Background:Both rare and common variants in the SRY-Box Transcription Factor 17 (SOX17) locus are associated with pulmonary arterial hypertension (PAH). SOX17 dysregulation leads to pulmonary artery endothelial cell (PAEC) dysfunction and the obstructive remodelling that characterises PAH. Hypothesis:Impaired SOX17 expression contributes to the pathogenesis of PAH. Restoring the function of SOX17 or its downstream targets using compounds that mimic its transcriptomic signature will rescue PAEC dysfunction and prevent PAH development. Methods and Results:We defined thousands of genes with direct SOX17 genomic binding sites and identified important potential binding partners, including ETS-transcription factors such as ERG by ChIP-seq in PAECs. Through the integration of three PAEC RNA-seq datasets involving overexpression and silencing of SOX17, we defined a robust SOX17 transcriptomic signature. In PAH patients, circulating plasma protein levels of 10 SOX17 signature genes were associated with the SOX17 common risk variants. This included EFNB2 and UNC5B; knockdown of these genes altered the viability and apoptosis of PAECs in response to TNFα treatment. The drug-transcriptome database Connectivity Map (CMap) was used to predict novel potential therapeutic compounds to correct the SOX17 transcriptomic signature. Five compounds were selected for in vitro testing and were able to partially reinstate SOX17 target gene expression in PAECs. One compound, BX-912, was selected for in vivo testing as it corrected the levels of multiple target genes, including suppressing Runt-related transcription factor-1 (RUNX1). BX-912 blocked the development of pulmonary hypertension in mice lacking the SOX17 enhancer associated with human disease. Conclusion:We have demonstrated the therapeutic potential of targeting SOX17 in PAH through correction of its gene targets, identifying BX-912 as a lead compound with in vivo efficacy.

Abstract Pulmonary arterial hypertension (PAH) is a severe, currently incurable lung disease characterized by endothelial injury and excessive repair, leading to arterial narrowing. However, the contributory mechanisms remain poorly understood. Here we show that Krüppel-like factor 6 (KLF6) is a feature of vascular pathology in PAH. KLF6 expression is elevated in human PAH and preclinical models of PAH and promotes endothelial repair and angiogenesis through transcriptomic remodelling, with effects distinct from those of KLF2 and KLF4. Endothelial KLF6 also stimulates vascular smooth muscle cell proliferation, which is attenuated by bosentan and imatinib. DisGeNET and spatial transcriptomic analyses of control and PAH lungs reveal elevated KLF6 in PAH endothelium, endothelial progenitor cells, and PAH with alveolar capillary dysplasia. In summary, KLF6 activation uniquely orchestrates endothelial repair and is a feature of the angio-proliferative vascular phenotype in PAH.

Transcription factor networks are crucial for the regulation of endothelial cell gene expression during vascular development and homeostasis. A recent analysis of 269 rare diseases in 77,539 individuals revealed an association between loss-of-function variants in ERG, encoding an ETS transcription factor, with primary lymphoedema. However, the pathogenicity of such variants and possible mechanisms of ERG-associated lymphatic vessel dysfunction remains to be established. Here, we have further identified and characterised lymphoedema-associated ERG genetic variants, revealing pathogenic mechanisms ranging from differential ERG subcellular localisation to altered DNA-binding and impaired transactivation. We confirm a role for ERG in regulating lymphangiogenesis using in vitro assays and a lymphatic endothelium-specific Erg deletion mouse model. Furthermore, we characterise the transcriptional and epigenomic landscape of human dermal lymphatic endothelial cells, identifying a unique role for ERG in regulating lymphatic gene programs, including the establishment of cooperative TF networks with PROX1 and GATA2. Our studies identify ERG as a master regulator of lymphatic endothelial cell transcriptional networks and uncover the mechanisms that underpin a novel causative gene for primary lymphoedema. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Infrastructure support for this research was provided by the NIHR Imperial Biomedical Research Centre (BRC), The Wellcome Trust, Cancer Research UK and the Medical Research Council (MRC). This study received financial support from the British Heart Foundation (BHF) through grants to GMB (PG/20/16/35047, PG/17/33/32990) and AMR (RG/11/17/29256, RG/17/4/32662). Rosetrees Trust (Seedcorn2022\100269) supported GMB & DP; DP is supported by the NIHR Imperial BRC; TV is funded by BHF PhD Studentship FS/4yPhD/F/23/34202 to GMB & IC; DN is funded by a BHF PhD Studentship FS/4yPhD/F/20/34128 to IC, AMR and GMB. IC is recipient of a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (224662/Z/21/Z). AS was jointly funded by EPSRC (EP/L015498/1) and BHF (RE/13/4/30184). SD, ES and PO were supported by the Swiss Federal National Fund for Scientific Research (CRSII5_177191/1). SM-A, SMa, KG, KO, SD and PO were supported by the MRC (MR/P011543/1) and BHF (RG/17/7/33217). DEA was funded by Qatar National Research Fund (QNRF) Graduate Scholarship Research Award (GSRA8-I-1-0210-21001). ADC acknowledges financial support from a UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S034757/1) and Economic and Social Research Council (ESRC) grant (ES/T013397/1). Computational analyses were performed at the Imperial College Research Computing Service (DOI: 10.14469/hpc/2232). This research was made possible through access to data in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: London Brighton and Sussex Research Ethics Committee gave ethical approval for this work (REC Ref: 14/LO/0753). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All source data are available within the article. Upon publication, all sequencing data generated in this study (RNA-seq, ATAC-seq, ChIP-seq) will be publicly available at the NCBI Gene Expression Omnibus as a super-series. Software code developed for this study will be available at GitHub. Any other data are available from the corresponding authors upon reasonable request.

