Reduced Folate Carrier Research Articles

Identification of IQ Motif Containing GTPase Activating Protein 2, IQGAP-2 as an interacting partner with the Human Colonic Thiamin Pyrophosphate Transporter (hcTPPT): Effects on physiology and Cell Biology of the Transporter

Background:Humans/mammals obtain vitamin B1 from two sources: the diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of thiamin pyrophosphate (TPP). We have previously shown that colonocytes process an effcient and specific carrier-mediated process for uptake of the generated TPP that involves the human colonic thiamine pyrophosphate transporter (hcTPPT, product of the SLC44A4 gene). We have also shown that expression of the cTPPT along the GI tract is restricted to the large intestine and have characterized different aspects of its regulation at both the transcriptional and post-transcriptional levels.Aim:To determine whether the hcTPPT has interacting protein partner(s) in human colonocytes that affect its functionality and/or cell biology.Methods: Yeast Two-Hybrid system (Y2H) was used to screen a random-primed normal human colonic cDNA library using the full-length SLC44A4 as a bait. Effect of knocking-down (with gene-specific siRNA) and over-expressing the putative interactor was examined using human-derived colonic epithelial NCM460 cells and human primary differentiated colonoid monolayers (obtained form organ donors). We also used confocal imaging, RT-qPCR and western blotting in our investigations. Results:Our Y2H screening have identified three putative interactors with the hcTPPT, namely: IQGAP-2, SNX-6, and DMXL-1. While all these putative interactors are expressed in human colonocytes, expression of the IQGAP-2 was the highest, and thus, we focused our investigation on this putative interactor. First, we verified the co-localization of IQGAP-2 with the hcTPPT in human colonic NCM460 cells using confocal imaging. We then examined the effect of expressing IQGAP-2 in NCM460 cells and in differentiated colonoids on carrier-mediated TPP uptake and observed significant induction with both models. Knock-down IQGAP-2 (with gene-specific siRNA), on the other hand, led to a significant decrease in TPP uptake by colonocytes. We also found that expressing the IQGAP-2 in colonocytes lead to a marked enhancement in hTPPT protein stability (increased protein half-life). Interestingly, we also found that expression of IQGAP-2 was suppressed by factors/conditions (e.g. lipopolysaccharide, pro-inflammatory cytokine TNF-α and hypoxia, respectively) that inhibit colonic TPP uptake.Conclusion: The accessory protein IQGAP-2 is an interacting partner with the hcTPPT in human colonocytes and that the interaction has physiological/cell biological consequences. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells.

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene (MLL) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in βHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2. Similar observations were made in MLL heterozygous knockout mice (MLL+/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.

Multiple genetic variants at the SLC30A8 locus affect local super-enhancer activity and influence pancreatic β-cell survival and function.

Variants at the SLC30A8 locus are associated with type 2 diabetes (T2D) risk. The lead variant, rs13266634, encodes an amino acid change, Arg325Trp (R325W), at the C-terminus of the secretory granule-enriched zinc transporter, ZnT8. Although this protein-coding variant was previously thought to be the sole driver of T2D risk at this locus, recent studies have provided evidence for lowered expression of SLC30A8 mRNA in protective allele carriers. In the present study, we examined multiple variants that influence SLC30A8 allele-specific expression. Epigenomic mapping has previously identified an islet-selective enhancer cluster at the SLC30A8 locus, hosting multiple T2D risk and cASE associations, which is spatially associated with the SLC30A8 promoter and additional neighboring genes. Here, we show that deletion of variant-bearing enhancer regions using CRISPR-Cas9 in human-derived EndoC-βH3 cells lowers the expression of SLC30A8 and several neighboring genes and improves glucose-stimulated insulin secretion. While downregulation of SLC30A8 had no effect on beta cell survival, loss of UTP23, RAD21, or MED30 markedly reduced cell viability. Although eQTL or cASE analyses in human islets did not support the association between these additional genes and diabetes risk, the transcriptional regulator JQ1 lowered the expression of multiple genes at the SLC30A8 locus and enhanced stimulated insulin secretion.

Polymorphism rs3733591 of the SLC2A9 gene and metabolic syndrome affect gout risk in Taiwan Biobank subjects.

