Rare Diseases Linked to Mutations in Vitamin Transporters Expressed in the Human Blood-Brain Barrier

Abstract

Recent advances have significantly enhanced our understanding of the role of membrane transporters in drug disposition, particularly focusing on their influence on pharmacokinetics and, consequently, pharmacodynamics. The relevance of these transporters in clinical pharmacology is well acknowledged. Recent research has also underscored the critical role of membrane transporters as targets in human diseases, including their involvement in rare genetic disorders. This review focuses on transporters for water-soluble B vitamins like thiamine, riboflavin, and biotin, essential cofactors for metabolic enzymes. Mutations in transporters such as SLC19A3 (thiamine), SLC52A2, and SLC52A3 (riboflavin), and SLC5A6 (multiple B vitamins including pantothenic acid and biotin) are linked to severe neurological disorders due to their role in the blood-brain barrier, which is crucial for brain vitamin supply. Current treatments, mainly involving vitamin supplementation, often result in variable response. This review also provides a short perspective on the role of the transporters in the blood-cerebrospinal fluid barrier and highlights the potential development of pharmacologic treatments for rare disorders associated with mutations in these transporters.

Introduction

Solute carrier transporters play important roles in the pharmacokinetics of many drugs and are major targets of drug-drug interactions. In the last decade many studies have led to the identification of important drug transporters as well as an understanding of the effects of genetic mutations in these transporters on drug disposition and response. In sharp contrast, there is much less information on SLC transporters that play a role in the absorption and disposition of macro- and micro- nutrients and in particular, water soluble vitamins. Within the brain, SLC transporters facilitate the transport of water soluble vitamins not only across the blood-brain barrier (BBB) but also the blood-cerebrospinal fluid barrier (BCSFB), and between astrocytes, neurons, oligodendrocytes, and microglial cells. The proper functioning of SLC transporters is crucial for maintaining normal brain function, and mutations in genes encoding vitamin transporters can lead to a range of neurological disorders through tissue specific vitamin deficiencies. Importantly, vitamin transporters can be inadvertently targeted by prescription drugs leading to drug-induced vitamin deficiencies. In this special issue focused on rare diseases, we will concentrate on genetic diseases associated with mutations in the vitamin B transporters in the BBB and describe drug-drug interactions that may phenocopy these genetic diseases through inhibition of BBB vitamin B transporters. Treatments including pharmacologic treatments will also be described.

SLC transporters in the human BBB

The SLC transporters in the human blood-brain barrier (BBB) play critical roles in the uptake of essential solutes including nutrients and compounds involved in energy metabolism from the systemic circulation to the brain (see review¹). Expression profiles of different SLC transporters in the human BBB can vary with some transporters such as GLUT1, which mediates the essential brain uptake of glucose in the Central Nervous System (CNS), being expressed at extremely high levels (see review²). The effects of mutations in BBB SLC transporters that are vital for CNS energy metabolism such as transporters for amino acid, glucose, thyroid, and lactate has recently been reviewed³. Vitamins are essential organic compounds not produced by the body; thus, they must be obtained from the diet and the brain must extract vitamins from the systemic circulation. Vitamin Bs are crucial for normal cellular functions, and act as coenzymes in many enzymatic processes, enhancing enzyme efficiency and diversity of reactions. For instance, vitamin B6 is vital for over 140 enzymes involved in amino acid metabolism, while pantothenic acid is necessary for about 4% of all mammalian enzymes (see review vitamin B⁴). The eight B vitamins, including thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), vitamin B6 (pyridoxal, pyridoxamine, pyridoxine), biotin (B7), folate (B9), and cobalamins (B12), are indispensable in various cellular coenzyme functions. Among these, thiamine, riboflavin, and biotin are essential for enzyme function in the brain and must cross the BBB to gain access to the brain. Mutations in these transporters are known to cause various diseases (Table 1 and Table S1). Herein, we provide an overview of each of these transporters in the human BBB, and describe mutations that cause rare diseases that affect neurological development, current treatments and treatment responses, and issues relevant to drug development. We also describe potential drug-vitamin interactions in which prescription drugs may interfere with vitamin transport, leading to symptoms associated with neurological deficiencies in the brain. Figure 1 illustrates the membrane localization of each vitamin transporter described in this review, highlighting their presence in major tissues including the endothelial cells of the blood-brain barrier.

