Region Of Gene Research Articles

Identification of a novel 145 kb deletion (Guigang deletion, -Guigang) in the alpha-globin gene cluster from a Chinese newborn using third-generation sequencing.

To describe a novel α-thalassemiadeletion identified from a newborn by third-generation sequencing (TGS). The proband, a newborn subject to neonatal capillary electrophoresis (CE) screening, exhibited suspected α0-thalassemia carrier status (Hb Bart's 3.0%). Notably, both parents had negative results on thalassemia screening during pregnancy. Multiplex ligation-dependent probe amplification (MLPA) presented a deletion between probes 364nt and 472 nt that extended from the HBZ gene to the downstream region of the RGS11 gene. Subsequently, TGS determined the approximated break position of this deletion, indicating a length exceeding 145 kb (chr16:127,815-273,190 del 145376 bp). Sanger sequencing validated the upstream and downstream breakpoints of this deletion. Only maternal data were available for pedigree analysis, with the father's sample lacking. MLPA showed no deletion in the mother, suggesting possible paternal inheritance. The deletion was named Guigang deletion (--Guigang) after the proband's city of origin, Guigang. We reported a novel α-thalassemiadeletion and provided insights into the hematological phenotype and molecular analysis. These findings have implications for genetic counseling and prenatal diagnosis.

Genetic Heterogeneity Across Dimensions of Alcohol Use Behaviors.

Increasingly large samples in genome-wide association studies (GWASs) for alcohol use behaviors (AUBs) have led to an influx of implicated genes, yet the clinical and functional understanding of these associations remains low, in part because most GWASs do not account for the complex and varied manifestations of AUBs. This study applied a multidimensional framework to investigate the latent genetic structure underlying heterogeneous dimensions of AUBs. Multimodal assessments (self-report, interview, electronic health records) were obtained from approximately 400,000 UK Biobank participants. GWAS was conducted for 18 distinct AUBs, including consumption, drinking patterns, alcohol problems, and clinical sequelae. Latent genetic factors were identified and carried forward to GWAS using genomic structural equation modeling, followed by functional annotation, genetic correlation, and enrichment analyses to interpret the genetic associations. Four latent factors were identified: Problems, Consumption, BeerPref (declining alcohol consumption with a preference for drinking beer), and AtypicalPref (drinking fortified wine and spirits). The latent factors were moderately correlated (rg values, 0.12-0.57) and had distinct patterns of associations, with BeerPref in particular implicating many novel genomic regions. Patterns of regional and cell type-specific gene expression in the brain also differed between the latent factors. Deep phenotyping is an important next step to improve understanding of the genetic etiology of AUBs, in addition to increasing sample size. Further effort is required to uncover the genetic heterogeneity underlying AUBs using methods that account for their complex, multidimensional nature.

Dynamic landscape of m6A modifications and related post-transcriptional events in muscle-invasive bladder cancer

BackgroundMuscle-invasive bladder carcinoma (MIBC) is a serious and more advanced stage of bladder carcinoma. N6-Methyladenosine (m6A) is a dynamic and reversible modifications that primarily affects RNA stability and alternative splicing. The dysregulation of m6A in MIBC can be potential target for clinical interventions, but there have been limited studies on m6A modifications in MIBC and their associations with post-transcriptional regulatory processes.MethodsPaired tumor and adjacent-normal tissues were obtained from three patients with MIBC following radical cystectomy. The additional paired tissues for validation were obtained from patients underwent transurethral resection. Utilizing Nanopore direct-RNA sequencing, we characterized the m6A RNA methylation landscape in MIBC, with a focus on identifying post-transcriptional events potentially affected by changes in m6A sites. This included an examination of differential transcript usage, polyadenylation signal sites, and variations in poly(A) tail length, providing insights into the broader impact of m6A alterations on RNA processing in MIBC.ResultsThe prognostic-related m6A genes and m6A-risk model constructed by machine learning enables the stratification of high and low-risk patients with precision. A novel m6A modification site in the 3’ untranslated region (3’UTR) of IGLL5 gene were identified, characterized by a lower m6A methylation ratio, elongated poly(A) tails, and a notable bias in transcript usage. Furthermore, we discovered two particular transcripts, VWA1-203 and CEBPB-201. VWA1-203 displayed diminished m6A methylation levels, a truncated 3’UTR, and an elongated poly(A) tail, whereas CEBPB-201 showed opposite trends, highlighting the complex interplay between m6A modifications and RNA processing. Source code was provided on GitHub (https://github.com/lelelililele/Nanopore-m6A-analysis).ConclusionsThe state-of-the-art Nanopore direct-RNA sequencing and machine learning techniques enables comprehensive identification of m6A modification and provided insights into the potential post-transcriptional regulation mechanisms on the development and progression in MIBC.

CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.

This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.

Comparison of intestinal and pharyngeal microbiota in preterm infants on the first day of life and the characteristics of pharyngeal microbiota in infants delivered by cesarean section or vaginally

BackgroundThis study aimed to explore the distribution of intestinal and pharyngeal microbiota on the first day of life in preterm infants and compare the composition of microbiota in infants delivered by cesarean section or vaginally.MethodsThis study included 44 late preterm infants with a gestational age of 34–36 + 6 weeks. Stool and throat swab samples were collected from the preterm infants on the first day of life. The infants were divided into cesarean section and vaginal delivery groups. Illumina NovaSeq high-throughput sequencing technology was used to sequence the V3-V4 hypervariable region of the 16S rRNA gene of all bacteria in the samples. Venn diagram was used to identify shared operational taxonomic units (OTUs) in the intestines and pharynges. Microbial analysis was conducted at the phylum and genus levels, and α and β diversity comparisons were performed.Results(1) Gestational age may have significantly affected the microbial colonization of the intestines and pharynges of preterm infants on the first day after birth (p ≤ 0.001). (2) More OTUs were detected in the pharynx than in the intestines, both have a total of 819 shared OTUs. Proteobacteria, Firmicutes, and Bacteroidota were the dominant phyla in both. At the genus level, Streptococcus had a lower relative abundance in stool samples (0.5%) compared to throat samples (0.5% vs. 22.2%, p = 0.003). 3) The relative abundance of Streptococcus in pharyngeal samples was 26.2% in the cesarean section group much higher than the 3.8% in the vaginal delivery group (p = 0.01).ConclusionThe early postnatal period is a critical time for the establishment of an infant's microbiota. Gestational age at birth may influence microbial colonization, while birth weight, gender, and mode of delivery do not. The intestinal and pharyngeal microbiota composition of preterm infants on the first day after birth showed high similarity, but larger samples are needed for further validation.

Lipid-lowering drugs and risk of rapid renal function decline: a mendelian randomization study.

Chronic kidney disease (CKD) patients face the risk of rapid kidney function decline leading to adverse outcomes like dialysis and mortality. Lipid metabolism might contribute to acute kidney function decline in CKD patients. Here, we utilized the Mendelian Randomization approach to investigate potential causal relationships between drug target-mediated lipid phenotypes and rapid renal function decline. In this study, we utilized two methodologies: summarized data-based Mendelian randomization (SMR) and inverse variance-weighted Mendelian randomization (IVW-MR), to approximate exposure to lipid-lowering drugs. This entailed leveraging expression quantitative trait loci (eQTL) for drug target genes and genetic variants proximal to drug target gene regions, which encode proteins associated with low-density lipoprotein (LDL) cholesterol, as identified in genome-wide association studies. The objective was to investigate causal associations with the progression of rapid kidney function decline. The SMR analysis revealed a potential association between high expression of PCSK9 and rapid kidney function decline (OR = 1.11, 95% CI= [1.001-1.23]; p = 0.044). Similarly, IVW-MR analysis demonstrated a negative association between LDL cholesterol mediated by HMGCR and kidney function decline (OR = 0.74, 95% CI = 0.60-0.90; p = 0.003). Genetically predicted inhibition of HMGCR is linked with the progression of kidney function decline, while genetically predicted PCSK9 inhibition is negatively associated with kidney function decline. Future research should incorporate clinical trials to validate the relevance of PCSK9 in preventing kidney function decline.

Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y chromosome segment lengths in phenotypic males with 46,XX testicular disorder/difference of sex development.

