Candidate Genes Research Articles

Abstract 4140108: A CRISPR-Activation CROP-seq Screen Identifies HMGN1 as a Dosage-Sensitive Regulator of Heart Defects in Down Syndrome

Introduction/Background: Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy accounts for nearly 15% of CHD and the most frequent form involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). CHD is present in ~50% of DS cases, with a 1000-fold enrichment of atrioventricular canal (AVC) defects that disrupt the junction of the atria and ventricles. Hypothesis: We hypothesize that the presence of a third copy of one or more genes on Ch21 underlies AVC and other cardiac defects; however, the dosage-sensitive CHD gene(s) on Ch21 causing this are not fully defined. Methods: We used human pluripotent stem cells derived from an individual mosaic for DS, allowing us to compare Ch21 trisomy cells with isogenic control cells disomic for Ch21. We performed single cell RNA-sequencing (scRNA-seq) to define the transcriptional dysregulation associated with DS in atrioventricular canal myocardial cells (AVCM). We then introduced a CRISPR-activation transgene to disomic cells and delivered small guide RNAs (sgRNAs) against 66 candidate Ch21 CHD genes. We developed a new machine learning algorithm to assign a trisomic score to CRISPR perturbed (CROP-seq) cells. Finally, we reduced the copy number of our top candidate gene in a mouse model of Down Syndrome and used microCT to assess incidence of cardiac septal defects. Results: Using human pluripotent stem cell and mouse models of DS, we identify HMGN1, a nucleosome-binding epigenetic factor on Ch21, as a dosage-sensitive regulator of cardiac defects in DS. Single cell transcriptomics revealed that human AVC cardiomyocytes shifted to a more ventricular myocardial state in trisomy 21 hiPS-derived cells. CRISPR-activation in isogenic disomic cells of 66 candidate Ch21 genes expressed in heart development, followed by single cell RNA-sequencing (CROP-seq) combined with machine learning predictions indicated that increased expression levels of HMGN1 altered the transcriptional state of atrioventricular cardiomyocytes similar to trisomy 21. Finally, reduction of Hmgn1 from three to two alleles in a mouse model of DS attenuated the incidence of cardiac septal defects. Conclusion: Our results identify HMGN1 as a dosage sensitive regulator of heart defects in DS and represent a generalizable approach for identifying candidate genes within areas of aneuploidy.

A genome-wide association study of the racing performance traits in Yili horses based on Blink and FarmCPU models.

Racing performance traits are the main indicators for evaluating the performance and value of sport horses. The aim of this study was to identify the key genes for racing performance traits in Yili horses by performing a genome-wide association study (GWAS). Breeding values for racing performance traits were calculated for Yili horses (n = 827) using an animal model. Genome-wide association analysis of racing performance traits in horses (n = 236) was carried out using the Blink, and FarmCPU models in GAPIT software, and genes within the significant regions were functionally annotated. The results of GWAS showed that a total of 24 significant SNP markers (P < 6.05 × 10- 9) and 22 suggestive SNP markers (P < 1.21 × 10- 7) were identified. Among them, the Blink associated 16 significant SNP loci and FarmCPU associated 12 significant SNP loci. A total of 127 candidate genes (50 significant) were annotated. Among these, CNTN6 (motor coordination), NIPA1 (neuronal development), and DCC (dopamine pathway maturation) may be the main candidate genes affecting speed traits. SHANK2 (neuronal synaptic regulation), ISCA1 (mitochondrial protein assembly), and KCNIP4 (neuronal excitability) may be the main candidate genes affecting ranking score traits. A common locus (ECA1: 22698579) was significantly associated with racing performance traits, and the function of the genes at this locus needs to be studied in depth. These findings will provide new insights into the detection and selection of genetic variants for racing performance and will help to accelerate the genetic improvement of Yili horses.

Two novel alleles of the MYB transcription factor BjA06.GL1 and BjB02.GL1 control leaf trichomes and enhance resistance to aphids in Brassica juncea