Over the past few decades, gout and diseases like metabolic syndrome (MetS) have become more prevalent. Attempts have been made in Taiwan to identify the genes responsible for gout. A few gene loci, among them SLC2A9, have been identified using Taiwan Biobank (TWB) data. We, therefore, examined whether MetS could also account for the association between polymorphism SLC2A9 rs3733591 and gout. The final analysis consisted of 73,558 subjects, of whom 2,709 had gout. To estimate the likelihood of gout occurrence based on rs3733591 and MetS, we used logistic regression models. Rs3733591-TC + CC compared to TT genotype was associated with gout (OR, 1.15; 95% CI, 1.06-1.25). Also associated with gout was MetS (OR, 1.21; 95% CI, 1.10-1.33). A significant interaction was seen between rs3733591 and MetS (p-value = 0.039). Using rs3733591-TT/no MetS as the reference group, the ORs (95% CI) for gout was 1.24 (1.11-1.38) for TC + CC/no MetS, 1.35 (1.17-1.56) for TT/MetS, and 1.39 (1.22-1.58) for TC + CC/MetS. However, subgroup analysis defined by sex showed no significant associations in women. In summary, metabolic syndrome and SLC2A9 rs3733591 genotypes were interactively associated with gout in Taiwanese men, but not women.

The positive feedback loop of the NAT10/Mybbp1a/p53 axis promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury

Ferroptosis is a nonapoptotic form of regulated cell death that has been reported to play a central role in cardiac ischemia‒reperfusion (I/R) injury. N-acetyltransferase 10 (NAT10) contributes to cardiomyocyte apoptosis by functioning as an RNA ac4c acetyltransferase, but its role in cardiomyocyte ferroptosis during I/R injury has not been determined. This study aimed to elucidate the role of NAT10 in cardiac ferroptosis as well as the underlying mechanism. The mRNA and protein levels of NAT10 were increased in mouse hearts after I/R and in cardiomyocytes that were exposed to hypoxia/reoxygenation. P53 acted as an endogenous activator of NAT10 during I/R in a transcription-dependent manner. Cardiac overexpression of NAT10 caused cardiomyocyte ferroptosis to exacerbate I/R injury, while cardiomyocyte-specific knockout of NAT10 or pharmacological inhibition of NAT10 with Remodelin had the opposite effects. The inhibition of cardiomyocyte ferroptosis by Fer-1 exerted superior cardioprotective effects against the NAT10-induced exacerbation of post-I/R cardiac damage than the inhibition of apoptosis by emricasan. Mechanistically, NAT10 induced the ac4C modification of Mybbp1a, increasing its stability, which in turn activated p53 and subsequently repressed the transcription of the anti-ferroptotic gene SLC7A11. Moreover, knockdown of Mybbp1a partially abolished the detrimental effects of NAT10 overexpression on cardiomyocyte ferroptosis and cardiac I/R injury. Collectively, our study revealed that p53 and NAT10 interdependently cooperate to form a positive feedback loop that promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury, suggesting that targeting the NAT10/Mybbp1a/p53 axis may be a novel approach for treating cardiac I/R.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

SLC44A4 Gene Deficiency: A Case Report Showcasing the Use of Supportive Photobiomodulation in a Congenital Conundrum with a Cerebral Palsy-Like Presentation

SLC44A4 Gene Deficiency: A Case Report Showcasing the Use of Supportive Photobiomodulation in a Congenital Conundrum with a Cerebral Palsy-Like Presentation

Investigation of the association between the polymorphism <i>SLC2A9</i> rs2280205 and gout in Vietnamese population

Gout, a predominant form of inflammatory arthritis, is characterized by the deposition of uric acid crystals in joints and other tissues. Genome-wide association studies (GWAS) have established the association between gout and genetic variants of solute carrier family 2 member 9 (SLC2A9). In this study, we assessed the association of the single nucleotide polymorphism SLC2A9 rs2280205 with gout susceptibility in the Vietnamese population. Genomic DNA was extracted from the peripheral blood of 477 samples (160 gout patients and 317 healthy controls). The polymorphism rs2280205 of the SLC2A9 gene was genotyped using the PCR-RFLP method. Results indicated that the allele distribution of this variant was in accordance with Hardy-Weinberg Equilibrium, and the genotype frequencies of rs2280205 GG/GA/AA were 0.65, 0.33, and 0.02, respectively. However, no relation was identified between SLC2A9 rs2280205 and gout in this studied population. This research contributed a preliminary insight into the genetic factors in the development of gout in the Vietnamese population.

