develop DM in their 40s. A genetic diagnosis helps in determining the choice of treatment, managing associated complications, and the need for further genetic counseling for the family. Insulin is the most appropriate treatment in CEL-MODY; however, other anti-DM drugs can also be used as seen in our patient. The risk of DM complications from CEL-MODY is unknown but patients can develop pancreatic cysts and clinicians should watch for these complications. Our case highlights the need for high suspicion for possible CEL-MODY and considering genetic testing in the setting of coexistent endocrine and exocrine pancreatic insufficiency. Presentation: Saturday, June 17, 2023

A case report of deleterious SGCE myoclonus: dystonia successfully treated with pallidal deep brain stimulation

SGCE (epsilon (ε)-sarcoglycan gene) myoclonus-dystonia (SGCE M-D) is a pleiotropic neuropsychiatric disorder with an autosomal dominant mode of inheritance, variable severity, and incomplete penetrance. There have been few reports of patients with SGCE M-D who have been successfully treated with deep brain stimulation (DBS). This case report presents a missense mutation (c.289>T) inherited in a Northeastern Asian family affected by SGCE M-D. The individuals with the condition exhibited clinical manifestations of generalized myoclonus accompanied by sustained cervical dystonia. A 38-year-old woman had a history of generalized dystonia myoclonus with paroxysmal jerks for the past 10 years. The symptoms gradually worsened over the years, affecting her entire body and interfering with most of her daily activities. Genetic testing identified a single base deletion in exon 3 of the SGCE gene, which was considered the genotype underlying her phenotypic symptoms. After failed attempts with oral medications as an outpatient, she underwent DBS targeting the globus pallidus internus (GPI). Her symptoms significantly improved after the activation of the stimulator. Our case supports the beneficial effect of GPI-targeted DBS in patients who are unresponsive to oral medications and have a genetically confirmed M-D phenotype.

Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids

BackgroundPrime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models.ResultspAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors.ConclusionTaken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

A systematic comparison of whole genome sequencing and targeted panel sequencing for precision oncology.

8546 Background: The analysis of the cancer genome provides clinically meaningful information such as actionable cancer driver mutations. Whole genome sequencing (WGS) provides a complete mutational landscape at an affordable cost thereby having increase clinical utility compared to the usual approach of targeted panel sequencing (TPS). To understand the capabilities of WGS in oncology, we conducted a study in lung adenocarcinomas. Methods: We produced WGS (surgically resected tumor tissues and matched blood; mean coverage 42x) and TPS (tumor only; SNUH panel encompassing 75 cancer genes; mean coverage 904x) through the Illumina sequencing platform and conducted comprehensive bioinformatic analyses. Oncogenic driver events, including single base substitution (SBS), insertion or deletion (INDEL), complex deletion-insertion, and fusion oncogene, were investigated in both datasets. Lastly, we compared the pattern-based mutational features, such as tumor mutational burden (TMB), mutational signatures, and copy number changes which is associated with genomic instability. Results: In 60 lung adenocarcinomas, the TPS method identified 120 driver mutations from the 75 targeted genes. Of these, 114 mutations were also detected by the WGS method, showing 95.0% sensitivity of WGS in detection of core driver mutations. In uncovered genomic regions by TPS, WGS captured 9 additional driver mutations (such as loss-of-function mutations in PIK3R2, ARID2, and APC) and 37 copy number variations (such as amplification of LMO and SMO, deletion of PIK3R1 and APC) in the cancer genes. TMB calculation is straightforward in WGS however, sophisticated considerations were necessary in TPS for TMB, to remove false positives and germline mutations. TPS showed a good capability for identifying hypermutator tumor samples, but a quantitative estimation was not always feasible due to the substantial background fluctuation, particularly for INDELs. Mutational signatures are unique combinations of mutation types in a given sample, which can give important clues to the mechanism underlying mutations. TPS showed adequate mutational signatures (cosine similarity > 0.7), but was not constantly satisfactory in tumors with high TMB ( > 20,000 genome wide SBS and INDELs). In TPS, approximately 30% of high TMB tumors represented marginal cosine similarity (0.5-0.7), and tobacco smoking related signature (SBS 4) found in WGS was captured only in 50% of the associated cases (9 out of 18). For DNA copy number changes and loss-of-heterozygosity analyses, WGS showed a much higher resolution. Conclusions: Our study demonstrates that WGS has comparable capabilities for the detection of driver mutations in core oncogenes and tumor suppressor genes. WGS was superior for pattern-based features, such as TMB, mutational signatures, and copy number changes. WGS is a medically necessary more comprehensive approach to precision oncology.

A homozygous loss-of-function C1S mutation is associated with Kikuchi-Fujimoto disease

BackgroundKikuchi-Fujimoto disease (KFD) is a self-limited inflammatory disease of unknown pathogenesis. Familial cases have been described and defects in classical complement components C1q and C4 have been identified in some patients. Material and methodsWe describe genetic and immune investigations of a 16 years old Omani male, a product of consanguineous marriage, who presented with typical clinical and histological features of KFD. ResultsWe identified a novel homozygous single base deletion in C1S (c.330del; p. Phe110LeufsTer23) resulting in a defect in the classical complement pathway. The patient was negative for all serological markers of SLE. In contrast, two female siblings (also homozygous for the C1S mutation), one has autoimmune thyroid disease (Hashimoto thyroiditis) and a positive ANA and the other sibling has serology consistent with SLE. ConclusionWe report the first association between C1s deficiency and KFD.

Heterokaryosis, the main obstacle in the generation of PPO1-edited Agaricus bisporus by CRISPR/Cas9 system

PPO1 gene editing in Agaricus bisporus was carried out by CRISPR/Cas9 through Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT to the gill tissue yielded 52 transformants, however, sequencing of which failed to detect any distinct PPO1 modification. Instead, two transformants, N9 and N14, were identified to have mixed sequences at the vicinity of the protospacer adjacent motif (PAM), which was attributed to the presence of multiple nuclei in the mycelial cell. Heterokaryosis of N9 was disrupted to generate 24 homokaryotic and 29 heterokaryotic isolates. PPO1 sequencing of the 53 isolates identified three edited homokaryotic isolates with two single base deletions (L18 and L22) and a four-base deletion (L41) at the 4th base from PAM, and six isolates with mixed sequences. The mixed sequences were resolved to consist of the edited and unedited PPO1s, indicating the presence of both edited and unedited nuclei, thus emphasizing that heterokaryosis is a major obstacle in generating edited nuclei in A. bisporus. Notably, the unedited PPO1s in the homokaryotic L32 and heterokaryotic L49 were discovered to be edited by single base deletion after prolonged incubation, suggesting the transfer of the Cas9-gRNA complex to neighboring nuclei in the common cytoplasm. Lastly, the PPO1-edited heterokaryon Het1, created by mating the edited homokaryotic L18 with the wild-type NH1, exhibited significantly less browning than the wild-type strain, indicating a functional role for PPO1 in the button mushroom browning.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.