Table 1.

The primary information and diseases associated with mutations in the four major vitamin B SLC transporters in the human blood-brain barrier (BBB) are outlined below. Due to the limited number of references permitted, Table S1 lists publications that support the presence of transporters in BBB and includes case reports or reviews of mutations in individuals with these transporter mutations. It also covers studies on knockout mice and the functional impacts of these mutations.

Gene (protein name)	Substrate	#Tissues other than blood brain barrier	Other substrate	#Known inhibitor	Rare Diseases	#Mouse	#Treatment
SLC5A6 (SMVT)	Pantothenic acid (vitamin B5) and Biotin (vitamin B7)	Liver, mammary tissue, placenta, choroid plexus https://www.gtexportal.org/home/gene/SLC5A6. https://www.proteinatlas.org/ENSG00000138074-SLC5A6/single+cell+type https://www.proteinatlas.org/ENSG00000138074-SLC5A6/brain	Lipoic acid		Peripheral motor neuropathy, childhood-onset, biotin-responsive; Sodium-dependent multivitamin transporter deficiency	Not embryonic lethal with nutrient supplement	High doses of biotin (15 mg a day) and pantothenic acid (100 mg a day) for SMVT deficiency.
SLC19A3 (ThTR2)	Thiamine (vitamin B1)	Intestine, mammary tissue, placenta, adipose tissue, choroid plexus https://www.gtexportal.org/home/gene/SLC19A3 https://www.proteinatlas.org/ENSG00000135917-SLC19A3/single+cell+type https://www.proteinatlas.org/ENSG00000135917-SLC19A3/brain	Pyridoxine (vitamin B6); trimethoprim, fedratinib, metformin	Clinical drug-nutrient interaction: Fedratinib; In vitro: trimethoprim, amprolium	Biotin-thiamine-responsive basal ganglia disease; Leigh Syndrome	Not embryonic lethal with nutrient supplement	Biotin and thiamine or thiamine 1500 mg alone
SLC52A2 (RFT2)	Riboflavin (vitamin B2)	Ubiquitous, brain, salivary gland, liver, pancreas, choroid plexus https://www.gtexportal.org/home/gene/SLC52A2 https://www.proteinatlas.org/ENSG00000185803-SLC52A2/single+cell+type https://www.proteinatlas.org/ENSG00000185803-SLC52A2/brain		In vitro: lumiflavin and lumichrome	Brown-Vialetto-Van Laere syndrome 2	Preweaning lethality, Complete penetrance	Riboflavin 7 to 70 mg/kg daily
SLC52A3 (RFT3)	Riboflavin (vitamin B2)	testis, small intestine, kidney, placenta https://www.gtexportal.org/home/gene/SLC52A3 https://www.proteinatlas.org/ENSG00000101276-SLC52A3/single+cell+type		In vitro: lumiflavin and lumichrome	Fazio-Londe disease; Brown-Vialetto-Van Laere syndrome 1	Not embryonic lethal with nutrient supplement	Riboflavin 7 to 70 mg/kg daily

^#This column in Table S1 has references to support the information.

Figure 1.

Vitamin B transporters in the human BBB and major organs involved in the absorption, distribution, clearance, and elimination of small molecules. The localization of SLC19A1 in the blood-brain barrier is not clear from the literature. However, one review places it on the luminal membrane (Alam et al. 2020, see Table S1 for reference).

Sodium-dependent multi-vitamin transporter, SMVT, in the BBB

The initial identification and functional analysis of the SMVT protein in rats and humans were reported in 1998⁵. This protein, encoded by the SLC5A6 gene, is composed of 635 amino acids and includes 12 transmembrane domains. Both the amino and carboxyl termini are oriented towards the cytoplasm. SMVT is broadly expressed across various organs including the liver, brain, placenta, intestine, heart, lung, kidney, cornea, retina and endothelial cells of the BBB. SMVT facilitates the transport of two vitamins: biotin (vitamin B7) and pantothenic acid (vitamin B5). Biotin is essential for glucose metabolism and hemostasis whereas pantothenic acid is crucial for the synthesis of coenzyme A (CoA), which is involved in the production of cholesterol, amino acids, phospholipids, and fatty acids. SMVT mediated transport of these negatively charged vitamins is electrogenic, driven by the sodium gradient and the electrical potential across the cellular membrane.