Folate gene polymorphisms CBS 844ins68 and RFC1 A80G and risk of Down syndrome offspring in young Iranian women: A cross-sectional study

Background: Cytogenetics and association studies showed that folate gene polymorphisms can increase the risk of chromosomal nondisjunction and aneuploidies. The folate-metabolizing gene polymorphisms in Down syndrome mothers (DSM) have been assessed in a variety of populations. Reduced folate carrier 1 (RFC1) and cystathionine beta-synthase (CBS) are key enzymes in folate metabolism.
 Objective: 2 common polymorphisms, CBS 844ins68 and RFC1 A80G, were analyzed to determine their probable risk for having Down syndrome (DS) babies in young mothers of Khuzestan province, Iran.
 Materials and Methods: This study was conducted on 100 mothers who had trisomy 21 DS children. 100 age- and ethnic-matched mothers with at least 2 healthy children and no history of abnormal pregnancies were considered as control. The samples were collected from all the mothers from June 2019 to April 2021. Genomic DNA was extracted from peripheral blood. The CBS-844ins68 and RFC1-A80G were genotyped using polymerase chain reaction-electrophoresis and restriction fragment length polymorphism, respectively.
 Results: The frequency of RFC1 AG and GG genotypes in DSM was significantly higher than the control mothers (odds ratio [OR] of 2.38 and 3.07, respectively). The heterozygote genotype of CBS 844ins68 was significantly more prevalent among DSM than the control (OR: 2.419). The OR was significantly increased to 6.667 when the homozygote of both variants was found together.
 Conclusion: Studying polymorphisms possibly increases the susceptibility of having a DS child. However, ethnicity, nutrition, and epistatic interactions are considerable factors to be evaluated in future studies.
 Key words: Down syndrome, Folic acid, Polymorphism, CBS, RFC1.

Abstract 5883: Role of major facilitative folate transporters SLC19A1 and SLC46A1 in cyclic dinucleotide transport and STING signaling

Abstract Activation of stimulator of interferon genes (STING) activates the transcription factor pIRF3, promoting gene transcription encoding type I interferons including IFNβ that stimulate broader immune responses. Targeting the STING pathway for cancer is especially compelling given its importance to the malignant phenotype. Cyclic dinucleotides (CDNs) function as STING agonists. Inspired by preclinical studies, two phase I clinical trials with CDNs including ADU-S100 were initiated but concluded early due to lack of anti-tumor efficacy. The reduced folate carrier (RFC; SLC19A1) has been identified as one of the primary means to transport CDNs through the cell membrane, although a study suggested that CDN and folate binding to RFC was as least partly distinct. While depletion of the proton-coupled folate transporter (PCFT; SLC46A1) in THP-1 cells had nominal effects on CDN stimulation, overexpressing PCFT increased STING signaling. Limited clinical responses may reflect the impact of physiologic variables governing RFC and PCFT not previously considered. Understanding the mechanisms of CDN uptake by RFC and PCFT under physiologic conditions may elucidate the role of CDNs on STING signaling. To study the impact of RFC levels on CDN transport and STING signaling, we used an engineered R1-11/Tet-on-RFC HeLa cell model induced with doxycycline. In R1-11/Tet-on-RFC cells, ADU-S100 showed a low affinity for binding to RFC, as measured by direct competition for transport with [3H]methotrexate. RFC dose-dependence of ADU-S100 uptake and STING activation was confirmed by monitoring IFN-β (qPCR) and pIRF3 (Westerns). Maximum induction of pIRF3/IFNβ was seen in THP-1 and R1-11/Tet-on-RFC cells by ADU-S100 treatment at pH 7.2 (RFC pH optimum) in the presence of 25 nM leucovorin. Induction of pIRF3/IFNβ by ADU-S100 in THP-1 and R1-11/Tet-on-RFC cells were partially abolished by treatment with 200 μM of leucovorin or 10 μM of PT523 (RFC-specific inhibitor), suggesting the transport of folates and CDNs may share common albeit distinct mechanisms. ADU-S100 treatment showed no pIRF3 or IFN-β production in an engineered R1-11/Tet-on-PCFT HeLa model expressing high levels of PCFT, suggesting no direct transport by PCFT, even under optimal pH conditions. Interestingly, pIRF3/IFN-β induction was enhanced in R1-11/Tet-on-RFC/PCFT cells expressing constitutive PCFT in addition to RFC, compared to R1-11/Tet-on-RFC cells that express comparable RFC without PCFT. This may reflect previously unrecognized functional interactions between RFC and PCFT. In conclusion, RFC mediates ADU-S100 uptake and STING activation under physiologic conditions and this process is enhanced by concomitant expression of PCFT. Additional characterization of key molecular and biochemical determinants of CDN-based cancer therapeutics may lead to improved approaches for CDN therapy based on enhanced cellular uptake. Citation Format: Holly McQuithey, Madelyn Brzezinski, Carrie O’Connor, Larry Matherly, Zhanjun Hou. Role of major facilitative folate transporters SLC19A1 and SLC46A1 in cyclic dinucleotide transport and STING signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5883.