Background and aims Steatotic liver disease (SLD), which encompasses various causes of fat accumulation in the liver, is a major cause of liver fibrosis. Understanding the specific mechanisms of lipotoxicity, dysregulated lipid metabolism, and the role of different hepatic cell types involved in fibrogenesis is crucial for therapy development. Methods We analysed liver tissue from SLD patients and 3 mouse models. We combined bulk/spatial lipidomics, transcriptomics, imaging mass cytometry (IMC) and analysis of published spatial and single-cell RNA sequencing (scRNA-seq) data to explore the metabolic microenvironment in fibrosis. Pharmacological inhibition of sphingolipid metabolism with myriocin, fumonisin B1, miglustat and D-PDMP was carried out in hepatic stellate cells (HSCs) and human precision cut liver slices (hPCLSs). Results Bulk lipidomics revealed increased glycosphingolipids, ether lipids and saturated phosphatidylcholines in fibrotic samples. Spatial lipidomics detected >40 lipid species enriched within fibrotic regions, notably sphingomyelin (SM) 34:1. Using bulk transcriptomics (mouse) and analysis of published spatial transcriptomics data (human) we found that sphingolipid metabolism was also dysregulated in fibrosis at transcriptome level, with increased gene expression for ceramide and glycosphingolipid synthesis. Analysis of human scRNA-seq data showed that sphingolipid-related genes were widely expressed in non-parenchymal cells. By integrating spatial lipidomics with IMC of hepatic cell markers, we found excellent spatial correlation between sphingolipids, such as SM(34:1), and myofibroblasts. Inhibiting sphingolipid metabolism resulted in anti-fibrotic effects in HSCs and hPCLSs. Conclusions Our spatial multi-omics approach suggests cell type-specific mechanisms of fibrogenesis involving sphingolipid metabolism. Importantly, sphingolipid metabolic pathways are modifiable targets, which may have potential as an anti-fibrotic therapeutic strategy.

Background/Aims Metabolic dysfunction-associated steatotic liver disease (MASLD) may progress to liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma. So far, genome-wide association studies explain a small fraction of MASLD heritability. Methods We sought to identify novel genetic determinants of MASLD by exploring interactions between genetic variants and body mass index (BMI). First, we examined genome-wide interactions with BMI for circulating alanine aminotransferase (ALT) levels using UK Biobank data. For identified loci, we next examined associations with hepatic proton density fat fraction (PDFF) in 35,146 independent UK Biobank participants. Associations with PDFF were replicated in four independent European cohorts, followed by a phenome-wide association study. Finally, we used human liver epigenomic maps and CRISPR/Cas9 experiments in vitro and in vivo to functionally characterize the CYP7A1 locus. Results Thirteen loci interact with BMI for ALT (P<5E-8), including eight well-known genetic modulators of MASLD. Two loci—UBXN2B/CYP7A1 and GIPR—are additionally associated with PDFF. For the intronic rs34783010 in GIPR, the minor T allele is associated with lower BMI and higher HbA1c and liver triglyceride content in humans. The UBXN2B/CYP7A1 locus is associated with PDFF in four additional European cohorts. Epigenomic data and in vitro experiments in human liver cells prioritise rs10504255 and CYP7A1 as the functional effectors in this locus. Perturbation of CYP7A1 orthologues using CRISPR/Cas9 results in less liver fat in 10-day-old, metabolically challenged zebrafish larvae. Conclusions A genome-wide single nucleotide polymorphism×BMI design fuelled identification of two MASLD genes: CYP7A1 and GIPR.