Studies of SMVT deficiency have been largely conducted in intestine specific knockout mice models, Smvt^−/−. The affected mice exhibit symptoms of growth delays, reduced bone density and length, and diminished biotin levels. Most die after a few months. Histopathological changes in the small bowel accompany the growth delays (including shortened villi and dysplasia) and cecum (marked by chronic active inflammation and dysplasia). Transport assays demonstrate a complete cessation of carrier-mediated biotin absorption in the knockout mice. Collectively the findings suggest that intestinal SMVT plays a crucial role in maintaining the integrity of the intestinal mucosa. Supplementation with biotin and pantothenic acid reverses the intestinal abnormalities in the knockout mice.

In brain endothelial cells, SMVT plays major roles in the transport of biotin and pantothenic acid in the CNS. In humans, genetic mutations in the SMVT gene can lead to various inherited disorders, such as childhood-onset peripheral motor neuropathy (COMNB) and sodium-dependent multivitamin transporter deficiency (SMVTD) (Table 1). SMVTD is a very rare genetic disorder, which has been reported so far in only 17 patients from 10 families (see references in Table S1). This condition is recessive, with affected individuals either being homozygous for the mutation or harboring heterozygous mutations. Table S1 provides a compilation of case reports on patients with sodium-dependent multivitamin transporter defects, and a recent review⁶ serves as a valuable reference. Mutations affecting both alleles of SLC5A6 result in SMVTD and COMNB, both of which respond favorably to nutrient replacement therapy. SMVTD typically manifests with a range of symptoms affecting multiple organs, such as gastrointestinal bleeding, brain atrophy, and global developmental delay, either at birth or during infancy. Without appropriate nutrient replacement therapy, SMVTD can be fatal in early childhood. COMNB, while clinically less severe, typically emerges around the age of 10 and primarily presents with peripheral motor neuropathy. In the absence of nutrient supplementation, this condition can also lead to early childhood fatality and developmental delays. In addition to the reported SMVTD and COMNB, there are other phenotypes within the SLC5A6-related spectrum that are attributed to biallelic variants in SLC5A6. These phenotypes include progressive neurodegenerative conditions, developmental delays, and epileptic encephalopathy.

Genome-wide association studies have demonstrated that common variants in SLC5A6 are associated with changes in plasma pantothenate levels, as well as other phenotypes such as lipid levels (https://www.ebi.ac.uk/gwas/genes/SLC5A6). However, studies are needed to determine the functional role of the common variants.

Potential Pharmacologic Treatments:

To date, supplementation of biotin and pantothenic acid represents the only treatment for these rare disorders. However, the transporter itself, SMVT, is being targeted for delivery of drugs for the treatment of other diseases. For example, SMVT has been targeted in retinal epithelia using biotin conjugated anti-oxidants for the treatment of age-related macular degeneration⁷. Many tumors also express SMVT, and biotin conjugates with anti-tumor drugs have been designed to target these tumors⁸. Importantly, the synthesis and development of hydrophobic vitamin prodrugs or formulations that circumvent the requirement for SMVT (or transporters in general) is a subject of much study⁹. Such vitamin prodrugs could be used to more efficiently deliver pantothenic acid or biotin to the systemic circulation and the CNS for the treatment of SMVTD and COMNB.