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Identification and validation of ferroptosis-related gene SLC2A1 as a novel prognostic biomarker in AKI.

Emerging evidence reveals the key role of ferroptosis in the pathophysiological process of acute kidney injury (AKI). Our study aimed to investigate the potential ferroptosis-related gene in AKI through bioinformatics and experimental validation. The AKI single-cell sequencing dataset was retrieved from the GEO database and ferroptosis-related genes were extracted from the GENECARD website. The potential differentially expressed ferroptosis-related genes of AKI were selected. Functional enrichment analysis was performed. Machine learning algorithms were used to identify key ferroptosis-related genes associated with AKI. A multi-factor Cox regression analysis was used to construct a risk score model. The accuracy of the risk score model was validated using receiver operating characteristic (ROC) curve analysis. We extensively explored the immune landscape of AKI using CIBERSORT tool. Finally, expressions of ferroptosis DEGs were validated in vivo and in vitro by Western blot, ICH and transfection experiments. Three hub genes (BAP1, MDM4, SLC2A1) were identified and validated by constructing drug regulatory network and subsequent screening using experimentally determined interactions. The risk mode showed the low-risk group had significantly better prognosis compared to high-risk group. The risk score was independently associated with overall survival. The ROC curve analysis showed that the prognosis model had good predictive ability. Additionally, CIBERSORT immune infiltration analysis suggest that the hub gene may influence cell recruitment and infiltration in AKI. Validation experiments revealed that SLC2A1 functions by regulating ferroptosis. In summary, our study not only identifies SLC2A1 as diagnostic biomarker for AKI, but also sheds light on the role of it in AKI progression, providing novel insights for the clinical diagnosis and treatment of AKI.

Cucurbitacin B induces ferroptosis in oral leukoplakia via the SLC7A11/mitochondrial oxidative stress pathway

BackgroundOral leukoplakia (OLK), characterized by abnormal epithelial hyperplasia, is the most common precancerous oral mucosa lesion and is closely related to oxidative stress. Cucurbitacin B (CuB), a tetracyclic triterpenoid molecule derived from plants, has shown promising anti-proliferative and antioxidant effects in preclinical studies. However, whether CuB can play an antiproliferative role in OLK by regulating oxidative stress remains elusive. PurposeTo investigate the role of CuB in inhibiting the malignant progression of oral leukoplakia and to further explore its underlying mechanisms of action. MethodsIn vitro, the effect of CuB on the proliferation, migration, apoptosis, and cell cycle of OLK cells DOK was detected. The core genes and key pathways of OLK and CuB were analyzed in the transcriptome database, by using immunofluorescence, qRT-PCR, and Western blot to evaluate the expression levels of the ferroptosis markers ROS, GSH, MDA, Fe2+, and marker genes SLC7A11, GPX4, and FTH1. Immunohistochemistry of human tissue was performed to investigate the expression of the SLC7A11. In vivo, the model of OLK was established in C57BL/6 mice and the biosafety of CuB treatment for OLK was further evaluated. ResultsCuB substantially suppressed the proliferation of DOK cells. Bioinformatics analysis showed that the core targets of OLK crossing with CuB include SLC7A11 and that the essential pathways involve ROS and ferroptosis. In vitro experiments indicated that CuB might promote ferroptosis by down-regulating the expression of SLC7A11. We observed a gradual increase in SLC7A11 expression levels during the progression from normal oral mucosa to oral leukoplakia with varying degrees of epithelial dysplasia. In vivo experiments demonstrated that CuB inhibited the malignant progression of OLK by promoting ferroptosis in OLK mice and exhibited a certain level of biosafety. ConclusionThis study demonstrated for the first time that CuB could effectively inhibit the malignant progression of OLK by inducing ferroptosis via activating the SLC7A11/ mitochondrial oxidative stress pathway. These findings indicate that CuB could serve as the lead compound for the future development of anti-oral leukoplakia drugs.