Abstract Leaf trichome formation is a very important agronomic trait as it confers resistance to biotic and abiotic stresses, but the causal genes involved in this process in B. juncea remain largely unexplored. In this study, we first characterized the haplotypes of BjB02.GL1 among different inbred lines with leaf trichomes or glabrous leaves. A comparative analysis of the number and density of leaf trichomes between the two mustard inbred lines was then performed. BSA analysis of leaves with trichomes and glabrous pools from the F2 segregating population mapped the candidate genes on Chr.A06 and Chr.B02. Two candidate genes, BjA06.GL1 and BjB02.GL1, were subsequently cloned. After sequence alignment of the BjGL1 genes, both SNPs and indel were identified in the BjA06.GL1 and BjB02.GL1 genes. And qRT–PCR analysis further confirmed that both the BjA06.GL1 and BjB02.GL1 genes were more highly expressed in leaves with trichomes than in glabrous leaves. As the leaf size increased, the leaf trichome density decreased. Gene editing of both BjA06.GL1 and BjB02.GL1 changed the leaf trichome to a glabrous leaf phenotype in mustard. In addition, plants with leaf trichomes presented greater resistance to aphids. Taken together, our results revealed that both BjA06.GL1 and BjB02.GL1 positively regulate leaf trichome formation and help increase aphid resistance in mustard. This study provides valuable resources and helps to elucidate the molecular mechanism of leaf trichome formation in B. juncea.

Abstract 4118949: Factors Influencing Left Ventricular Function Recovery Following Left Ventricular Assist Device Implantation in Pediatric Dilated Cardiomyopathy: Insights from Single-Cell RNA Sequencing Analysis

Background and Objective: Heart transplantation is a recognized treatment for pediatric severe heart failure, but long-term issues necessitate alternative treatments. Some dilated cardiomyopathy (DCM) cases show left ventricular function recovery after LVAD implantation. This occurrence could lead to new treatments, but mechanisms are unclear. This study explores preoperative molecular factors interacting with LVAD's effects using single-cell transcriptomics on clinical samples. Methods: Twenty-five pediatric DCM patients underwent Berlin Heart EXCOR (BHE) implantation at our institution since 2013. Those with left ventricular ejection fraction (LVEF) improving by &gt;20% were in the Recovery group (R), others in Non-Recovery (NR). Single-cell RNA-sequence was performed on myocardial tissue from six patients (three per group). Differential gene expression and pathway analysis were conducted for each cell type. Findings were validated by comparing them with histological evaluation or clinical examination in all 25 cases. Results: R: 10 patients; NR: 15. R's weight was 6.3 kg, NR's 9.9 kg (p=0.049); LVEF: R 23%, NR 16.5% (p=0.61). In pseudobulk analysis of cardiomyocytes, upregulated genes in R included NPPB, MYL7, PCDH9. KEGG analysis indicated activation of Tight Junction and ECM receptor pathways in R. In fibroblasts, major extracellular matrix proteins were higher in R, GSL gene expression in NR. Plasma Brain Natriuretic Peptide (BNP) at BHE implantation was significantly higher in R; 2913 [1836-3990 (95% CI)] vs 1278 [667-1888] pg/ml (Image1,2). No differences in tissue fibrosis rates were found between the groups. Conclusion: Alterations in gene expression patterns in some cell clusters, including cardiomyocytes, may contribute to left ventricular function recovery post-BHE implantation in pediatric DCM patients. Plasma BNP levels correlated with gene expression alterations, suggesting both a potentially protective effect on cardiomyocytes and a useful marker of LV functional recovery. Further validation of these candidate genes may elucidate intracellular molecular mechanisms.

Abstract 4134619: A Novel Missense Mutation in TNNT2 Gene in a Lebanese Pedigree With Ebstein Anomaly And Wolf-Parkinson-White Syndrome: A Case Report

Background: Ebstein anomaly (EA) is a rare congenital heart defect occurring in 1.2 to 5 in 100,000 live births, characterized by a downward displacement of the tricuspid valve, thin-walled right ventricle, and tricuspid valve regurgitation. It can present variably from asymptomatic cases to severe symptoms like arrhythmias and right-sided heart failure. EA is often associated with other anomalies such as interatrial communication and mitral valve prolapse. The condition can lead to accessory atrioventricular pathways, frequently resulting in Wolff-Parkinson-White syndrome (WPW), which involves abnormal heart electrical activity and increases the risk of sudden cardiac death. While the genetic basis of EA is not fully understood, it appears to involve multiple genes like FLNA and NKX2-5, MYH6, MYH7 suggesting a complex polygenic inheritance pattern. TNNT2 is known to be associated with Cardiomyopathy but has not been previously associated with EA and WPW. Case: In this report, we present findings from a lebanese family with EA, comprising 2 affected individuals. Whole exome sequencing in the affected individuals identified a pathogenic variant in the TNNT2 gene at coding strain position 260 (a missense mutation: C to T), resulting in the substitution of Proline with Leucine at position 87 in affected individuals (Figure 1). No variants were detected in any other candidate gene examined. Individuals I:1 and II:2 were found to be normal, with no EA findings on echocardiography. Methods: Whole exome sequencing in the patients involved collecting blood samples after obtaining consent. These samples were sent to Centogene lab, where genomic DNA is enzymatically fragmented. The regions of interest are then enriched using DNA capture probes, facilitating detailed genetic analysis. Results: The whole exome sequencing identified a heterozygous missense mutation in both the father and his daughter. This nonsynonymous variant is located on chromosome 1 (GRCh37) in the TNNT2 gene region. The specific variant, NM_001276345.1:c.260C&gt;T p.(Pro87Leu), was found in both individuals. Conclusion: In conclusion, the genetic basis of EA is rather complex and remains poorly understood. It is established that mutations in the TNNT2 gene are linked to several cardiomyopathies, none of them overlap with the described phenotype of our patients. In this report, we therefore confirm the potential role of TNNT2 gene mutation in the genetic basis of familial EA with WPW.