Folate transporters and expressions in human BBB

Folate, including several vitamers (e.g. dihydrofolate, 5-methyltetrahydrofolate) that function as coenzymes in cellular one-carbon metabolism. Folate is crucial for synthesizing thymidine, purines, myelin, and neurotransmitters, as well as for metabolizing amino acids like homocysteine, methionine, serine, and glycine. The remethylation of homocysteine to methionine facilitates over 100 methylation reactions through S-adenosylmethionine. Two primary folate transporters are critical for folic acid absorption and distribution: the reduced folate carrier (RFC), encoded by SLC19A1, and the proton-coupled folate transporter protein (PCFT), encoded by SLC46A1¹⁰. Both transporters are expressed in greater abundance in the blood-CSF barrier, in comparison to the BBB. In fact proteomic data indicate that PCFT is not expressed in the human BBB¹¹. The primary route for folate transport into the brain occurs at the choroid plexus via folate receptor alpha (FOLR1) and PCFT. Mutations that result in loss of function in FOLR1 and PCFT1 cause childhood neurological disorders, such as cerebral folate deficiency and hereditary folate malabsorption respectively¹². However, because the transporter is not in the human BBB, we elected not to include further details in this mini-review, which is focused on Vitamin B transporters in the BBB. Conversely, RFC is widely expressed throughput the body, including in the human BBB (Table S1) and choroid plexus. However, mutations in SLC19A1 result primarily in megaloblastic anemia, rather than in disorders of the CNS. Therefore, we do not discuss the transporter further, but references may be found in Table S1.

Clinical relevance and genetic implications of mutations in SLC19A3

SLC19A3 is the third member in the SLC19 family and was cloned and characterized in 2001¹³. SLC19A3, which shows high homology to SLC19A1 (reduced folate carrier, RFC) and SLC19A2 (thiamine carrier, THTR1), was discovered to take up thiamine but not methotrexate or folic acid and shares functional characteristics with SLC19A2. Thiamine uptake via SLC19A3 is pH-dependent, exhibiting a rise in activity with increasing pH and reaching a maximum at approximately pH 7.4. The uptake kinetics of thiamine are saturable at higher concentrations of thiamine (> 3 μM)¹⁴. Due to its critical endogenous role, a nutrient transporter has a narrower substrate specificity in comparison to a typical drug transporter involved in drug absorption and disposition. However, members in the SLC19 family have been shown to transport several other substrates. For example, SLC19A3 transports pyridoxine¹⁵, as well as several clinically used drugs such as metformin, trimethoprim, and fedratinib^{14, 16}. In addition, studies have shown that SLC19A3 is also inhibited by several prescription drugs¹⁷ (see Table 1). SLC19A3 is widely expressed at low levels in many human tissues. Transcriptomic and single-cell RNA sequencing have demonstrated a significant presence of SLC19A3 in the blood-brain barrier (Table 1). Our group recently published a global proteomics study of the human blood-brain barrier using samples from developing ages, children, and adults¹¹. Although the protein expression of SLC19A3 is not as abundant as that of several well-known SLC transporters, such as SLC2A1 (glucose transporter, GLUT1) and SLC7A5 (large amino acid transporter, LAT1), the SLC19A3 protein levels are detectable in all samples.

SLC19A3 appears to play a primary role in thiamine intestinal absorption. Intestines of THTR-2-deficient mice (Slc19a3^−/−) exhibit a significantly reduced uptake of thiamine compared to those of their wild-type littermates, consistent with a role of the transporter in intestinal absorption of this vitamin. Importantly, Slc19a3 knockout mice, fed a thiamine-restricted diet, die within a few days due to significantly decreased thiamine concentrations in the blood and brain, and exhibit neurodegenerative disorders¹⁸. Some data suggest that there may be a transporter in the BBB for the active derivative of thiamine, thiamine pyrophosphate. Notably in humanized mice expressing human SLC19A3 in their intestines on a background of Slc19a3 deficiency, thiamine plasma levels are lower than in wildtype mice, yet brain thiamine pyrophosphate levels remain normal consistent with the expression of an independent transporter for the derivative in the BBB or other compensatory pathways¹⁹.