Genetic diversity of variants involved in drug response among Tunisian and Italian populations toward personalized medicine

Adverse drug reactions (ADR) represent a significant contributor to morbidity and mortality, imposing a substantial financial burden. Genetic ancestry plays a crucial role in drug response. The aim of this study is to characterize the genetic variability of selected pharmacogenes involved with ADR in Tunisians and Italians, with a comparative analysis against global populations. A cohort of 135 healthy Tunisians and 737 Italians were genotyped using a SNP array. Variants located in 25 Very Important Pharmacogenes implicated in ADR were extracted from the genotyping data. Distribution analysis of common variants in Tunisian and Italian populations in comparison to 24 publicly available worldwide populations was performed using PLINK and R software. Results from Principle Component and ADMIXTURE analyses showed a high genetic similarity among Mediterranean populations, distinguishing them from Sub-Saharan African and Asian populations. The Fst comparative analysis identified 27 variants exhibiting significant differentiation between the studied populations. Among these variants, four SNPs rs622342, rs3846662, rs7294, rs5215 located in SLC22A1, HMGCR, VKORC1 and KCNJ11 genes respectively, are reported to be associated with ethnic variability in drug responses. In conclusion, correlating the frequencies of genotype risk variants with their associated ADRs would enhance drug outcomes and the implementation of personalized medicine in the studied populations.

Distribution of the SLC2A9 genotype and its association with the uric acid level in patients with arterial hypertension and atrial fibrillation

Asymptomatic hyperuricemia (HU) is widespread in the population and is one of the main risk factors for cardiovascular diseases. Evidence of numerous studies supports the association between increased uric acid (UA) levels and the risk of arterial hypertension (HTN) and/or atrial fibrillation (AF). One of the causes for an increase in UA levels and a significant risk factor for HU is the polymorphism of the SLC2A9 gene, a highly specific urate transporter in proximal renal tubule cells, encoding the GLUT9 protein.The aim of the study is to investigate the frequency of genotypes and alleles of the SLC2A9 gene rs734553 polymorphic variant and its association with the UA level in patients with HTN and AF, as well as in healthy individuals. 141 patients, including 50 healthy patients (group 1), 68 – with HTN and AF (group 2), 23 – with HTN (group 3) were examined.In the overall group of patients, the frequencies of the SLC2A9 gene rs734553 polymorphism were as follows: AA – 46.1 % (n = 65), AC – 36.2 % (n = 51), CC – 17.7 % (n = 25). According to the genotyping results of patients, in group 1 the AA genotype was 64 % (n = 32), AC – 22 % (n = 11), CC – 14 % (n = 7); in group 2 – 32.4 % (n = 22), 48.5 % (n = 33) and 19.1 % (n = 13); in group 3 47.8 % (n = 11), 30.4 % (n = 7) and 21.7 % (n = 5), respectively. The CC genotype patients (420 [413; 424] μmol/l) and the AC genotype patients (330 [284; 412] μmol/l) had higher uricemia values than those with the AA genotype (310 [281; 341] μmol/l) ( p = 0,003). HU with the AA genotype in group 2 was diagnosed in 2 (2.9 %) patients, with the AC genotype in 12 (17.6 %), and with the CC genotype in 10 (14.7 %) patients; in group 3 – 1 (4.3 %), 1 (4.3 %) and 3 (13 %), respectively. One patient (2 %) with HU in group 1 had the CC genotype.The distribution of genotype and allele frequencies of the SLC2A9 gene rs734553 polymorphic variant and its association with the UA level in patients with HTN and AF, as well as in healthy individuals were established. In patients with HTN and AF, the AC genotype occurred 3 times more often and the CC genotype occurred 2 times more often than in healthy patients. HU occurred significantly more often ( р = 0.003) in the group of patients with HTN and AF and the CC genotype, as well as in patients with the CC genotype.

