Mutant Allele Research Articles

POLE-mutated uterine carcinosarcomas: a clinicopathologic and molecular study of 11 cases

Uterine carcinosarcomas (UCS) are high-grade biphasic neoplasms with generally poor outcomes. Based on TCGA molecular classification of endometrial carcinomas, the majority of UCS are classified as copy-number high/serous-like (p53 abnormal); however, a small subset represent other molecular subtypes, including those that harbor POLE mutations. We identified 11 POLE-mutated (POLEmut) UCS across three institutions and assessed the clinical, histopathologic, immunohistochemical and molecular features of these tumors. POLEmut UCS occurred in adult women (median age 64 years, range 48 to 79 years) and usually presented as FIGO (2009) clinical stage IA (n = 4) or IB (n=3). Almost all tumors were predominantly carcinomatous (n =10), with most showing endometrioid morphology (n = 7), followed by ambiguous (n=4) and serous (n = 3) histotypes. By immunohistochemistry, 7 tumors showed aberrant or subclonally aberrant expression of p53, 6 of which harbored pathogenic mutations in TP53 by sequencing. Other frequent mutations included PIK3CA (10/11), PTEN (8/11), RB1 (7/11), ARID1A (7/11), ATM (6/11), PIK3RA (5/11) and FBXW7 (4/11). Two tumors demonstrated loss of mismatch repair protein expression and one had subclonal loss. Heterologous differentiation was uncommon and only chondrosarcomatous type (n = 2) was observed. Mean and median follow-up were 24.3 and 14.1 months, respectively (range 1.4 to 61.1 months). Ten patients (91%) had no recurrences or death from disease, though 3 of these had follow-up periods <1 year. One patient, with the subclonal POLE variant, presented with stage IV disease and died 1.4 months after surgery. In conclusion, POLEmut UCS demonstrate unique morphologic and immunohistochemical features compared to their p53-abnormal counterparts and may have significant prognostic differences. Our study supports full molecular classification of UCS. We also raise awareness for potentially assessing POLE mutation allele fraction and clonality in the consideration of classifying a tumor as POLEmut.

European EHBP1L1 Genotyping Survey of Dyserythropoietic Anemia and Myopathy Syndrome in English Springer Spaniels

Dyserythropoietic anemia and myopathy syndrome (DAMS) with neonatal losses was recently characterized as an autosomal recessive disorder caused by an EHBP1L1 frameshift variant in English Springer Spaniels (ESSPs). The frequency and dissemination of the mutation remained unknown. The EHBP1L1 protein is essential for muscle function, and the Rab8/10-EHBP1L1-Bin1-dynamin axis participates in nuclear polarization during the enucleation of erythroblasts. Lack of EHBP1L1 function decreases enucleation, leading to increased numbers of nucleated erythrocytes, which are characteristic of DAMS. A genotyping survey for the EHBP1L1 variant was conducted based upon submitted samples of ESSPs from Europe. DNA was extracted, and a real-time PCR assay, with allele-specific TaqMan probes for EHBP1L1 wild-type and frameshift deletion, was applied. Between September 2022 and August 2024, 803 samples were received from 18 European countries. The EHBP1L1 mutant allele frequency was 9.7%, including 4 homozygous dogs and 148 heterozygotes. The mutant EHBP1L1 allele was found in 13 countries. A total of 6 homozygous and 73 heterozygous ESSPs reported on an open database could be tracked to an original common ancestor. Although the survey is biased, it indicates that the mutant EHBP1L1 variant is disseminated in the breed and across Europe. The genotyping of ESSPs is recommended to diagnose DAMS and guide breeders.