46,XX testicular disorder/difference of sex development (46,XX DSD) is a rare congenital condition, characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of individuals with 46,XX DSD, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment and the genome-wide effects remain elusive. We performed long-read DNA sequencing, RNA sequencing and DNA methylation analyses on blood samples from 46,XX DSD (n = 11), male controls (46,XY; variable cohort sizes) and female controls (46,XX; variable cohort sizes), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measurements on all 46,XX DSD and a subset of 46,XY (n = 10). We identified variation in the translocated Y chromosome segments, enabling subcategorization into 46,XX DSD (1) lacking Y chromosome material (n = 1), (2) with short Yp arms (breakpoint at 2.7-2.8Mb, n = 2), (3) with medium Yp arms (breakpoint at 7.3Mb, n = 1), and (4) with long Yp arms (n = 7), including deletions of AMELY, TBLY1 and in some cases PRKY. We also identified variable expression of the X-Y homologues PRKY and PRKX. The Y-chromosomal transcriptome and methylome reflected the Y chromosome segment lengths, while changes to autosomal and X-chromosomal regions indicated global effects. Furthermore, transcriptional changes tentatively correlated with phenotypic traits of 46,XX DSD, including reduced height, lean mass and testicular size. This study refines our understanding of the genetic composition in 46,XX DSD, describing the translocated Y chromosome segment in more detail than previously and linking variability herein to genome-wide changes in the transcriptome and methylome.

Serological and molecular characterization of novel ABO variants including an interesting B(A) subgroup.

ABO grouping is the most important pretransfusion testing that is directly related to the safety of blood transfusion. A weak ABO subgroup is one of the important causes of an ABO grouping discrepancy. Here, we investigated the characterization of four novel ABO variants including a novel B(A) subgroup. RBCs were phenotyped by standard serology methods. The full coding regions of the ABO gene and the erythroid cell-specific regulatory elements in intron one were sequenced. The effect of the possible splice site variant was predicted by Alamut software. The 3D structural modeling of three relative B(A) enzymes (p.Met214Thr, p.Met214Val, and p.Met214Leu) were performed by PyMOL software. Four novel ABO alleles were identified with weak ABO expression in this study, in which two would lead to premature terminations, and two resulted in amino acid changes. In silico analysis revealed that the splice site variant c.155G>T had the potential to alter splice transcripts. 3D structural view shown that the variant amino acid position 214 was spatially adjacent to the donor recognition pocket residues (266Met and 268Ala) and just next to the 211DVD213 motif. The size of the side chain of Thr and Val is the smallest, Leu is medium, and Met is the largest, and the size changes in the critical position 214 may affect the donor recognition pocket. Four ABO subgroup alleles were newly linked to different kinds of ABO variants and the possible mechanism through which they produce weak ABO subgroups was analyzed in silico.

Potato E3 Ubiquitin Ligase StXERICO1 Positively Regulates Drought Resistance by Enhancing ABA Accumulation in Potato and Tobacco and Interacts with the miRNA Novel-miR1730-3p and Proteins StUBC and StTLP

Potato (Solanum tuberosum L.) is sensitive to drought, which severely impacts tuber yield and quality. In this study, we characterized a XERICO gene, encoding a RING-H2 type E3 ubiquitin ligase, StXERICO1, from a diploid potato, investigated its role in enhancing drought resistance and ABA accumulation, and identified its interaction with the miRNA novel-miR1730-3p, as well as its protein interactions with StUBC and StTLP. StXERICO1, with a complete Open Reading Frame (ORF) of 459 bp encoding 152 amino acids, was highly responsive to drought, ABA treatment, and abiotic stresses in potato plants. Overexpression of the StXERICO1 significantly enhanced drought resistance and ABA accumulation in transgenic potato and tobacco plants and exhibited greater sensitivity to ABA treatment, which was associated with the upregulation of expression of ABA biosynthetic genes NCED and CYP707A. Furthermore, our results revealed that StXERICO1 and its encoding protein interacted with miRNAs and other proteins. 5′ RLM-RACE (cDNA terminal rapid amplification) experiment showed that the miRNA novel-miR1730-3p targets 5′ UTR region of the StXERICO1 gene. Dual luciferase assay and virus-based miRNA silencing experiment showed that the novel-miR1730-3p negatively regulates StXERICO1 expression. Moreover, yeast two-hybrid assay indicated that StXERICO1 interacts with StUBC (an E2 ubiquitin ligase) and StTLP (a Tubby-like protein), suggesting that StXERICO1 might function on ABA homeostasis at the post-translational level. These findings elucidate the molecular mechanisms by which StXERICO1, a RING-H2 type E3 ubiquitin ligase, enhances drought resistance through increased ABA accumulation, how its expression is regulated by miRNA, and how it exerts its function through interactions with other proteins. The results also provide a potential candidate gene for subsequent precision molecular breeding aimed at improving crop drought resistance.