Rare mutations in SLC19A3 lead to biotin-thiamine-responsive basal ganglia disease (BTBGD), which is characterized by subacute encephalopathy, seizures, and other neurological symptoms that can manifest in childhood, early infancy, or adulthood (see review²⁰). Magnetic resonance imaging results in patients with BTBGD show abnormal signal intensities in the caudate and putamen, along with widespread involvement throughout the brain, including the cortical and subcortical white matter, and the infratentorial region. According to the review of rare diseases discussed in this article, BTBGD occurs with a relatively high prevalence of about 1 in 215,000 live births²¹. The mutation p.Thr422Ala is one of the most prevalent harmful mutations found in patients with BTBGD. This condition shows improvement when treated with high doses of biotin (5–10 mg/kg/day), and additional benefits are observed when thiamine is combined with biotin therapy. Prompt administration of both vitamins is crucial for achieving normal outcomes. Although biotin is not a known substrate of SLC19A3, the mechanisms by which biotin and thiamine produce a synergistic effect in alleviating symptoms of BTBGD remain unclear. In addition to BTBGD, other rare diseases are associated with thiamine transporter deficiency. These include Leigh syndrome, Wernicke encephalopathy, and infantile beriberi (see review²⁰). Early infantile Leigh syndrome is the most severe SLC19A3-related phenotype²². Leigh syndrome is an early-onset, often fatal progressive neurodegenerative disorder caused by mutations in mitochondrial or nuclear DNA. There are more than 35 genes reported to cause Leigh syndrome, including SLC19A3.

Potential Pharmacologic Treatments:

Because of its high water solubility, attempts have been made to develop hydrophobic analogs of thiamine for the treatment of BTBGD and other diseases related to SLC19A3 mutations. Interestingly, benfotiamine is a thiamine prodrug, which raises systemic levels of thiamine (see review²³). Because thiamine deficiency is associated with Alzheimer’s disease, the drug is currently in clinical trials for the treatment of Alzheimer’s disease (see NCT06223360). Although thiamine and benfotiamine have been shown to benefit various diseases, including those associated with thiamine deficiency (see review²³), no studies have been reported on the use of benfotiamine in individuals with BTBGD and other diseases associated with mutations in SLC19A3.

In addition to drugs that can be used to treat BTBGD and other diseases associated with SLC19A3 deficiency, prescription drugs may also phenocopy the clinical phenotypes of mutations in SLC19A3. This observation was made in 2012 during the development of fedratinib for the treatment of myelofibrosis. Notably, the clinical trials were halted due to an unexpected incidence of Wernicke’s encephalopathy, a serious neurological disorder caused by thiamine (vitamin B1) deficiency. Wernicke’s encephalopathy is characterized by symptoms such as confusion, lack of muscle coordination (ataxia), and vision changes. The halting of the trials was a precautionary measure to ensure patient safety. Later, after thorough in vitro evaluation showed that fedratinib inhibits SLC19A3²⁴ and measures were taken to mitigate the risk of thiamine deficiency, the trials were resumed, and fedratinib was eventually approved for the treatment of myelofibrosis. Currently, the product inserts states that thiamine levels must be determined prior to the initiation of the drug.

Characteristics of SLC52A2 and SLC52A3 and genetic consequences of their mutations

Another important vitamin B is riboflavin. For many years, riboflavin transport mechanisms remained unknown. However, in 2008, Yonezawa and colleagues identified the rat and human riboflavin transporters, which were termed RFT1. Further research by Yamamoto and Yao in 2009 and 2010 led to the discovery of RFT2 and RFT3 (see review²⁵). These transporters have been reclassified into the new SLC52A family, with RFT1, RFT2, and RFT3 being renamed to RFVT1/SLC52A1, RFVT2/SLC52A2, and RFVT3/SLC52A3, respectively. The genes SLC52A2 and SLC52A3 encode for RFVT2 and RFVT3, containing 445 and 469 amino acids. Research indicates that all three RFVT transporters mediate riboflavin uptake and are independent of Na+ and Cl− ions. However, RFVT3’s activity is unique as its uptake of riboflavin is pH-dependent, increasing as the pH decreases. This suggests that an inward-directed H+ gradient may serve as the driving force for RFVT3, distinguishing it from RFVT1 and RFVT2, which facilitate riboflavin transport through different mechanisms. Riboflavin is vital for the synthesis of the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential for metabolic processes in carbohydrates, proteins, and lipids. The Michaelis-Menten constants for riboflavin are notably higher than typical human plasma levels, measuring 0.33 μM for RFVT2 and 0.98 μM for RFVT3. RFVT3 plays a crucial role in riboflavin absorption within the small intestine, reabsorption in the kidneys, and fetal transport via the placenta. On the other hand, RFVT2 is primarily responsible for distributing riboflavin from the bloodstream into various tissues. These details underscore the specific and crucial roles of these transporters in riboflavin physiology.