Background & Aims: Liver sinusoidal endothelial cells (LSECs) are highly specialized components of the hepatic vascular niche, regulating liver function and disease pathogenesis through angiocrine signaling. Recently, we identified GATA4 as a key transcription factor controlling LSEC development and protecting against liver fibrosis. As the transcription factor c-Maf was strongly downregulated in Gata4-deficient LSECs, we hypothesized that c-Maf might be an important downstream effector of GATA4 in LSEC differentiation and liver fibrogenesis. Methods: Clec4g-iCre/Maffl/fl (MafLSEC-KO) mice with LSEC-specific Maf deficiency were generated and liver tissue was analyzed histologically. LSECs were isolated for bulk RNA-seq, ATAC-seq, and single-cell (sc) RNA-seq analysis. MafLSEC-KO livers were analyzed after MASH diet feeding. The expression of MAF and its targets was analyzed in published human scRNA-seq data. Results: Endothelial Maf deficiency resulted in perisinusoidal liver fibrosis (Sirius red 0.46% vs. 2.92%; p <0.05) without affecting metabolic liver zonation, accompanied by a switch from sinusoidal to continuous endothelial cell identity, which was aggravated upon MASH diet feeding (p <0.01). Furthermore, endothelial Maf deficiency caused LSEC proliferation (p <0.05) and expression of profibrotic angiocrine factors including Pdgfb, Igfbp5, Flrt2, and Cxcl12, among which FLRT2 (p <0.01) and CXCL12 (p <0.001) activated hepatic stellate cells in vitro. scRNA-seq revealed replacement of zonated LSEC subpopulations with capillarized, proliferative, sprouting and secretory endothelial cell subsets that promote liver fibrogenesis and angiogenesis. This fundamental dysregulation of LSEC gene expression and differentiation was caused by changes in chromatin accessibility and transcription factor activity following loss of Maf. Notably, endothelial MAF expression was also significantly reduced in human cirrhotic livers (p <0.0001). Conclusions: Hepatic endothelial c-Maf protects against metabolic dysfunction-associated steatohepatitis-like liver fibrosis and regulates endothelial differentiation and zonation by controlling chromatin opening. Impact and implications: This work builds on the known importance of liver sinusoidal endothelial cells in liver function and disease. Here, transcription factor c-Maf is identified as a master regulator in maintaining normal differentiation and zonation of liver sinusoidal endothelial cells, thereby protecting against the development of liver fibrosis/cirrhosis. The findings are significant for researchers and clinicians focusing on liver disease, as they suggest potential new targets for therapeutic intervention. These findings could instruct the development of novel preventive treatment options and antifibrotic therapy regimens as well as liver repair strategies, benefiting patients, clinicians and policy makers in the management of liver disease.

Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder characterized by reduced or absent hair and dystrophic nails. PHNED is caused by pathogenic variants in genes involved in hair and nail development, including HOXC13. Previously reported biallelic HOXC13 pathogenic variants led to PHNED by either disrupting protein expression through nonsense-mediated decay or altering the DNA-binding affinity of the homeobox domain of HOXC13. Here, we report a case of HOXC13-related PHNED with a rare homozygous variant, c.931C>T, p.Arg311Trp. Similarly to previously reported missense variants, p.Arg311Trp resides in the homeobox domain of HOXC13 and was assumed to lead to the decreased transcriptional activity of target genes. However, in contrast with previously reported variants, in vitro overexpression assays revealed that the p.Arg311Trp variant decreases HOXC13 protein stability, which is corroborated by a series of in silico predictions. Computational models further suggest that p.Arg311Trp results in a structural rearrangement with loss of interhelical connection between Arg311 in α-helix 3 and Glu276 in α-helix 1. Altogether, our results suggest a novel molecular mechanism causative of PHNED, whereby biallelic pathogenic variants in HOXC13 may result in decreased protein stability and consequently decreased transcriptional activity of target genes essential for hair and nail development.