Genome-Wide Scan for Selective Sweeps Reveals Novel Loci Associated with Prolificacy in Iranian Sheep Breeds in Comparison with Highly Prolific Exotic Breed

Domestication and selection significantly changed phenotypic traits in modern domestic animals. To identify the genomic regions associated with prolificacy in this study, 837 ewes from three Iranian indigenous sheep breeds, consisting of Baluchi, Lori-Bakhtiari, and Zandi uniparous breeds, and one Greek highly prolific dairy sheep, namely Chios, were genotyped using OvineSNP50K arrays. Statistical tests were then performed using different and complementary methods based on either site frequency (FST) and haplotype (hapFLK) between populations, followed by a pathway analysis of the genes contained in the selected regions. The results revealed that for the top 0.01 percentile of the obtained FST values, 16 genomic regions on chromosomes 2, 3, 4, 7, 8, 9, 13, 14, 16, 18, 19, and 20, and for hapFLK values, 3 regions located on chromosomes 3, 7, and 13, were under selection. A bioinformatic analysis of these genomic regions showed that these loci overlapped with potential candidate genes associated with prolificacy in sheep including GNAQ, COL5A2, COL3A1, HECW1, FBN1, COMMD3, RYR1, CCL28, SERPINA14, and HSPA2. These regions also overlapped with some quantitative trait loci (QTLs) linked to prolificacy traits, milk yield, and body weight. These findings suggest that future research could further link these genomic regions to prolificacy traits in sheep.

Abstract 4136781: Stethoscopes to Sequencers: How Next Generation Sequencing Aids Pediatric Cardiac Intensive Care Diagnosis

Background: Despite a high burden of rare genetic diseases among children admitted to cardiac intensive care units (CICUs), comprehensive guidelines for genetic testing in children with heart disease, particularly those that are critically ill, are lacking. Traditional testing methods, such as karyotype, chromosomal microarray (CMA) and candidate gene panels, are often employed based on suspected diagnoses. Rapid advancements in next-generation sequencing (NGS) technologies allow for more comprehensive genetic evaluation which is already recommended for patients with ventricular dysfunction, primary arrhythmias, and pulmonary hypertension (PH). Broad application of NGS in intensive care settings for pediatric heart disease has not been extensively investigated. Aims: Evaluate diagnostic efficacy of NGS in critically ill pediatric patients with cardiac disease. Methods: Retrospective cohort of patients who underwent exome or genome sequencing in a pediatric CICU between January 2020 and August 2023. Results: Exome or genome sequencing was performed on 49 patients with median testing age of 46 days (IQR 7-609). Primary cardiac phenotypes included congenital heart disease, ventricular dysfunction, arrhythmia, and PH. Forty-two pathogenic or likely pathogenic variants were identified and 23 (46.9%) resulted in a genetic diagnosis, of those, 16 (69.6%) were associated with a cardiac phenotype. Positivity rate did not differ between primary cardiac phenotypes but was significantly higher when the patient had associated multiple congenital anomalies (Fig. 1). Genome sequencing trended towards a higher positivity rate (54.5%) than exome (31.3%), though did not meet statistical significance (Fig. 1). Notably, 84.6% and 38.5% of the identified pathogenic variants would have remained undetectable by CMA and many commercially available gene panels, respectively. Conclusion: Exome and genome sequencing demonstrate high diagnostic yield across isolated and non-isolated cardiac disease and various cardiac phenotypes with diagnostic efficacy surpassing CMA and many commercial gene panels. As NGS technologies evolve, they promise to streamline diagnoses and enhance therapeutic decision-making for this patient population.