The study by Jin C. et al. (2021, Table S1) explores the physiological role of RFVT2 in Slc52a2-mutant mice. Slc52a2 knockout mice exhibit preweaning lethality with complete penetrance and further, appear to exhibit embryonic lethality. That is, homozygous mutant (Slc52a2^−/−) offspring of Slc52a2^+/− are not found, indicating that a lack of the Slc52a2 gene leads to early embryonic lethality. These findings underscore the critical role of RFVT2 in growth and development, suggesting that its absence may adversely affect embryonic viability.

In Slc52a3^−/− mice, levels of brain riboflavin, FMN, and FAD were notably lower compared to wild-type (WT) animals, leading to abnormal brain morphology due to the deficiency in riboflavin (Table S1). An analysis of cerebral cortex development in neonatal Slc52a3^−/− mice with and without riboflavin supplementation showed that additional riboflavin ameliorated changes in the structure of the lateral ventricles, cerebral cortex, striatum, and hippocampus. Furthermore, over-supplementation significantly enhanced the thickness of the cortical layers, increased the number of neurons in these layers in the Slc52a3^−/− mice. Intestinal-specific knockout mice deficient in Slc52a3 demonstrate an essential role of RFVT3 in riboflavin absorption. These knockout mice displayed symptoms of riboflavin deficiency, significant growth retardation, and a high mortality rate between 6 and 12 weeks. Administering pharmacological doses of riboflavin mitigated the growth deficits and restored normal gene expression levels related to oxidative stress in their intestines. These results highlight the critical function of RFVT3 in facilitating riboflavin uptake and sustaining normal physiological balance in the mouse intestine.

In humans, Brown-Vialetto-Van Laere (BVVL) syndrome, a rare and progressively worsening motor neuron disorder, is inherited in an autosomal recessive manner. This condition stems from defects in the riboflavin transporter genes SLC52A2 and SLC52A3, now recognized collectively as Riboflavin Transporter Deficiency (RTD) (see review²⁶). Initially identified by Dr. Charles Henry Brown in 1894 as a familial form of infantile amyotrophic lateral sclerosis, subsequent reports have noted a predominance in females and an autosomal recessive inheritance pattern. BVVL syndrome is notably marked by sensorineural hearing loss, progressive pontobulbar palsy affecting the motor functions of cranial nerves seven, and nine through twelve, along with progressive muscle weakness that leads to respiratory issues, limb weakness, and upper motor neuron signs. While the onset typically occurs in the second decade of life, variations in onset age, symptom severity, and disease duration have been documented. The syndrome shows genetic variability: BVVL1 is linked to mutations in the SLC52A3 gene on chromosome 20p13, while BVVL2 is associated with mutations in SLC52A2 on chromosome 8q. Additionally, Fazio-Londe syndrome, similar to BVVL but without hearing loss, also falls under the RTD spectrum. Some patients displayed abnormalities in plasma flavin levels and acylcarnitine profiles.

The transcript levels of RFVT2 in the blood-brain barrier were first reported in rats (see Table S1). However, the protein levels of RFVT2 and RFVT3 in the human blood-brain barrier have not been previously reported. Our recent findings from a global proteomic study of microvessels prepared from human brain samples showed that both RFVT2 and RFVT3 are expressed, but RFVT2 is more highly expressed compared to RFVT3¹¹. Interestingly, protein levels of RFVT2 were expressed in brain endothelial cells in samples from early development (infants), children, and adults; however, RFVT2 is not expressed in samples from older adults above 60 years old. Conversely, RFVT3 is only expressed in samples from older adults above 60 years old. There are several cases of patients with RTD who have mutations in SLC52A3 (RFVT3) and can experience a variable age of onset, with some even occurring in later adulthood²⁷. The higher expression of RFVT3 in the BBB in older adults may be reason why mutations in SLC52A3 manifest phenotypically in later adulthood.

