Folate is a crucial nutrient that supports important physiological functions such as DNA synthesis, cell division and substrate methylation. Low folate level caused by suboptimal intake, transport and cellular utilization of folate, is a risk factor for diseases such as spina bifida and cardiovascular diseases . Intracellular uptake of folate is in part mediated by the reduced folate carrier protein 1 (RFC1), encoded by the human solute carrier family 19, member 1 (SLC19A1) gene. RFC1 is a high-capacity, bi-directional transporter of 5-methyl-tetrahydrofolate and thiamine monophosphate. RFC1 also actively transports antifolate chemotherapeutic agents, such as methotrexate (MTX), into cells [2–9]. In addition to its role in folate uptake, RFC1 plays critical role in folate homeostasis of mammalian cells, where it is down regulated in response to folate deficiency .
SLC19A1 is found on chromosome 21 (21q22.3). Several groups of investigators have cloned cDNAs (approximately 2.7 kb in length) encoding the 591-amino acid human SLC19A1 from lymphoblast, placenta, and small intestine cDNA libraries [2–5]. The SLC19A1 gene from human lymphoblasts was shown to contain five exons (exon 2 to exon 6), which code for RFC1 protein [11–13]. There are at least four 5-prime alternative exons, which are used in the production of RFC1 mRNA transcript in lymphoblast cells. These are spliced in a mutually exclusive manner to exon 2, which contains the translational start site for the RFC1 protein. Semi-quantitative PCR shows that exon 1 is preferentially incorporated in the transcript . A separate study showed that RFC1 exhibits alternative splicing . Specifically, three splice variants of RFC1 were identified from a human liver genomic library, and resulted from the incorporation of three alternatives of exon 1 and different 3' sequences. Functional deletion analysis of the region upstream of the transcriptional start site of the SLC19A1 gene led to the identification of two TATA-less promoters, each of which showed significant differences in the efficiency of transcription.
A humanized mouse model for the reduced-function folate carrier has been created . Expressing human SLC19A1 in transport-deficient Chinese hamster ovary cells results in restoration of MTX transport and MTX sensitivity . Several point mutations have been identified in SLC19A1 and down regulation of SLC19A1 mRNA has also been associated with impaired MTX transport and MTX resistance [16–19]. A reverse correlation between the RFC promoter methylation and its mRNA level in cancer cell lines has been described . These constitute important factors in the development of resistance to anti-folate chemotherapeutic agents. A recent study in polymorphism and expressions of folate pathway genes showed that SLC19A1 expression correlated with the sensitivity of several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs) in the NCI-60 cancer cell lines . Furthermore, some groups have also found association between the expression of RFC1 and accumulation of methotrexate in Acute Lymphocytic Leukemias (ALL) cells [22, 23]. The SLC19A1 mRNA expression from ALL blasts isolated from newly diagnosed children were higher in certain lineage ALL such as the hyperdiploid B-lineage compared to nonhyperdiploid ALL. In addition, the accumulation of methotrexate polyglutamates was highest in the hyperdiploid B-lineage which indicated that higher SLC19A1 expression plays important role in MTX accumulation [22, 23].