Transcriptomic profiling of backfat and muscle in Lijiang pigs with divergent body size across growth stages.

Backfat thickness is an important economic trait that affects pork quality and flavor. The Lijiang pig (LJP), a local breed in Yunnan province, China, exhibits variations in growth and body composition. However, the molecular basis for these variations is unclear. This study aimed to analyze transcriptome profiles of backfat at different growth stages in LJP with discrepant body size: two months (M2), four months, and six months. Firstly, we analyzed the gene expression differences and discovered a significantly highest number of differentially expressed genes (DEGs) at the M2 stage. Secondly, we identified four gene profiles with reverse expression trends in LJP populations with different body sizes, and the related genes mainly associated with immune response functionality. Thirdly, we observed a lower correlation in LJP with large body size at the M2 stage, with a specific enrichment of DEGs with high genetic differentiation related to neural activity. Finally, we correlated transcriptome profiles of muscle and backfat and discovered the lowest correlation at the M2 stage. Highly correlated genes exhibited more significant differences and were prominently enriched for immune response processes. This study unveils candidate genes linked to backfat growth in LJP, emphasizing the specificity of the early growth stage of backfat.

Transcriptome analyses reveal molecular mechanisms of novel compound heterozygous ACO2 variants causing infantile cerebellar retinal degeneration

Background and purposeInfantile cerebellar retinal degeneration (ICRD) (OMIM #614559) is a rare autosomal recessive inherited disease associated with mutations in the aconitase 2 (ACO2) gene. We report a Chinese girl with novel compound heterozygous variants in ACO2, who presented at 7 months of age with psychomotor retardation, truncal hypotonia, and ophthalmologic abnormalities. This study aims to investigate the potential molecular mechanisms underlying ACO2 deficiency-induced neuropathy.MethodsWhole exome sequencing was performed on family members to screen for potential pathogenic mutations, followed by Sanger sequencing for validation. Mitochondrial aconitase activity and mitochondrial DNA (mtDNA) copy number were measured using an aconitase activity detection kit and quantitative PCR, respectively. Transcriptome expression profiles from patient cells, and cerebellar and retinal organoids retrieved from the GEO database were integrated. Functional enrichment analysis and protein-protein interaction networks were used to identify key molecules, and their expression levels were validated using Western blot analysis.ResultsGenetic testing revealed novel compound heterozygous variations in the proband's ACO2 gene (NM:001098), including c.854A&amp;gt;G (p.Asn285Ser) and c.1183C&amp;gt;T (p.Arg395Cys). Predictive analysis of the tertiary structure of the ACO2 protein suggests that both p.Asn285Ser and p.Arg395Cys affect the binding ability of ACO2 to ligands. The mitochondrial aconitase activity and mtDNA copy number in the proband's leukocytes were significantly reduced. Transcriptomic data analysis identified 80 key candidate genes involved in ACO2-related neuropathy. Among these, LRP8 and ANK3, whose gene expression levels were significantly positively correlated with ACO2, were further validated by Western blot analysis.ConclusionsThis study expands the spectrum of pathogenic ACO2 variants, elucidates the potential molecular mechanisms underlying ACO2-related neuropathy, provides in-depth support for the pathogenicity of ACO2 genetic variations, and offers new insights into the pathogenesis of ICRD.

Integrated eQTL mapping approach reveals genomic regions regulating candidate genes of the E8-r3 locus in soybean

Deciphering the gene regulatory networks of critical quantitative trait loci associated with early maturity provides information for breeders to unlock soybean’s (Glycine max (L.) Merr.) northern potential and expand its cultivation range. The E8-r3 locus is a genomic region regulating the number of days to maturity under constant short-day photoperiodic conditions in two early-maturing soybean populations (QS15524F2:F3 and QS15544RIL) belonging to maturity groups MG00 and MG000. In this study, we developed a combinatorial expression quantitative trait loci mapping approach using three algorithms (ICIM, IM, and GCIM) to identify the regions that regulate three candidate genes of the E8-r3 locus (Glyma.04G167900/GmLHCA4a, Glyma.04G166300/GmPRR1a, and Glyma.04G159300/GmMDE04). Using this approach, a total of 2,218 trans (2,061 genes)/7 cis (7 genes) and 4,073 trans (2,842 genes)/3,083 cis (2,418 genes) interactions were mapped in the QS15524F2:F3 and QS15544RIL populations, respectively. From these interactions, we successfully identified two hotspots (F2_GM15:49,385,092-49,442,237 and F2_GM18:1,434,182-1,935,386) and three minor regions (RIL_GM04:17,227,512-20,251,662, RIL_GM04:31,408,946-31,525,671 and RIL_GM13:37,289,785-38,620,690) regulating the candidate genes of E8-r3 and several of their homologs. Based on co-expression network and single nucleotide variant analyses, we identified ALTERED PHLOEM DEVELOPMENT (Glyma.15G263700) and DOMAIN-CONTAINING PROTEIN 21 (Glyma.18G025600) as the best candidates for the F2_GM15:49,385,092-49,442,237 and F2_GM18:1,434,182-1,935,386 hotspots. These findings demonstrate that a few key regions are involved in the regulation of the E8-r3 candidates GmLHCA4a, GmPRR1a, and GmMDE04.