Potential Pharmacologic Treatments:

Currently the only treatments for RTD is high dose riboflavin administration. A recent review of the literature suggests that 81% of patients treated with riboflavin supplementation show overall improvements in their conditions (motor neuron function)²⁸. However, despite treatment, many patients still exhibit severe hearing loss and require assistance in ambulation. This residual disease suggests an important gap and a need for the development of pharmacologic treatments of RTD.

Expression of Vitamin B Transporters at the Blood-CSF Barrier (BCSFB).

Another pathway for micronutrients and vitamins to gain an entry into the brain is through the transport across the BCSFB formed by choroid plexus (CP) epithelial cells located in the four brain ventricles²⁹. The CP has long been thought to be an important site for folate (vitamin B9) and ascorbic acid (vitamin C) transport in the brain; and concentrations of these vitamins in the CSF are much higher than their circulating concentrations in the blood. Indeed, the reduced folate carrier 1 (SLC19A1) and the proton-coupled folate transporter (SLC46A1) are highly expressed in the human and rodent BCSFB¹². Compared to the BBB, little is known regarding the BCSFB expression and cellular localization of SLC5A6, SLC19A3, SLC52A2, and SLC52A3. The human protein atlas and COVID19 scRNAseq (https://twc-stanford.shinyapps.io/scrna_brain_covid19/) reveals significant expressions of SLC5A6, SLC19A3, and SLC52A2 transcripts in the human CP. A proteomic analysis in porcine CP detected SMVT protein at an expression level comparable to that in the BBB. It is likely that SMVT at the BCSFB could play an additional role in influencing brain homeostasis of biotin and pantothenic acid.

Treatment available to treat rare diseases associated with transporter deficiency

To date, vitamin supplementation represents a primary treatment for these rare disorders discussed above. However, clinical response to vitamin supplement is highly variable in most of these disorders. Although much higher doses of vitamins are needed (see Table 1), toxicities related to thiamine, biotin, pantothenic acid, and riboflavin are rare, and very few cases of significant adverse effects have been reported. The minimal toxicity observed with these water-soluble B vitamins is due to their quick elimination through urine when their blood levels are excessively high. However, occasionally toxic effects have been reported following intravenous doses of thiamine or when taken orally at 3 grams per day, potentially causing ganglionic blockage³⁰. Similarly, consuming large amounts of riboflavin may lead to gastrointestinal discomfort.

The development and clinical trials of more hydrophobic prodrugs of B vitamins represent an important avenue for advancing treatment and for reducing the requirement for high doses of these vitamins, which as noted for RTD, often leave the patients with residual disease. Further, mutations in transporters generally affect expression levels of the transporter protein on the plasma membrane. Treatment options which enhance the expression levels of transporters affected by mutations are currently under study for other transporter associated diseases such as Allan-Herndon-Dudley Syndrome, which is associated with mutations in SLC16A2 (MCT8) (ClinicalTrials.gov NCT05019417). In particular, analogs of the deficient transporter substrate, such as tetraiodothyroacetic acid (Triac), a T3 thyroid hormone analog, is in clinical trials for Allan-Herndon-Dudley Syndrome (NCT02060474). Since B vitamins are critical for glycolytic energy metabolism in the brain and other tissues, the development of molecules such as analogs for beta oxidation of fatty acids energy, which rely less on enzymes that require vitamin B, represents another strategy for treatment of vitamin B transporter deficiencies. For example, triheptanoin (UX007), a triglyceride of odd-chain fatty acids, has demonstrated therapeutic potential in treating metabolic and neurodegenerative diseases, such as citrate transporter deficiency (mutations in SLC13A5, NaCT) and GLUT1 (SLC2A1) deficiency syndrome (NCT02500082, NCT02036853, NCT01993186, NCT02014883). These disorders result in metabolic and neurological impairments that may be alleviated by triheptanoin, which helps supply energy to improve neuronal signaling or fatty acid degradation³¹.