SPIRRIG is required for BRICK1 stability and salt stress induced root hair developmental plasticity in Arabidopsis

Developmental plasticity is critical for plants to adapt to constantly changing environments. Plant root hairs display dramatic plasticity under different environments and therefore play crucial roles in defense against environmental stressors. Here, we report the isolation of an Arabidopsis mutant, salinityover-sensitivemutant 1–1 (som1-1), also exhibiting root hair developmental defects. Map-based cloning and allelic analyses confirmed that som1-1 is a new mutant allele of SPIRRIG (SPI), which encodes a Beige and Chediak Higashi (BEACH) domain-containing protein. SPI has been reported to facilitate actin dependent root hair development by temporally and spatially regulating the expression of BRICK1 (BRK1), a subunit of the SCAR/WAVE actin nucleating promoting complex. Our living cell imaging examinations revealed that salt stress induces an altered actin organization in root hair that mimics those in the spi mutant, implying SPI may respond to salt stress induced root hair plasticity by modulating actin cytoskeleton organization. Furthermore, we found BRK1 is also involved in root hair developmental change under salt stress, and overexpression of BRK1 resulted in root hairs over-sensitive to salt stress as those in spi mutant. Moreover, based on biochemical analyses, we found BRK1 is unstable and SPI mediates BRK1 stability. Functional loss of SPI results in the accumulation of steady-state of BRK1.

Congenital diarrhoea due to microvillus inclusion disease: a case report

Inherited diarrheal diseases are group of disorders which cause significant morbidity with dependence on parental nutrition. Microvillus inclusion disease (MVID) is a secretory enteropathy with autosomal recessive inheritance associated with bi allelic mutations in MYO5B gene or more rarely STX3 gene. We report a newborn, which developed diarrhea since birth and whose exome sequencing showed the rare mutation of biallelic STX3. There is a possibility of familial involvement (two other siblings having similar symptoms involved, but not evaluated and died) in this particular case. All three had antenatal polyhydramnios and dilated bowel loops. This case report describes an infant diagnosed with congenital diarrhea due to a genetic mutation, explores the diagnostic process, and outlines the management strategies and outcomes. The report emphasizes the importance of early diagnosis and tailored treatment in improving patient outcomes.

A Novel Heterozygous NFKB2 Variant in a Multiplex Family with Common Variable Immune Deficiency and Autoantibodies Against Type I IFNs.

We studied a family with three male individuals across two generations affected by common variable immune deficiency (CVID). We identified a novel missense heterozygous variant (c.2602T>A:p.Y868N) of NFKB2 in all patients and not in healthy relatives. Functional studies of the mutant allele in an overexpression system and of the patients' cells confirmed the deleteriousness of the NFKB2 variant and genotype, respectively, on the activation of the non-canonical NF-κB signaling pathway. Impaired processing of p100 into p52 underlies p100 accumulation, which results in gain-of-function (GOF) of IκBδ inhibitory activity and loss-of-function (LOF) of p52 transcriptional activity. The three patients' plasma contained autoantibodies that neutralized IFN-α2 and/or IFN-ω, accounting for the severe or recurrent viral diseases of the patients, including influenza pneumonia in one sibling, and severe COVID-19 and recurrent herpes labialis in another. Our results confirm that NFKB2 alleles that are IκBδ GOF and p52 LOF can underlie CVID and drive the production of autoantibodies neutralizing type I IFNs, thereby predisposing to severe viral diseases.

Transcriptome profiling of maize transcription factor mutants to probe gene regulatory network predictions.

Transcription factors (TFs) play important roles in regulation of gene expression and phenotype. A variety of approaches have been utilized to develop gene-regulatory networks (GRNs) to predict the regulatory targets for each TF, such as yeast-one-hybrid (Y1H) screens and gene co-expression network (GCN) analysis. Here we identified potential TF targets and used a reverse genetics approach to test the predictions of several GRNs in maize. Loss-of-function mutant alleles were isolated for 22 maize TFs. These mutants did not exhibit obvious morphological phenotypes. However, transcriptomic profiling identified differentially expressed genes in each of the mutant genotypes, and targeted metabolic profiling indicated variable phenolic accumulation in some mutants. An analysis of expression levels for predicted target genes based on Y1H screens identified a small subset of predicted targets that exhibit altered expression levels. The analysis of predicted targets from GCN-based methods found significant enrichments for prediction sets of some TFs, but most predicted targets did not exhibit altered expression. This could result from false-positive GCN predictions, a TF with a secondary regulatory role resulting in minor effects on gene regulation, or redundant gene regulation by other TFs. Collectively, these findings suggest that loss-of-function for single uncharacterized TFs might have limited phenotypic impacts but can reveal subsets of GRN predicted targets with altered expression.