Advancing Chickpea Breeding: Omics Insights for Targeted Abiotic Stress Mitigation and Genetic Enhancement.

Chickpea is a major source of proteins and is considered the most economically vital food legume. Chickpea production is threatened by several abiotic and biotic factors worldwide. The main constraints limiting worldwide chickpea production are abiotic conditions such as drought, heat, salinity, and cold. It is clear that chickpea is treasured for its nutritive value, in particular its high protein content, and hence study of problems like drought, cold and salinity stresses are very important concerning chickpeas. In this regard, several physiological, biochemical, and molecular mechanisms are reviewed to confer tolerance to abiotic stress. The most crippling economic losses in agriculture occur due to these abiotic stressors, which affect plants in many ways. All these abiotic stresses affect the water relations of the plant, both at the cellular level as well as the whole-plant level, causing both specific and non-specific reactions, damage and adaptation reactions. These stresses share common features. Breeding programs use a huge collection of over 100,000 chickpea accessions as their foundation. Significant advancements in conventional breeding, including mutagenesis, gene/allele introgression, and germplasm introduction, have been made through this method. Abiotic tolerance and yield component selection are made easier by creating unique DNA markers for the genus Cicer, which has been made possible by developments in high-throughput sequencing and molecular biology. Transcriptomics, proteomics, and metabolomics have also made it possible to identify particular genes, proteins, and metabolites linked to chickpea tolerance to abiotic stress. Chickpea abiotic stress tolerance has been directly and potentially improved by biotechnological applications, which are covered by all 'Omics' approaches. It requires information on the abiotic stress response at the different molecular levels, which comprises gene expression analysis for metabolites or proteins and its impact on phenotype. Studies on chickpea genome-wide expression profiling have been conducted to determine important candidate genes and their regulatory networks for abiotic stress response. This study aimed to offer a detailed overview of the diverse 'Omics' approaches for resilience's to abiotic stresses on chickpea plants.

ACVR2B polymorphism, Adiponectin, and GDF-15 levels as biomarkers for cachexia in gastrointestinal cancer.

Reversing cancer cachexia remains a challenge. Genetic biomarkers for the early detection of the disease have been explored in order to enable the implementation of preventive measures. We therefore genotyped candidate genes based on cachexia phenotype and quantified adiponectin and GDF-15 levels in cachectic patients with gastrointestinal cancer. Patients with a diagnosis of gastrointestinal cancer were divided into a cachectic and a non-cachectic group after the start of chemotherapy. A control group (no cancer) was also included. We genotyped the following single nucleotide polymorphisms (SNPs) by quantitative PCR: FOXO3 (rs1935949), FOXO3 (rs4946935), ACVR2B (rs2268757), and SELP (rs6136). In addition, we quantified adiponectin and GDF-15 levels by ELISA. The rs2268757 SNP in the ACVR2B gene was associated with the weight loss phenotype in cachectic patients with gastrointestinal cancer (non-cachectic, P = 0.004). Plasma adiponectin levels were higher in cachectic patients compared to controls (P = 0.01) and non-cachectic patients (P = 0.004). GDF-15 was also elevated in cachectic patients compared to controls (P < 0.0001) and non-cachectic patients (P = 0.001). Analysis by sex showed elevated adiponectin levels in men (control, P = 0.01) and cachectic women (control, P = 0.04; non-cachectic, P = 0.01), as well as elevated GDF-15 levels in men (control, P = 0.002) and cachectic women (control, P = 0.002; non-cachectic, P = 0.007). However, there was no significant difference in the levels of these cytokines between cachectic men and women. The results suggest the rs2268757 SNP in the ACVR2B gene, adiponectin, and GDF-15 as potential biomarkers of cachexia in gastrointestinal cancer.

Genome-wide scan for selection signatures in Mexican Sardo Negro Zebu cattle.