Background The cost-free increase in statistical power of using imputation to infer missing genotypes is undoubtedly appealing, but is it hazard-free? This case study of three type-2 diabetes (T2D) loci demonstrates that it is not; it sheds light on why this is so and raises concerns as to the shortcomings of imputation at disease loci, where haplotypes differ between cases and reference panel. Results T2D-associated variants were previously identified using targeted sequencing. We removed these significantly associated SNPs and used neighbouring SNPs to infer them by imputation. We compared imputed with observed genotypes, examined the altered pattern of T2D-SNP association, and investigated the cause of imputation errors by studying haplotype structure. Most T2D variants were incorrectly imputed with a low density of scaffold SNPs, but the majority failed to impute even at high density, despite obtaining high certainty scores. Missing and discordant imputation errors, which were observed disproportionately for the risk alleles, produced monomorphic genotype calls or false-negative associations. We show that haplotypes carrying risk alleles are considerably more common in the T2D cases than the reference panel, for all loci. Conclusions Imputation is not a panacea for fine mapping, nor for meta-analysing multiple GWAS based on different arrays and different populations. A total of 80% of the SNPs we have tested are not included in array platforms, explaining why these and other such associated variants may previously have been missed. Regardless of the choice of software and reference haplotypes, imputation drives genotype inference towards the reference panel, introducing errors at disease loci.

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic β-cell identity and function. Elimination of Lkb1 from the β-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of β cell-selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from β-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors (TFs) important for β-cell identity, such as FOXA, MAFA or RFX6, and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates β-cell identity and function.

Background Pulmonary arterial hypertension (PAH) is a severe lung condition with an unmet clinical need. Endothelial damage is followed by excessive repair and narrowing of lung arteries, but the mechanisms are unclear. Methods We investigated the role of Krüppel-like transcription factor 6 (KLF6), known for its involvement in tissue injury response and cancer onset, in PAH through functional and expression analyses of human pulmonary artery endothelial cells (HPAECs) and PAH lung tissues. KLF6 activation patterns and transcriptional programs were compared with those regulated by KLF2 and KLF4, previously linked to PAH. Results KLF6 expression increased due to hypoxic and inflammatory triggers in early rodent PAH. KLF6 overexpression improved endothelial survival and induced angiogenesis in cultured HPAECs and human pulmonary arterial explants. Transcriptomic analysis of KLF6- overexpressing HPAECs revealed its role in regulating endothelial homeostasis and expression of arterial identity genes, such as SOX17, ERG, BMPR2, KDR, TEK, ENG, ACVRL1 and CDH5. DisGeNET analysis showed a significant association of KLF6-regulated genes with PAH. Spatial transcriptomic analysis of PAH lung vascular tissues (n=6/group) revealed significant enrichment of KLF6-regulated genes in angiogenesis, cell motility, VEGFR signalling, and extracellular matrix organization. KLF6+ Erg+ vWF+ cells accumulated in vascular channels of remodelled PAH lungs and. KLF6 expression was elevated in PAH blood-derived endothelial progenitor cells (n=5) and pulmonary arterial cells in PAH associated with Alveolar Capillary Dysplasia. Conclusions KLF6 uniquely orchestrates endothelial repair but its sustained activation promotes development of the apoptosis-resistant, angio-proliferative vascular phenotype in human PAH. Dysregulation of KLF6 signalling may have broader implications for pulmonary vascular disease. ### Competing Interest Statement The authors have declared no competing interest.

The liver plays a key role in sensing nutritional and hormonal inputs to maintain metabolic homeostasis. Recent studies into pre-mRNA splicing and alternative splicing (AS) and their effects on gene expression have revealed considerable transcriptional complexity in the liver, both in health and disease. While the contribution of these mechanisms to cell and tissue identity is widely accepted, their role in physiological and pathological contexts within tissues is just beginning to be appreciated. In this review, we showcase recent studies on the splicing and AS of key genes in metabolic pathways in the liver, the effect of metabolic signals on the spliceosome, and therapeutic intervention points based on RNA splicing.

Liver sinusoidal endothelial cells (LSECs) are highly specialised endothelial cells that form the liver microvasculature. LSECs maintain liver homeostasis, scavenging bloodborne molecules, regulating immune response, and actively promoting hepatic stellate cell quiescence. These diverse functions are underpinned by a suite of unique phenotypical attributes distinct from other blood vessels. In recent years, studies have begun to reveal the specific contributions of LSECs to liver metabolic homeostasis and how LSEC dysfunction associates with disease aetiology. This has been particularly evident in the context of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, which is associated with the loss of key LSEC phenotypical characteristics and molecular identity. Comparative transcriptome studies of LSECs and other endothelial cells, together with rodent knockout models, have revealed that loss of LSEC identity through disruption of core transcription factor activity leads to impaired metabolic homeostasis and to hallmarks of liver disease. This review explores the current knowledge of LSEC transcription factors, covering their roles in LSEC development and maintenance of key phenotypic features, which, when disturbed, lead to loss of liver metabolic homeostasis and promote features of chronic liver diseases, such as non-alcoholic liver disease.