Genetic variants in mitochondrial sirtuins associated with brain tumor risk: a case-control study.

Previous studies on brain tumors have been performed on the nuclear genome, but limited studies have been reported on the mitochondrial genome. The mitochondrial sirtuin (SIRT3/SIRT4/SIRT5) has been mutated in different cancers. Limited studies have been performed on brain tumors. Isocitrate dehydrogenase (IDH) is an important marker, and polymorphism in the IDH gene has been reported to differentiate the brain tumor subtypes. The present study was designed to screen mitochondrial sirtuins and IDH polymorphisms in brain tumor patients. One thousand blood samples were collected (500 brain tumor patients and 500 controls). Two SNPs for each gene SIRT3 (rs12226697, rs570591), SIRT4 (rs184496260, 1925909), SIRT5 (rs2841522, rs2841523), and one SNP for IDH (rs11554137) was screened using Tetra-ARMS PCR. Logistic regression showed that the mutant genotype of selected SNPs was associated with increased disease incidence compared to wild type. Haplotype analysis and linkage disequilibrium (LD) showed a strong LD in brain tumor patients. Kaplan-Meier analysis showed that mutant allele frequency was found to be associated with a significant decrease in the survival of brain tumor patients. The present study showed that the mutant allele of selected mitochondrial sirtuins' SNP was associated with increased brain tumor risk.

Endothelial Rho kinase controls blood vessel integrity and angiogenesis.

The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. We generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo . Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

Generation of viable hypomorphic and null mutant plants via CRISPR-Cas9 targeting mRNA splicing sites.

Genetic analysis is important for modern plant molecular biology, and in this regard, the existence of specific mutants is crucial. While genome editing technologies, particularly CRISPR-Cas9, have revolutionized plant molecular biology by enabling precise gene disruption, knockout methods are ineffective for lethal genes, necessitating alternatives like gene knockdown. This study demonstrates the practical generation of a hypomorphic mutant allele, alongside severe null mutant alleles, via the targeting of mRNA splicing sites using CRISPR-Cas9. The Arabidopsis HIGH PLOIDY 2 (HPY2) encodes a yeast NSE2 ortholog, part of the conserved eukaryotic SMC5/6 complex, with SUMO E3 ligase activity essential for cell cycle progression and plant development. Loss-of-function HPY2 mutants exhibit severe dwarfism and seedling lethality, making functional analysis challenging. To overcome these limitations, we created HPY2 knockdown mutants as novel tools to investigate gene function. Of the three mutant alleles, the hpy2-cr1 and hpy2-cr2 mutants resembled the existing severe hpy2-1 allele, both harboring a single base pair insertion in one exon, causing significant root shortening and seedling lethality. In contrast, the hypomorphic mutant hpy2-cr3, which has a five bp deletion at an intron-exon junction, showed relatively longer root growth and survived until the reproductive stage. RT-PCR analysis of hpy2-cr3 revealed atypical mRNAs producing truncated polypeptides that retained some HPY2 function, explaining the milder phenotype. These results establish the successful generation of novel hypomorphic mutant alleles critical for studying the lethal gene HPY2, and demonstrate the usefulness of CRISPR-Cas9 for producing viable hypomorphic mutants for investigating complex genetic interactions.

Effects of Co-Exposure to Benzene, Toluene, and Xylene, Polymorphisms of microRNA Genes, and Their Interactions on Genetic Damage in Chinese Petrochemical Workers.