Identification of novel mutations in TPK1 and SLC19A3 genes in families exhibiting thiamine metabolism dysfunction syndrome

Background and aimsThe occurrence of thiamine metabolism dysfunction syndrome (THMD), a rare autosomal recessive condition, may be linked to various mutations found in the TPK1 and SLC19A3 genes. The disease chiefly manifests through ataxia, muscle hypotonia, abrupt or subacute onset encephalopathy, and a decline in developmental milestones achieved during the early stages of infancy. We present findings from an investigation that involved two individuals from Iran, both of whom experienced seizures along with ataxia and hypotonia. The underlying genetic causes were found with the use of next-generation sequencing (NGS) technology, which has facilitated the detection of causal changes in a variety of genetic disorders. Material and methodsThe selection of cases for this study was based on the phenotypic and genetic information that was obtainable from the Center for Comprehensive Genetic Services. The genetic basis for the problems observed among the participants was determined through the application of whole-exome sequencing (WES). Subsequently, sanger sequencing was employed as a means of validating any identified variations suspected to be causative. ResultsThe first patient exhibited a homozygous mutation in the TPK1 gene, NM_022445.4:c.224 T > A:p.I75 N, resulting in the substitution of isoleucine for asparagine at position 75 (p.I75 N). In our investigation, patient 2 exhibited a homozygous variant, NM_025243.4:c.1385dupA:pY462X, within the SLC19A3 gene. ConclusionsCollectively, when presented with patients showcasing ataxia, encephalopathy, and basal ganglia necrosis, it is essential to account for thiamine deficiency in light of the potential advantages of prompt intervention. At times, it may be feasible to rectify this deficiency through the timely administration of thiamine dosages. Accordingly, based on the results of the current investigation, these variations may be useful for the diagnosis and management of patients with THMD.

Altered placental immune cell composition and gene expression with isolated fetal spina bifida.

Fetal spina bifida (SB) is more common in pregnant people with folate deficiency or anomalies of folate metabolism. It is also known that fetuses with SB have a higher risk of low birthweight, a condition that is typically placental-mediated. We therefore hypothesized that fetal SB would associate with altered expression of key placental folate transporters and an increase in Hofbauer cells (HBCs), which are folate-dependent placental macrophages. Folate receptor-α (FRα), proton coupled folate receptor (PCFT), and reduced folate carrier (RFC) protein localization and expression (immunohistochemistry) and HBC phenotypes (HBC abundance and folate receptor-β [FRβ] expression; RNA in situ hybridization) were assessed in placentae from fetuses with SB (cases; n=12) and in term (n=10) and gestational age (GA) - and maternal body mass index - matched (n=12) controls without congenital anomalies. Cases had a higher proportion of placental villous cells that were HBCs (6.9%vs. 2.4%, p=.0001) and higher average HBC FRβ expression (3.2 mRNA molecules per HBC vs. 2.3, p=.03) than GA-matched controls. HBCs in cases were largely polarized to a regulatory phenotype (median 92.1% of HBCs). In sex-stratified analyses, only male cases had higher HBC levels and HBC FRβ expression than GA-matched controls. There were no differences between groups in the total percent of syncytium and stromal cells that were positive for FRα, PCFT, or RFC protein immunolabeling. HBC abundance and FRβ expression by HBCs are increased in placentae of fetuses with SB, suggesting immune-mediated dysregulation in placental phenotype, and could contribute to SB-associated comorbidities.

РАСПРЕДЕЛЕНИЕ ГЕНОТИПОВ ГЕНА SLC2A9, УРОВЕНЬ МОЧЕВОЙ КИСЛОТЫ И МЕТАБОЛИТЫ ПУРИНОВОГО ОБМЕНА У ПАЦИЕНТОВ С АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИЕЙ И ФИБРИЛЛЯЦИЕЙ ПРЕДСЕРДИЙ