Reporting three rare pathogenic variants at the CFTR gene in two unrelated Iranian Azeri children with cystic fibrosis

BackgroundCystic fibrosis (CF) is an autosomal recessive inherited life-threatening disease that causes changes in the electrolyte transport system, leading to high absorption of sodium and water. Disease-causing variants of the CFTR gene are responsible for this disease. In the present study, three rare pathogenic variants were identified in two unrelated Iranian Azeri children with CF.Case presentationOne child affected with CF was found to have two rare variants, c.1545_1546delTA and c.3196C > T, detected through Sanger sequencing of the entire coding region and promoter of the CFTR gene. Another patient was identified as compound heterozygous for the variant c.1545_1546delTA and a rare variant, c.2998delA, which has not been previously reported. The variants were found to be in trans, as both parents were heterozygous for the variants.ConclusionThe rare variant c.1545_1546delTA has been previously reported as a known variant in certain populations, including Azeris in northwest Iran. Our results, along with previous findings, suggest that this variant may be considered a founder mutation in specific geographical regions. The variant c.2998delA has not been previously reported as pathogenic. Following the guidelines of the American College of Genetics and Genomics and considering the proband's symptoms, we classified this variant as pathogenic based on criteria PVS1, PS4, PM3, PP1, and PM2. The identified variants in the CFTR gene as well as the previously reported variants could serve as a basis for future genetic counseling and prenatal diagnosis in Iran.

Molecular Diagnosis and Typing of Cryptosporidium spp. Species in Human Stools with Diarrhea.

This study was conducted to molecularly identify and classify Cryptosporidium spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. In this study, the positivity of Cryptosporidium spp. in fecal samples was investigated using direct microscopy, Kinyoun's acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, Cryptosporidium spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S rRNA gene region. The sequencing results identified the isolates as C. parvum. Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the Cryptosporidium parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of Cryptosporidium species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to Cryptosporidium in both humans and animals in Türkiye.

Hypermethylation in Ovarian Cancer of Long Non-Coding RNA Genes: <i>HOTAIR</i>, <i>GAS5</i>, <i>LINC00472</i>, <i>LINC00886</i>, <i>TUG1</i>

Recently, more and more data have been accumulating indicating the role of long non-coding RNAs (lncRNAs) in the regulation of biological processes in cells, as well as in the mechanisms of cancer development and progression. Aberrant methylation of promoter regions of both protein genes and lncRNA genes can disrupt their expression and functional activity. Using bioinformatics databases, six lncRNA genes (GAS5, HOTAIR, LINC00472, LINC00886, SNHG17 and TUG1) with CpG islands, differentially expressed and presumably hypermethylated in tumors of patients with ovarian cancer (OC) were selected. A statistically significant (p 0.05) increase in the methylation level in tumours was demonstrated in a sample of 93 OC specimens using methylation-specific real-time PCR assay. Moreover, for the genes LINC00472, LINC00886, SNHG17 and TUG1, hypermethylation in OC was detected for the first time. 5 genes (except SNHG17) showed a further increase in methylation levels at a more advanced stage, and 4 genes (except SNHG17 and LINC00886) showed a significant association with metastasis. Using real-time RT-PCR, differential changes in the expression level of the GAS5, HOTAIR, SNHG17 and TUG1 genes and a significant correlation of methylation with expression for the GAS5 gene were shown. Thus, hypermethylation associated with the progression and/or development of OC was detected for six lncRNA genes, which is important for elucidating the epigenetic processes involved in the pathogenesis of OC and can be used as new biomarkers of OC.