The Sardo Negro cattle (SN) is the only zebu cattle breed developed in Mexico. Since its development, the selection could have led to an increase in the homozygosity level in some regions of the genome and made differentiation with other cattle populations. We aimed to identify and characterize selection signatures in SN using medium-density SNP data using four approaches: 1) Runs of homozygosity (ROH) 2) Nucleotide Diversity 3) Tajima's D and 4) the Wright's fixation index (FST). A sample of 555 SN animals genotyped for 65k SNPs was used to obtain ROH segments considered regions under selection. The FST values were estimated by comparing the sample of genotyped SN animals with samples of genotyped animals from the Gir, Brahman, and Ongole breeds. Only one region mapped to 35.78-42.51 Mb on BTA6 was considered a selection signature by the ROH method. This selection signature overlapped with the lowest diversity, negative values of Tajima's D and a diversification region between SN and the other Zebu breeds by FST. We found several candidate genes (LCORL, NCAPG, and SLIT2) related to growth and other economically important productive traits in this common region. Using the FST method, different regions, such as regions on BTA8 (8:93.4-93.9 Mb), BTA11 (11:99.2-99.7), and BTA14 (14: 26.1-26.8) related to growth and milk traits also were defined as candidate selection signatures. The selective signals identified in this study reflected the direction of the selection pressure that primarily involves the increase of live weight traits in the Sardo Negro cattle breeding program.

Mining of Candidate Genes Associated with Leaf Shape Traits in Grapes

As the most important organ for photosynthesis, leaves provide the main energy source for plant growth. Leaf traits affect light energy utilization and, thus, plant development and biomass. Given the high morphological variability of leaves between and within grape genotypes, phenotypic analysis is challenging. This study first evaluated leaf shape trait parameters using a specific leaf profile and area analyzer, along with genome-wide association study (GWAS) analyses, to identify additional candidate genes related to grape leaf shape traits. In the two-year analysis, 89 single-nucleotide polymorphisms (SNPs) were found to be significantly associated with leaf shape traits. These SNP loci were distributed on 18 chromosomes, of which chromosome 15 had the most relevant SNPs. We found that leaf shape-associated genes included mainly plant hormone-, ubiquitin ligase-, serine/threonine protein kinase-, transcription factor-, and cell wall metabolism-related genes. By analyzing the expression of these candidate genes on the chip, we found that they exhibited diverse expression levels in leaves at different developmental stages (young, mature, and senescent). This suggests that these genes could be considered candidates for grape leaf improvement.

Identification of novel variants and candidate genes in women with 46,XX complete gonadal dysgenesis.

46,XX complete gonadal dysgenesis (46,XX-CGD) is a rare disorder of sexual development (DSD) characterized by primary amenorrhea and a lack of spontaneous pubertal development in individuals with a 46,XX karyotype despite the presence of female internal and external genitalia due to failure of bilateral ovarian development. The condition is genetically heterogeneous, and in most cases, its etiology is unknown. Determining the genetic cause would provide insights into potential targets for genetic diagnosis and counseling. To clarify the molecular mechanisms of 46,XX complete gonadal dysgenesis in the population of China, whole-exome sequencing (WES) was performed on DNA samples from patients with 46,XX-CGD. In silico analysis was conducted to predict the pathogenicity of the variants. We recruited 20 patients with 46,XX-CGD and identified 8 variants in 6 genes, including three homozygous variants in MCM9, POF1B, and PSMC3IP; compound heterozygous variants in TWNK; and three heterozygous variants in TP63 and INSRR, from 7 patients. These variants included 3 recurrent variants and 5 novel variants. This study identified several novel variants, broadening the variant spectrum of 46,XX-CGD. 46,XX-CGD is a genetically heterogeneous condition, and WES is a powerful tool for determining its genetic etiology. The results of this study will aid researchers and clinicians in genetic counseling and suggest that WES is valuable for detecting 46,XX-CGD, which may lead to early interventions for patients.

Fine mapping of a major co-localized QTL associated with self-incompatibility identified in two F2 populations (broccoli × cauliflower and cauliflower × Chinese kale).