Sequencing of SLC19A1 in healthy subjects
The SLC19A1 gene is highly polymorphic in humans. The proximal promoter region, exons 3 to 5 and their flanking intronic regions of the SLC19A1 gene has been resequenced in an ethnically diverse population of 276 individuals as part of the Pharmacogenetics of Membrane Transporters (PMT) project (http://pharmacogenetics.ucsf.edu/cgi-bin/Study.py). This cohort includes unrelated healthy individuals from the San Francisco Bay Area (80 African Americans, 80 European Americans, 60 Asian Americans, 50 Mexican Americans and 6 Pacific Islanders). A total of 6 non-synonymous SNPs were found in exons 3 to 5 of SLC19A1 gene in this cohort. (https://www.pharmgkb.org/do/serve?objId=PA327&objCls=Gene#tabview=tab2 and in http://pharmacogenetics.ucsf.edu/). Among the non-synonymous SNPs, there are 4 singletons (only found on one chromosome from the sequenced SOPHIE cohort) and they are Leu338Phe (1012C>T, rs59638403, 0.6% in African American); Gly341Asp (1022G>A; rs56822323, 0.6% in European American); Cys458Gly (1372T>G; rs58227024, 0.6% in European American) and Asp522Asn (1564G>A; rs58836581, 0.6% in African American) and two rare variants with minor allele frequency of 1% (Arg456Gln; 1367G>A; rs59841046 and Ala469Val; 1406C>T; rs7278825). One common non-synonymous variant (Ala558Val; 1792C>T; rs35786590) is reported in the dbSNP (dbSNP 130) with total allele frequency of 49.9% and it is found across the four HapMap populations (CEU, HCB, JPT and YRI) (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=35786590). A frameshift as a result of deletion of 52bp in Exon 3 and give rise to a synonymous SNP (Ala324Ala; 972G>A; rs56138890) is found in the Asian American (12.7 %) and in Mexican American (3 %) populations of the SOPHIE cohort (http://pharmacogenetics.ucsf.edu/) and also reported in the Indian population in India (dbSNP Build 131, http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=56138890). A common non-synonymous SNP in Exon 2 of the gene, Arg27His (80G>A; rs1051266), which was not sequenced in the PMT project, but it is reported in the dbSNP, have total minor allele frequency of 44% and is found across all ethnic groups. In addition, the sequencing of SLC19A1 promoter region in 72 healthy individuals in Australia revealed a 61-bp insertion polymorphisms in 78% of these individuals. Functional studies conducted by this group showed that this 61-bp insertion resulted in higher luciferase activity as a result from the additional binding sites for AP-2 and Mzf-1 transcription factors in this 61-bp insertion region .
Sequencing of SLC19A1 in resistant cell lines and tumor specimens
Several SLC19A1 gene mutations leading to antifolate-resistant phenotype are observed in rodent and human cell lines [6, 16, 24]. In one study, L1210 mouse leukemia cells were selected for resistance to the anti-folate (6R)-5,10-dideazatetrahydrofolate . The resistant mouse leukemia cells were shown to have two point mutations in SLC19A1, I48F and W105G, which contributed to the drug-resistant phenotype. Since these mutations dramatically altered the kinetics of folate transport, they were reported as participating in substrate interaction and likely constitute the substrate recognition domain of RFC1. Since the L1210/D3 resistant cells were shown to express the mutant transporter but the wild-type allele was still present in the genomic DNA, it seems that the wild-type allele was silenced during the development of resistance. Another point mutation in SLC19A1 was identified in a methotrexate transport-defective human T-ALL cell line known as CCRF-CEM . This cell line has a lysine to glutamic acid substitution at codon 45 (E45K) and has been identified to be resistant to other anti-folate [16, 25]. Although this nonsynonymous variant could lead to methotrexate resistance due to decreased membrane transport, and have altered binding affinities and transport of folate substrates, there was no evidence that this mutation occurred in ALL samples from children . Other point mutations have been discovered by sequence analysis in human CEM leukemia cells which is resistant to a thymidylate synthase inhibitor (GW1843) and methotrexate. The mutations appeared to be Val29Leu, Glu45Lys and Ser46Ile in the first transmembrane domain of the SLC19A1 and conferred resistance to GW1843 in cells transfected with these mutations . Similarly, other point mutations in SLC19A1 have also been reported in human leukemia cells after cellular exposure to gradually increasing PT523, a potent dihydrofolate reductase inhibitor. DNA sequencing of these cells revealed mutations in SLC19A1: Leu143Pro, Ala147Val, Arg148Gly, Gln150Stop and these mutations resulted in decreased RFC protein levels and showed impaired methotrexate transport . On the other hand, a few studies have been conducted where RFC1 were resequenced in tumor samples. In one study, 203 B-precursor ALL specimens, 32 T-lineage ALL specimens and 11 AML specimens were resequenced for its RCF1. Only 3 B-precursor ALL specimens were found to have nonsynonymous variants, i.e. Asp56His and Asp522Asn . The study showed that the methotrexate uptake is lower in the blast cells from B-precursor ALL patient with the Asp522Asn mutation compared with cells from the wild type carriers. In another study, RFC1 was resequenced in 162 osteosarcoma samples, and the study has identified new variants, such as Ser46Asn, Glu21Lys, Ala7Val and Ser4Pro, in addition to the common variant Arg27His. . In addition, the clinical significance of these genetic polymorphisms in terms of methotrexate plasma level and resistance has not been determined.
Table 1 summarizes the functional effect of genetic variants in SLC19A1 gene identified through sequencing project in healthy subjects, in patients and in antifolate resistant cells.