Benzene, toluene, and xylene (BTX) co-exist in human environments, yet their individual and combined effects on genetic damage at low exposure levels are not fully understood. Additionally, single nucleotide polymorphisms in microRNAs (mirSNPs) might be involved in cancer etiology by affecting the related early health damage. To investigate the influence of BTX exposure, mirSNPs, and their interactions on genetic damage, we conducted a cross-sectional study in 1083 Chinese petrochemical workers, quantifying the BTX cumulative exposure levels and multiple genetic damage biomarkers. Additionally, we genotyped multiple common mirSNPs. Benzene and a BTX mixture were positive associated with the olive tail moment (OTM) and tail DNA% (p < 0.05). Higher levels of toluene and xylene enhanced the association of benzene with genetic damage levels. Genotypes and/or mutant allele counts of miR-4482-related rs11191980, miR-4433-related rs136547, miR-27a-related rs2594716, miR-3130-related rs725980, and miR-3928-related rs878718 might significantly influence genetic damage levels. Stronger effect estimates of benzene/BTX exposure were found in carriers of miR-196a-2-related rs11614913 heterozygotes and of wild homozygotes of miR-1269b-related rs12451747, miR-612-related rs12803915, and miR-4804-related rs266437. Our findings provide further support of the involvement of BTX co-exposure, mirSNPs, and their gene-environment interactions in determining the severity of DNA strand break in a complex manner.

Association of BRAF V600E allele frequency with clinicopathologic outcomes in papillary thyroid cancer.

BRAF V600E mutation is the most common genetic driver of papillary thyroid cancer (PTC), where it is found with various allele frequency (AF), reflecting the proportion of cells carrying the mutant and wild-type gene alleles. To determine whether BRAF V600E AF can improve prognostication and inform initial surgical management of PTC. Retrospective cohort study (2016-2019). UCLA health. Consecutive patients with Bethesda V/VI nodules and isolated BRAF V600E mutation who underwent surgery with histopathology showing PTC. Blinded ThyroSeq v3 molecular analysis after completion of initial management and follow-up. Aggressive histopathology and cancer persistence/recurrence. Of 73 patients, the median BRAF V600E AF was 25.5% (IQR, 16.7-34.3%). Higher median AF was seen in patients classified as American Thyroid Association (ATA) high-risk (37%) vs. intermediate-risk (25.3%, p<0.01) and low-risk (24.7%, p<0.01), largely attributed to higher AF in patients with gross extrathyroidal extension (ETE) (40.1% vs. 25.2% without gross ETE, p=0.02). No differences in AF were observed on the basis of lymph node positivity or presence of aggressive variants of PTC. A higher BRAF V600E AF was also found in patients with tumors ≥2cm vs. <2cm (median 32.0% vs. 24.4%, p<0.01). Over 4.1 years of follow-up, disease persistence/recurrence was found in 7 patients (9.4%) and was associated with higher median AF than those without recurrence (35.3% vs. 25.2%, p=0.02). Higher AF was associated with poorer recurrence-free survival (AF≥35%, HR 7.40, CI 1.4-38.1). Higher AF was associated with gross ETE and increased recurrence risk. This may inform initial management in patients with PTC harboring an isolated BRAF V600E mutation.

Abstract B057: Base-error model (BEM) for improved detection of ctDNA in lymph samples of HPV-negative head and neck cancer patients