Functional genomic analyses reveal a complex relationship between the COBLL1 gene and metabolic health, as well as how the same variant can both reduce body fat and increase the risk of type 2 diabetes.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis -regulatory elements as contributing mechanisms to ICP susceptibility.

This study investigated the potential genetic mechanisms which underlie adipose tissue mitochondrial dysfunction in Type 2 diabetes (T2D), by systematically identifying nuclear-encoded mitochondrial genes (NEMGs) among the genes regulated by T2D-associated genetic loci. The target genes of these ‘disease loci’ were identified by mapping genetic loci associated with both disease and gene expression levels (expression quantitative trait loci, eQTL) using high resolution genetic maps, with independent estimates co-locating to within a small genetic distance. These co-locating signals were defined as T2D-eQTL and the target genes as T2D cis-genes. In total, 763 cis-genes were associated with T2D-eQTL, of which 50 were NEMGs. Independent gene expression datasets for T2D and insulin resistant cases and controls confirmed that the cis-genes and cis-NEMGs were enriched for differential expression in cases, providing independent validation that genetic maps can identify informative functional genes. Two additional results were consistent with a potential role of T2D-eQTL in regulating the 50 identified cis-NEMGs in the context of T2D risk: (1) the 50 cis-NEMGs showed greater differential expression compared to other NEMGs and (2) other NEMGs showed a trend towards significantly decreased expression if their expression levels correlated more highly with the subset of 50 cis-NEMGs. These 50 cis-NEMGs, which are differentially expressed and associated with mapped T2D disease loci, encode proteins acting within key mitochondrial pathways, including some of current therapeutic interest such as the metabolism of branched-chain amino acids, GABA and biotin.

Insulin resistance (IR) is largely recognized as a unifying feature that underlies metabolic dysfunction. Both lifestyle and genetic factors contribute to IR. Work from recent years has demonstrated that the epigenome may constitute an interface where different signals may converge to promote IR gene expression programs. Here, we review the current knowledge of the role of epigenetics in hepatic IR, focusing on the roles of DNA methylation and histone post-translational modifications. We discuss the broad epigenetic changes observed in the insulin resistant liver and its associated pathophysiological states and leverage on the wealth of 'omics' studies performed to discuss efforts in pinpointing specific loci that are disrupted by these changes. We envision that future studies, with increased genomic resolution and larger cohorts, will further the identification of biomarkers of early onset hepatic IR and assist the development of targeted interventions. Furthermore, there is growing evidence to suggest that persistent epigenetic marks may be acquired over prolonged exposure to disease or deleterious exposures, highlighting the need for preventative medicine and long-term lifestyle adjustments to avoid irreversible or long-term alterations in gene expression.

Vitamin B12 deficiency is common among older adults. However, the most commonly used marker of deficiency, total serum vitamin B12 (B12), is not sensitive enough to diagnose true deficiency in a significant proportion of the population. The combined indicator of B12 status (cB12), formulated as a composite score of various biomarkers of vitamin B12 status which also accounts for folate status and age, has been shown to offer a more robust and powerful test to diagnose B12 deficiency. There are no epidemiological studies of cB12 variability in older adults. We carried out a twin study to characterise the relative contribution of heritable (h2) and environmental factors to the observed variability in cB12 score in an older adult population (n = 378). Furthermore, we tested for association between variability in cB12 and candidate polymorphisms and genes previously associated with serum B12 levels characterized in-silico the mechanism linking the genetic variants and cB12 variability. We found the variability in cB12 and its constituents to be highly heritable (h2= 55%-64%). The SNP rs291466 in HIBCH, previously associated with variation in MMA, was significantly associated with cB12 (R2= 5%|P= 5E-04). Furthermore, variants in MTRR, MMAB, and MUT, underlying inborn errors of B12 metabolism, were nominally associated with variation in cB12. Pathway accompanied by expression quantitative trait loci (eQTL) analysis revealed that HIBCH rs291466 influences the concentration of MMA via the valine degradation pathway. Our study provides etiological insight into how B12 deficiency can manifest into impaired mitochondrial function through perturbations in mitochondrial “fuel” usage.