The Complete Chloroplast Genome of Meconopsis simplicifolia and Its Genetic Comparison to Other Meconopsis Species

Background: Chloroplasts, due to their high conservation and lack of recombination, serve as important genetic resources for the classification and evolutionary analysis of closely related species that are difficult to distinguish based on their morphological features. Meconopsis simplicifolia (M. simplicifolia), an endangered herb within the Meconopsis genus, has demonstrated therapeutic potential in treating various diseases. However, the highly polymorphic morphology of this species poses a challenge for accurate identification. Methods: In this study, the complete chloroplast genome of M. simplicifolia was sequenced and assembled using Illumina sequencing technology. Simple sequence repeats (SSRs) and repetitive sequences were characterized. In addition, a comparative analysis was conducted with the chloroplast genomes of six other Meconopsis species. Results: The chloroplast genome of M. simplicifolia has a quadripartite circular structure with a total length of 152,772 bp. It consists of a large single-copy region of 83,824 bp and a small single-copy region of 17,646 bp, separated by a pair of inverted repeat sequences (IRa and IRb, 25,651 bp). The genome contains 131 genes, 33 SSRs, and 27 long repetitive sequences. Comparative analysis with six other chloroplast genomes of Meconopsis revealed that M. simplicifolia is closely related to M. betonicifolia and that the rpl2 (ribosomal protein L2) gene in the IRb region has been deleted. This deletion is of significant importance for future taxonomic studies of M. simplicifolia. Conclusions: This study provides a valuable reference for the identification of M. simplicifolia and contributes to a deeper understanding of the phylogeny and evolution of the Meconopsis genus.

Genomic Sequencing to Detect Cross-Breeding Quality in Dogs: An Example Studying Disorders in Sexual Development

Disorders of sexual development (DSDs) in dogs, similar to humans, arise from genetic mutations, gonadal differentiation, or phenotypic sex development. The French Bulldog, a breed that has seen a surge in popularity and demand, has also shown a marked increase in DSD incidence. This study aims to characterize the genetic underpinnings of DSDs in a French Bulldog named Brutus, exhibiting ambiguous genitalia and internal sexual anatomy, and to explore the impact of breeding practices on genetic diversity within the breed. We utilized a comprehensive approach combining conventional cytogenetics, molecular techniques, and deep sequencing to investigate the genetic profile of Brutus. The sequence data were compared to three other male French Bulldogs’ genome sequences with typical reproductive anatomy, including Brutus’s father and the canine reference genome (CanFam6). We found a Robertsonian fusion involving chromosome 23 previously reported in dogs as a causative mutation responsible for sex reversal syndrome. Our findings revealed a 22% mosaicism (78,XX/77,XX), the absence of the sex-determining region (SRY) gene, and the presence of 43 unique Single Nucleotide Variants (SNVs) not inherited from the father. Notably, the run of homozygosity (ROH) analysis showed Brutus has a higher number of homozygous segments compared to other Bulldogs, with a total length of these fragments 50% greater than the average, strongly suggesting this dog is the product of the mating between siblings. Although no direct causative genes for the DSD phenotype were identified, four candidate loci warrant further investigation. Our study highlighted the need for a better annotated and curated reference dog genome to define genes causative of any specific phenotype, suggests a potential genetic basis for the DSD phenotype in dogs, and underscores the consequences of uncontrolled breeding practices in French Bulldogs. These findings highlight the importance of implementing strategic genetic management to preserve genetic health and diversity in canine populations.

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

Characterization and expression analysis of the MADS-box gene AGL8 in cotton: insights into gene function differentiation in plant growth and stress resistance.

AGAMOUS-LIKE 8 (AGL8) belongs to the MADS-box family, which plays important roles in transcriptional regulation, sequence-specific DNA binding and other biological processes and molecular functions. The genome of cotton, a representative polyploid plant, contains multiple AGL8 genes. However, their functional differentiation is still unclear. In this study, a comprehensive genomic analysis of AGL8 genes was conducted. Cotton AGL8s were subdivided into four subgroups (Groups 1, 2, 3, and 4) based on phylogenetic analysis, and different subgroups of AGL8s presented different characteristics, including different structures and conserved motifs. With respect to the promoter regions of the GhAGL8 genes, we successfully predicted cis-elements that respond to phytohormone signal transduction and the stress response of plants. Transcriptome data and real-time quantitative PCR validation indicated that three genes, namely, GH_D07G0744, GH_A03G0856 and GH_A07G0749, were highly induced by methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA), which indicated that they function in plant resistance to abiotic and biotic stresses. The information from the gene structure, number and types of conserved domains, tissue-specific expression levels, and expression patterns under different treatments highlights the differences in sequence and function of the cotton AGL8 genes. Different AGL8s play roles in vegetative growth, reproductive development, and plant stress resistance. These results lay a foundation for further study of GhAGL8s in cotton.