A major QTL responsible for self-incompatibility was stably identified in two F2 populations. Through fine mapping and qRT-PCR analysis, ARK3 emerged as the most promising candidate gene, playing a pivotal role in regulating self-incompatibility in Brassica oleracea. Self-incompatibility (SI) is a common phenomenon in Brassica oleracea species, which can maintain genetic diversity but will also limit seed production. Although the S locus has been extensively studied in Arabidopsis and some Brassicaceae crops, map-based cloning of self-incompatibility genes has not been conducted in Brassica oleracea, such as cauliflower and broccoli. In the present study, we identified a major co-localized QTL on chromosome C6 that control SI in two F2 populations derived from intervarietal crosses: broccoli × cauliflower (CL_F2) and cauliflower × Chinese kale (CJ_F2). Subsequently, this QTL was narrowed down to 168.5 Kb through fine mapping using 3,429 F2:3 progenies and 12 available KASP markers. Within this 168.5 Kb region, BolC6t39084H, a homologue of Arabidopsis ARK3, could be a candidate gene that plays a key role in regulating SI in B. oleracea species. This finding can pave the way for an in-depth understanding of the molecular mechanisms underlying SI, and will contribute to the seed production of B. oleracea vegetables.

TMET-08. RADIATION-INDUCED SLC25A22 CONTRIBUTES TO GLIOBLASTOMA RADIORESISTANCE ACQUISITION THROUGH REMODELING GLUTAMATE METABOLISM

Abstract Glioblastoma Multiforme (GBM), a malignant primary brain tumor, is treated by surgical resection followed by chemo- and radiotherapy. Nevertheless, most GBM patients show dismal prognosis due to the radioresistance acquisition of GBM cells via metabolic rewiring. Therefore, it is urgently required to discover how metabolic rewiring attributes to GBM radioresistance, and its targeting strategies. To identify GBM radioresistance driver genes, we previously established radioresistance GBM cells (RGCs) via repetitive in vivo selection, followed by mRNA sequencing, and selected candidate genes that are altered in RGCs (fold change&amp;gt;2 or &amp;lt;0.5, p-value&amp;lt;0.05) and also related with GBM patients’ prognosis. Here, we focused on amino acid transmembrane transporter (Gene Ontology: 0003333) and found that SLC25A22 was the most increased in RGCs. SLC25A22 exhibits uni-directional properties, exporting glutamate to cytosol, resulting in cytosolic glutamate accumulation in RGCs. Using 13C glutamine isotope tracing analysis following mitochondrial fractionation, we quantified the mitochondrial metabolic flux of glutamine. Despite decreased 13C glutamate efflux from RGCs mitochondria after genetic inhibition of SLC25A22, glutamine metabolites (a-ketoglutarate, succinate, malate, fumarate) remained constant. Cytosolic glutamate, as a substrate, is incorporated into various biosynthetic pathways, especially glutathione (GSH) and proline synthesis pathways in radioresistant GBM cells. GSH protects GBM cells by scavenging radiation-induced reactive oxygen species. Whereas, proline, a rate-limiting substrate for collagen biosynthesis, leads GBM cells to show an invasiveness phenotype via extracellular matrix remodeling. Genetic inhibition of SLC25A22 using short hairpin RNA (shRNA) or microRNA-184 mimic suppresses RGCs radioresistance and aggressiveness. Additionally, intranasal administration of microRNA-184 mimic prolonged the survival (median survival: 26 days after irradiation) of GBM-bearing mice, compared with non-target control (median survival: 16 days after irradiation). Together, our study demonstrates that SLC25A22 upregulation confers GBM radioresistance by inducing cytosolic glutamate-driven biosynthesis and suggests SLC25A22 as a therapeutic target to overcome GBM radioresistance.

Genetic Diversity and Genome-Wide Association Study of Pleurotus pulmonarius Germplasm

Pleurotus pulmonarius is prized by consumers for its distinct flavor, strong aroma, and dense, crispy texture. Although China has extensive germplasm resources for P. pulmonarius, only a limited number of cultivars are commercially available. A comprehensive evaluation and detailed analysis of P. pulmonarius germplasm, alongside the exploration of superior germplasm resources, are essential for developing new varieties. In this study, we resequenced the genomes of 47 P. pulmonarius strains collected nationwide, identifying a total of 4,430,948 single nucleotide polymorphism (SNP) loci. After filtering based on minor allele frequency and data integrity, 181,731 high-quality SNP markers were retained. Phylogenetic analysis grouped the strains into six clusters, with strains from similar geographical regions clustering together. Most CBS strains formed a single cluster; cultivated varieties exhibited higher genetic similarity, whereas wild strains displayed greater diversity. Principal component analysis (PCA) and population structure analyses, using the same SNP markers, corroborated the phylogenetic findings. DNA fingerprinting, derived from 369 core SNPs, further underscored the genetic diversity among strains. Significant morphological variation was observed, with strains in groups ZP, CBS, and WHLJ exhibiting notably higher yields and cap widths compared to other groups. Correlation analysis revealed associations among various phenotypes, while genome-wide association study (GWAS) identified multiple SNP markers within candidate genes linked to agronomic traits, most of which were controlled by multiple genes. This research offers a molecular-level characterization and evaluation of P. pulmonarius germplasm resources, providing a scientific basis for enriching available germplasm and advancing breeding materials.