Abstract Introduction: Previously we demonstrated that ctDNA present in lymphatic exudate collected via surgical drains (“lymph”) outperformed plasma for detecting minimal residual disease (MRD) in head and neck squamous cell carcinoma (HNSCC) patients through a targeted sequencing (TS) approach. However, artifacts introduced during library preparation and sequencing remain challenges to sensitive low allelic fraction mutation detection. To enhance the performance of the TS approach, we characterized the background noise in lymph samples and developed a base-error model (BEM) that reduces sequencing artifacts and maximizes ctDNA signal. Methods: Lymph and blood were collected from 44 HPV-negative HNSCC patients postoperatively at 24 hours along with resected tumor. Cell-free DNA was extracted from lymph and sequenced using a custom TS panel with unique molecular identifiers (UMI). Somatic mutations were identified by tumor and blood exome sequencing (200x). Five patients had &amp;lt;2 mutations in tumor and were excluded. Two patients were censored due to lack of clinical data, yielding 17 patients with disease recurrence (REC) and 20 with no evidence of disease (NED) with &amp;gt;1 year of follow-up. Tumor-specific variants were force-called in lymph using a custom pipeline. A BEM of each tumor variant was built using a series of high-quality lymph reference samples to quantify the background noise. BEM was fit by Weibull distribution if more than 5 non-zero non-reference alleles were observed at a tumor variant position across the reference samples. Otherwise, Gaussian distribution was used to fit BEM. Force-called variants were considered artifacts if the variant allele fraction (VAF) was not greater than BEM cutoff controlled by false discovery rate. Patients were considered MRD positive if the mean VAF was greater than the estimated limit of detection. The Kaplan-Meier (KM) estimator with log-rank test and Cox proportional-hazards model were used for survival analyses. Results: Background noise was observed in reference lymph samples across all 12 nucleotide substitution classes. Out of 299 single nucleotide variants (SNVs), 94 were determined as artifacts by BEM. Among the artifacts, 77 were C/G to A/T mutations, indicating that the majority of recurrent background artifact in TS is likely the result of oxidative damage. The artifacts had VAFs ranging from 0.004% to 0.12% with median of 0.02%. Incorporation of BEM in lymph TS showed significantly improved specificity (sensitivity (SN) = 71%, specificity (SP) = 65%; p = 0.027, Hazard ratio (HR) = 3.07) compared to the same cohort without BEM (SN = 76%, SP = 30%, p = 0.60, HR = 1.34). Conclusions: BEM is a sensitive and robust method to improve ctDNA detection accuracy in TS. We demonstrated its utility in improving recurrence prediction in a HNSCC cohort. Given its advantages, we envision that BEM will prove useful as a general strategy for deep sequencing applications requiring precise digital quantification of low frequency alleles in TS. Citation Format: Zhuosheng Gu, Maciej Pacula, Seka Lazare, Noah Earland, Marra S. Francis, Adam Harmon, Megan Long, Aadel A. Chaudhuri, Jose P. Zevallos, Wendy Winckler. Base-error model (BEM) for improved detection of ctDNA in lymph samples of HPV-negative head and neck cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B057.

Abstract B025: Monitoring response to immunotherapy in lung cancer using cell-free DNA fragmentomes

Abstract BACKGROUND: Immune checkpoint inhibition (ICI) continues to be a promising area of research with treatments continuing to show efficacy in late-stage cancers. The detection of disease progression for patients undergoing treatment with this modality remains challenging however given the lack of reliable biomarkers for which to monitor disease. Current methods including imaging can be challenging to evaluate as they do not always capture the timing and nature of clinical responses. Liquid biopsies may offer a solution by allowing for minimally invasive tumor monitoring during therapy. We previously reported on the DELFI Tumor Fraction (DELFI-TF) algorithm, as a tumor- and mutation-naive approach to monitoring using cell-free DNA (cfDNA) fragmentomes (Alipanahi et al., AACR 2023). In this study we examine the performance of DELFI-TF in a cohort of patients with metastatic NSCLC treated with ICI. METHODS: Longitudinal blood samples (n=323) were collected from NSCLC patients (n=109) which received ICI therapy. Whole genome sequencing at low coverage (∼4x) was performed following cfDNA extraction. DELFI-TF scores, predicted using fragmentome features, were used to monitor changes in circulating tumor DNA (ctDNA) levels and associated variations in disease burden. Predictions from DELFI-TF were compared to maximum observed mutation allele frequencies (maxMAF) from a targeted sequencing gene panel for the same samples. In order to determine the prognostic performance of DELFI-TF we evaluated ctDNA changes at baseline and within 3 to 9 weeks of ICI initiation. The presence of undetectable DELFI-TF, defined as below the observed limit of blank (LOB) within 3 to 9 weeks of ICI initiation, indicated a molecular response (mR). In contrast, the persistence of DELFI-TF within this interval signified molecular disease progression (mPD). RESULTS: We observed a strong correlation between DELFI-TF predictions and maxMAF (r=0.93, p&amp;lt;0.001). Further, changes in ctDNA predictions from baseline to the first post-treatment timepoint showed a high correlation between DELFI-TF and maxMAF (r=0.94, p&amp;lt;0.001). Patients with a higher disease burden prior to immunotherapy, as predicted by DELFI-TF and maxMAF, had shorter overall survival (OS) compared to patients with a lower disease burden (DELFI-TF: 11.4 v 33.0 months, p=0.006; maxMAF: 7.82 vs low 33.0 months, log-rank p=0.001). Among 79 patients evaluable for molecular response, 28 (35%) were classified as mPD and 51 (65%) were classified in the mR category. Patients with mR attained longer progression free survival (PFS;16.04 v 2.70 months, log-rank p&amp;lt;0.001) compared to those in the mPD group. Furthermore, assessment of landmark PFS at 6 months revealed a significant association between ctDNA molecular response and durable clinical benefit (DCB; p &amp;lt; 0.001, Fisher’s exact test). CONCLUSIONS: DELFI-TF is a low-cost, tumor and mutation naive approach that accurately monitors longitudinal dynamic changes in tumor burden and captures durable therapeutic responses in patients with metastatic NSCLC receiving immunotherapy. Citation Format: Lavanya Sivapalan, Bahar Alipanahi, Jamie E Medina, Bridget Tripp, Zachary L Skidmore, Paola Ghanem, Gavin Pereira, Nisha Rao, Kavya Velliangiri, Caitlin Schonewolf, Noushin Niknafs, Bryan Chesnick, Jennifer Tom, Stephen Cristiano, Peter Bach, Nicholas C Dracopoli, Robert B Scharpf, Victor Velculescu, Lorenzo Rinaldi, Valsamo Anagnostou. Monitoring response to immunotherapy in lung cancer using cell-free DNA fragmentomes [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B025.