MhGeneS: An Analytical Pipeline to Allow for Robust Microhaplotype Genotyping.

Microhaplotypes are small linked genomic regions comprising two or more single-nucleotide polymorphisms (SNPs) that are being applied in forensics and are emerging in wildlife monitoring studies and genomic epidemiology. Typically, targeted in non-coding regions, microhaplotypes in exonic regions can be designed with larger amplicons to capture functional non-synonymous sites and minimise insertion/deletion (indel) polymorphisms. Quality control is an important first step for high-confidence genotyping to counteract such false-positive variants. As genetic markers with higher polymorphism compared to biallelic SNPs, it is critical to ensure sequencing errors across the microhaplotype amplicon are filtered out to avoid introducing false-haplotypes. We developed the MhGeneS pipeline which works in tandem with Seq2Sat to help validate microhaplotype genotyping of the coding region of genes, with broader applicability to any microhaplotype profiling. We genotyped microhaplotype regions of the Zfx (≅ 160 bp) and Zfy (≅ 140 bp) genes, as well as an exon of the prion protein (Prnp) gene (≅ 370 bp) in caribou (Rangifer tarandus) using paired-end Illumina technology. As important quality metrics affecting microhaplotype calling, we identified the sequencing error rate profile related to the overlap or non-overlap of paired-end reads as well as the read depth as significant. In the case of Prnp, we achieved confident microhaplotype calling through MhGeneS by removing small sections of the 5' and 3' amplicons and using a minimum read depth of 20. Read depth and sequence trimming may be locus-specific, and validation of these parameters is recommended before the high-throughput profiling of samples.

The coevolution of fungus-ant agriculture.

Fungus-farming ants cultivate multiple lineages of fungi for food, but, because fungal cultivar relationships are largely unresolved, the history of fungus-ant coevolution remains poorly known. We designed probes targeting >2000 gene regions to generate a dated evolutionary tree for 475 fungi and combined it with a similarly generated tree for 276 ants. We found that fungus-ant agriculture originated ~66 million years ago when the end-of-Cretaceous asteroid impact temporarily interrupted photosynthesis, causing global mass extinctions but favoring the proliferation of fungi. Subsequently, ~27 million years ago, one ancestral fungal cultivar population became domesticated, i.e., obligately mutualistic, when seasonally dry habitats expanded in South America, likely isolating the cultivar population from its free-living, wet forest-dwelling conspecifics. By revealing these and other major transitions in fungus-ant coevolution, our results clarify the historical processes that shaped a model system for nonhuman agriculture.

Maintenance of X chromosome inactivation after T cell activation requires NF-κB signaling.

X chromosome inactivation (XCI) balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of X-inactive specific transcript (Xist) RNA and heterochromatic modifications on the inactive X chromosome (Xi), which are involved in maintenance of XCI, and these modifications return to the Xi after stimulation. Here, we examined allele-specific gene expression and epigenomic profiles of the Xi in T cells. We found that the Xi in unstimulated T cells is largely dosage compensated and enriched with the repressive H3K27me3 modification but not the H2AK119-ubiquitin (Ub) mark. Upon T cell stimulation mediated by both CD3 and CD28, the Xi accumulated H2AK119-Ub at gene regions of previous H3K27me3 enrichment. T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of the TCR, was required for Xist RNA localization to the Xi. Disruption of NF-κB signaling in mouse and human T cells using genetic deletion, chemical inhibitors, and patients with immunodeficiencies prevented Xist/XIST RNA accumulation at the Xi and altered X-linked gene expression. Our findings reveal a previously undescribed connection between NF-κB signaling pathways, which affects XCI maintenance in T cells in females.