Prediction of causal genes at GWAS loci with pleiotropic gene regulatory effects using sets of correlated instrumental variables.

Multivariate Mendelian randomization (MVMR) is a statistical technique that uses sets of genetic instruments to estimate the direct causal effects of multiple exposures on an outcome of interest. At genomic loci with pleiotropic gene regulatory effects, that is, loci where the same genetic variants are associated to multiple nearby genes, MVMR can potentially be used to predict candidate causal genes. However, consensus in the field dictates that the genetic instruments in MVMR must be independent (not in linkage disequilibrium), which is usually not possible when considering a group of candidate genes from the same locus. Here we used causal inference theory to show that MVMR with correlated instruments satisfies the instrumental set condition. This is a classical result by Brito and Pearl (2002) for structural equation models that guarantees the identifiability of individual causal effects in situations where multiple exposures collectively, but not individually, separate a set of instrumental variables from an outcome variable. Extensive simulations confirmed the validity and usefulness of these theoretical results. Importantly, the causal effect estimates remained unbiased and their variance small even when instruments are highly correlated, while bias introduced by horizontal pleiotropy or LD matrix sampling error was comparable to standard MR. We applied MVMR with correlated instrumental variable sets at genome-wide significant loci for coronary artery disease (CAD) risk using expression Quantitative Trait Loci (eQTL) data from seven vascular and metabolic tissues in the STARNET study. Our method predicts causal genes at twelve loci, each associated with multiple colocated genes in multiple tissues. We confirm causal roles for PHACTR1 and ADAMTS7 in arterial tissues, among others. However, the extensive degree of regulatory pleiotropy across tissues and the limited number of causal variants in each locus still require that MVMR is run on a tissue-by-tissue basis, and testing all gene-tissue pairs with cis-eQTL associations at a given locus in a single model to predict causal gene-tissue combinations remains infeasible. Our results show that within tissues, MVMR with dependent, as opposed to independent, sets of instrumental variables significantly expands the scope for predicting causal genes in disease risk loci with pleiotropic regulatory effects. However, considering risk loci with regulatory pleiotropy that also spans across tissues remains an unsolved problem.

EXTH-16. UNBIASED DISCOVERY OF SYNERGISTIC VULNERABILITIES AGAINST GLIOBLASTOMA: AN INNOVATIVEIN VIVO CRISPR PROFILING PLATFORM TO UNLEASH THE POWER OF TUMOR-TARGETED CYTOKINE THERAPY

Abstract BACKGROUND Glioblastoma poses a formidable challenge due to its intricate intra- and intertumoral heterogeneities and its associated immunosuppressive milieu. The standards of care have limited activity, and no major advances have been made in decades. One promising avenue is tumor-targeted cytokine-based therapies, particularly L19-TNF, which is undergoing clinical trials in Europe and the US for patients with newly diagnosed and recurrent glioblastoma. However, there is still an opportunity to find safer and more tolerable combination partners to improve the efficacy of L19-TNF immunotherapy. To address this challenge, we employed a cutting-edge in vivo CRISPR screening approach to identify synergistic vulnerabilities with a focus on druggable targets. METHODS We systematically investigated the dependencies of tumor growth and key signaling pathways under the pressure of L19-TNF immunotherapy in the CT-2A orthotopic murine glioma model. We used an innovative in vivo CRISPR screening approach that enables timed sgRNA activation to mitigate off-target effects and overcome orthotopic tumor cell implantation challenges, such as the bottleneck of cell engraftment. RESULTS We first conducted a genome-wide in vivo screen and identified 400 top-scoring candidate genes, which we subsequently tested again in a targeted in vivo screen to refine our drug selection process. Through this, we uncovered novel and previously unexplored druggable targets for glioblastoma. Out of these, we validated four promising target-specific drugs in preclinical glioblastoma models in downstream in vivo experiments. In addition, we investigated modes of action and resistance. CONCLUSION Our research marks a significant advancement in pursuing novel treatment strategies for glioblastoma. We have unveiled a range of synergistic combinations and paved the way for the rational design of combinatory treatment approaches to maximize the therapeutic efficacy of L19-TNF immunotherapy, laying the groundwork for future clinical translation.