Novel Allelic Mutations in Dw3 Gene That Affect the Height of Sorghum Plants.

Breeding for dwarfing traits in sorghum is crucial. However, only three genes (Dw1-Dw3) that control plant height have been mapped. In this study, 634 sorghum cultivars were collected to investigate plant height and genotypes. Four were genotyped Dw1DW2Dw3 (wild type) but with different plant heights, and they were selected to construct two populations and map new dwarf genes. Bulked segregant analysis with whole-genome resequencing of the two populations identified the candidate gene in one same genomic region-on chromosome 7. Then, it was narrowed down to a region containing nine genes. Amino acid and DNA sequence analysis of the parent and offspring plants revealed that two novel allelic mutations in the Dw3 gene play a role in reducing the plant height-8R262 or 8R417, including 1 bp substitution and 2 bp deletions. Furthermore, we sequenced 19 cultivars that primarily exhibited a "one-dwarf" hybrid or wild-type and presumed another allelic mutation via the amino acid alignment of 8R019, 8R100, and 8R402, which was another one-base substitution. These results indicate that multiple types of allelic mutations in the Dw3 gene should be considered when identified or applied.

Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

Identification of β-globin gene mutations among transfusion-dependent β-thalassemia patients

Abstract BACKGROUND: β-thalassemias are widely distributed in Mediterranean and Middle Eastern countries, including Iraq. There are more than 400 transfusion-dependent β-thalassemia patients registered in the thalassemia center. β-thalassemia is a significant problem in Karbala as well as other regions of Iraq. The detection of the most frequent mutations is significant to the implementation of an effective preventive program in this area because of the significant burden it places on the local health authorities, patients, and their families. OBJECTIVES: To define the most common mutations and their frequencies among patients with transfusion-dependent β-thalassemia and to evaluate the reverse hybridization strip assay method for the detection of β-thalassemia mutations. PATIENTS, MATERIALS AND METHODS: Sixty transfusion-dependent β-thalassemia patients were recruited from the thalassemia center in Karbala. Blood samples were aspirated from each patient just before blood transfusions for CBC, reticulocyte count, DNA extraction, PCR amplification, and identification of the mutations by reverse hybridization technique using the β-Globin strip assay method. RESULTS: A total of 60 patients with 120 chromosomes were studied, searching for the most common mutations causing β-thalassemia. Among the twelve identified mutations, the six most frequent mutations represented 79.16% of all β-globin defects. These mutations were IVSII-1 (30.83%), IVSI-110 (15.83%), Codon 5 (10.83%), Codon 44 (8.33%), IVSI-1 (6.67%), and IVSI-5 (6.67%). The detection rate of the method used in our population was 96.66%. CONCLUSION: The most frequent mutations encountered were IVSII.1 and IVSI-110, while IVS 2.745 was the least common mutant allele. Reverse hybridization strip assay molecular techniques used in the current study provide an extremely quick, precise, and simple to carry out molecular diagnostic technique for the detection of β-thalassemia mutations.