Resources available to characterization and studying of mutations in rare disease

Resources for characterizing and studying mutations in rare diseases have become increasingly resourceful and accessible. Scientists use these resources to obtain in silico predictions of the effects of missense mutations, determining whether they are pathogenic. Examples include PolyPhen-2, REVEL, and CADD (see Table S2 and http://database.liulab.science/dbNSFP). Key tools for annotating rare variants associated with specific diseases and genes include:

• Geno2mp - This resource helps link phenotypic and genotypic data, facilitating the understanding of how genetic variations contribute to rare diseases.

• LOVD database - The Leiden Open Variation Database provides a platform for sharing genetic variants found in individuals, aiding researchers in discovering novel variants and their associated effects.

• ClinVar - This NIH-funded database aggregates information about genomic variations and their relationship to human health, providing valuable insights into the potential clinical significance of rare mutations. Tables S3, S4, S5, and S6 present lists of mutations reported in patients, as downloaded from ClinVar.

Additionally, several disease-specific foundations offer tailored resources that provide specialized information and community support. For instance:

Cure RTD Foundation (https://curertd.org/) focuses on SLC52A2 and SLC52A3 mutations.

Examples of the names of non-profit organizations dedicated to rare variants in SLC transporters include: SLC6A1 Connect (https://slc6a1connect.org/) (see Perspective in this Special Issue), TESS Research Foundation for SLC13A5 (https://www.tessresearch.org/), GLUT1 Deficiency Foundation for SLC2A1 (https://www.g1dfoundation.org/), Association for Creatine Deficiencies for SLC6A8 (https://creatineinfo.org/), and Cystinuria Support Network for SLC7A9 (https://cystinuria.org/), provide valuable resources for families and researchers, including active registries of individuals with the transporter deficiency, research updates, and community forums.

Functional data from mutated Vitamin B transporters found in patients with rare diseases (referenced in this review; see Table S1 for a list of publications), revealed either no uptake or some residual uptake. This could be due to low expression levels of the transporter protein within the cell membrane, resulting from incorrect protein processing and trafficking, or due to SLC transporter proteins being functionally impaired on the cell membrane. Splicing variants that result in truncated proteins are frequently observed in these transporters. Although experimental validation is crucial for understanding the mechanism of a deleterious variant, an increasing number of studies are relying on in silico analysis to predict the impact of these variants. Table S2 presents a list of in silico tools used to predict the function of missense variants. For example ESM1b (Table S2) database offer predictions for all human missense variants and amino acid substitutions. Similarly, a comprehensive dataset (AlphaMissense) also predict missense variant pathogenicity, which can be accessed via Google Cloud Storage (https://console.cloud.google.com/storage/browser/dm_alphamissense), providing critical insights into potential disease mechanisms through computational predictions. These resources collectively enhance the research community’s ability to study rare diseases at a molecular level, leading to better diagnostic tools and therapeutic strategies. High throughput experimental approaches such as deep mutational scanning offer a method to rapidly scan all potential mutations of proteins and have recently been applied to mutations in SLC transporters (see Yee et al.³²).

Conclusion.

In conclusion, research on solute carrier (SLC) transporters, particularly in the context of the blood-brain barrier, highlights their critical role in absorbing and transporting vitamin B, which is vital for brain health. This review focuses on four vitamin B transporters expressed at the blood-brain barrier: SMVT, THTR2, RFVT2, and RFVT3. Genetic mutations in these transporters can lead to severe neurological disorders, underscoring the importance of maintaining transporter function for neurological health. Furthermore, unintended interactions between prescription drugs and these transporters can lead to drug-induced vitamin deficiencies, emphasizing a crucial area for pharmacological consideration. While vitamin supplementation remains a fundamental treatment for rare diseases associated with these transporter deficiencies, its effectiveness varies widely. Advanced therapies, including hydrophobic prodrugs and compounds that enhance transporter expression, are emerging and promise more effective management of these conditions. Although the majority of patients treated with high doses of vitamin B show improvement, none of the diseases associated with these transporter mutations are included in newborn screening, due to their rarity and the high costs of sequencing at a population level. Advances in technologies, robust resources, and novel computational tools are now available, improving the characterization of mutations and supporting the development of targeted treatments. These advancements, along with increased access to genetic databases and patient registries, are enhancing our understanding of rare diseases at a molecular level, fostering better diagnostics and more personalized therapeutic approaches.