The aim is to investigate the frequency of genotypes and alleles of the SLC2A9 gene rs734553 polymorphism, the level of serum uric acid (sUA) and metabolites of purine metabolism in patients with arterial hypertension (AH) and atrial fibrillation (AF), as well as in healthy individuals. Material and methods. The study included 154 patients: 50 were healthy individuals (group 0), of which 22 (44%) were men and 28 (56%) were women aged 50 [45;53] years and 104 were patients with AH and AF (main group), of which 94 (90.4%) were men and 10 (9.6%) were women aged 55 [45; 61] years. The main group was divided into subgroups: subgroup I – patients with AF without a history of AH and other rhythm disorders (n=13); subgroup II – patients with AH in combination with AF (n=68); subgroup III – patients with AH without a history of AF or other rhythm disturbances (n=23). Hyperuricemia was detected in 34 (22.1%) patients, normal uric acid levels were revealed in 120 (77.9%) patients. All patients were studied using clinical, laboratory, instrumental and molecular genetic research methods. The level of sUA was determined by the enzymatic colorimetric method. Serum xanthine oxidase was measured using a method based on a solid phase sandwich-type enzyme-linked immunosorbent assay. Metabolites of purine metabolism in blood plasma were measured using the method of high-performance liquid chromatography. The SLC2A9 gene rs734553 polymorphism was determined using the polymerase chain reaction with real-time detection of results. Results. The patients with AH and AF, as compared to healthy individuals, had more severe disturbances of purine metabolism, characterized by higher concentration of sUA (330 [283; 412] µmol/l and 197 [161; 229] µmol/l (p<0.001), respectively). Also, in contrast to the group of healthy individuals, the group of patients with AH and AF demonstrated an increase in the level of adenosine (p=0.001), and a decrease in the levels of hypoxanthine and xanthine (p<0.001). There were no statistically significant differences in xanthine oxidase activity level (p>0.05), however, in 54% of patients in the main group it was higher than normal values. The dominant allele A and the dominant genotype A/A of the SLC2A9 gene rs734553 polymorphism (75%, p=0.005; 64%, p=0.001, respectively) occurred significantly more often in healthy individuals, while the recessive allele C and the heterozygous genotype A/C were found significantly more often in the group of patients with AH and AF (41.3%, p=0.005; 48.1%, p=0.003, respectively). The C/C genotype (41.7%, p=0.001) was significantly more common in patients with AH in combination with AF and hyperuricemia, compared to patients with AH combined with AF without hyperuricemia as well as healthy individuals (6.8%; 14%, p=0.001, respectively). Patients with AH in combination with AF and the C/C genotype were characterized by a significantly higher level of sUA (p=0.003) compared to patients with the A/A genotype. Conclusions. A statistically significant predominance of the recessive allele C of the SLC2A9 gene rs734553 polymorphism was established in patients with AH and AF compared with healthy individuals (p=0.005). In patients with AH in combination with AF and hyperuricemia, the C/C genotype was significantly more common (41.7%, p=0.001). Patients with AH in combination with AF and the C/C genotype were characterized by a significantly higher level of sUA (p=0.003) compared to patients with the A/A genotype.

Influence of SLC22A1 gene polymorphisms on gastrointestinal adverse effects with metformin therapy in South Indian type 2 diabetes mellitus patients

Background: Metformin, a first-line agent in Type 2 diabetes mellitus, causes gastrointestinal adverse effects in 20-30% of patients, leading to discontinuation in 5-10% of them. Organic cation transporter 1 (OCT1) encoded by SLC22A1, transports metformin from the enterocytes into the bloodstream. Reduced function OCT1 variants could lead to increased luminal concentration of metformin leading to gastrointestinal adverse effects. Two single nucleotide polymorphisms in the SLC22A1 gene were studied in this cross-sectional study with cases and controls. Objective was to determine the association between genetic polymorphisms rs628031 (1222A>G) and rs622342 (1386C>A) in SLC22A1 gene and gastrointestinal adverse effects to metformin therapy in South Indian type 2 diabetes mellitus patients. Methods: The study was conducted in JIPMER, Puducherry, India in T2DM patients (n=300) of South Indian origin, who were categorized into case (N=100) and control (N=200) groups, based on their gastrointestinal tolerance to metformin. DNA was extracted from the patients using whole blood by phenol-chloroform method and genotyping was done using real-time PCR. Results: Minor allele frequency of rs628031 (A allele) and rs622342 (C allele) were 33.8% and 26.5% respectively. Genotype frequencies did not differ significantly between the case and control groups (rs628031, p=0.45, rs622342, p=0.28). Female gender (AOR 3.77; 95% CI 2.07, 6.85; p<0.001) and proton pump inhibitor usage (AOR 7.66; 95% CI 3.01, 19.47; p<0.001) had higher association with metformin intolerance. Conclusions: No significant association was found between the genotypes of single nucleotide polymorphisms (rs628031 and rs622342) in the SLC22A1 gene and gastrointestinal adverse effects to metformin therapy in South Indian type 2 diabetes mellitus patients.

Mitochondrial and Cytosolic One-Carbon Metabolism Is a Targetable Metabolic Vulnerability in Cisplatin-Resistant Ovarian Cancer.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared with normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin-sensitive (A2780, CaOV3, IGROV1) and cisplatin-resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359, and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism, which offers a promising new platform for therapy.