A plastidial lipoyl synthase LIP1p plays a crucial role in phosphate homeostasis in Arabidopsis.

Phosphate (Pi) homeostasis is important for plant growth and adaptation to the dynamic environment, which requires the precise regulation of phosphate transporter (PHT) trafficking from the endoplasmic reticulum to the plasma membrane. LIPOYL SYNTHASE 1p (LIP1p) is known as a key enzyme in plastids to catalyze lipoylation of pyruvate dehydrogenase complex for de novo fatty acid synthesis. It is unknown whether this process is involved in regulating Pi homeostasis. Here, we demonstrate a new role of LIP1p in controlling Pi homeostasis by regulating PHT1 trafficking. We recovered a weak mutant allele of LIP1p in Arabidopsis that accumulates much less Pi and has enhanced expression of phosphate starvation-induced genes. LIP1p mutation alters the lipid profile and compromises vesicle trafficking of PHT1 to the plasma membrane to impair Pi uptake. Beside phosphorus, the homeostasis of a series of mineral nutrients was also perturbed in lip1p mutant. Our findings provide powerful genetic evidence to support the linkage between lipoylation and ion homeostasis in plants.

Efficient gene editing of pig embryos by combining electroporation and lipofection

Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. Keywords: electroporation, guide RNA sequence, lipofection, pig embryo.

Impact of the COL1A1 (RS1107946) gene polymorphism on anterior cruciate ligament rupture in professional soccer players

Objectives To describe the allelic frequency of rs1107946, belonging to the COL1A1 gene in professional soccer players and compare these findings with data from continental populations described in the 1000 Genomes database, in order to evaluate the frequency of genotypes highly associated with genetic susceptibility to anterior cruciate ligament (ACL) injuries in the literature. Methods The DNA of the 38 professional soccer players was collected from oral mucosa samples. DNA extraction was performed using the PureLinkTMGenomicDNAMini Kit, according to the manufacturer's protocol. For genotyping and allelic discrimination of the selected SNP (rs1107946), real-time PCR was used with TaqMan® SNP GenotypingAssay specific probes (AppliedBiosystems, Foster City, CA, USA) on the QuantStudio 6 Flex Realtime PCR system. The MMA was consistently identified in all specimens. In 26 of 33 (82%) specimens, the MMA branched directly from the popliteal artery, while in 6, the MMA shared a common trunk with the inferior medial genicular artery (IMGA). The MMA was easily distinguishable from the superior MGA, inferior MGA, and MGA, given its direct course to the medial joint line with terminal branches to the posterior horn of the medial meniscus and deep to the semimembranosus tendon. The MMA exhibited a smaller vessel diameter relative to the genicular arteries. Results Our findings demonstrated that the frequency of the wild-type allele C was 65%, while the frequency of the mutant allele A was 45%, highlighting that despite the wild-type allele, which confers protection against the risk of ACL injury, is predominantly distributed in the sample, the mutant allele is significantly present in the individuals investigated. When comparing our findings with data from continental populations described in 1000 Genomes, we found statistically significant allelic and genotypic distributions (Student's T Test p = 0.021), demonstrating that the athletes investigated have a different genetic profile. In our findings, the individuals studied presented a statistically significant allelic frequency of the mutation when compared to the European population, inferring that the frequency of this mutation is higher in European individuals than in mixed-race individuals from Brazil. Conclusion The prevalence of the rs1107946 polymorphism of the COL1A1 gene in athletes may have an important role in the development of ACL injuries.

Zebrafish are resilient to the loss of major diacylglycerol acyltransferase enzymes

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). Export of lipid from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Though triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth, or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways.