Other entities represented in this entry:
HGNC Approved Gene Symbol: DHFR
SNOMEDCT: 866092006;
Cytogenetic location: 5q14.1 Genomic coordinates (GRCh38): 5:80,626,226-80,654,983 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 5q14.1 | Megaloblastic anemia due to dihydrofolate reductase deficiency | 613839 | Autosomal recessive | 3 |
Dihydrofolate reductase (EC 1.5.1.3) converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids. DHFR is inhibited by methotrexate (MTX), a folate analog used as an antineoplastic and immunosuppressive agent. In addition to the functional DHFR gene, there are at least 4 intronless genes that are probably pseudogenes. Each of the 5 is on a separate chromosome. The pseudogenes do not undergo amplification when such occurs at the functional locus (Anagnou et al., 1984).
The functional DHFR gene was cloned and characterized by Chen et al. (1982) and Chen et al. (1984).
Banka et al. (2011) found that DHFR was widely expressed in fetal and adult human brain and whole blood; expression was higher in adult brain than fetal brain.
Chen et al. (1984) determined that the DHFR gene is about 30 kb long and consists of 6 exons separated by 5 introns.
From comparisons of eukaryotic gene sequences and protein sequences of homologous enzymes from bacterial and mammalian organisms, Craik et al. (1983) noted that intron-exon junctions often coincide with variable surface loops of the protein structure. Proteins studied included DHFR, trypsin, and chymotrypsin. They pointed out that altered surface structures can account for functional differences among the members of a family, e.g., the serine proteases. 'Sliding' of the intron-exon junctions may constitute a mechanism for generating length polymorphisms and divergent sequences. Different function can thus be achieved without disrupting the stability of the protein core.
From DNA transfer hybridization analyses using a human DHFR cDNA probe on genomic DNA from human-mouse and human-Chinese hamster cell hybrids segregating human chromosomes, Maurer et al. (1984) concluded that chromosome 5 carries the DHFR locus. The assignment was confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin (126150), a chromosome 5 marker, in 2 human-mouse cell hybrids selected for resistance to the toxin. By means of somatic cell hybrids of human fetal fibroblasts and a DHFR-deficient Chinese hamster ovary cell line, Funanage et al. (1984) assigned the DHFR gene to chromosome 5 and, with cell lines carrying chromosome 5 aberrations, further narrowed the assignment to 5q11-q22. The assignment to chromosome 5 is also supported by homology of synteny (Myoda and Funanage, 1983).
Maurer et al. (1985) assigned DHFR to 5q23, which is inconsistent with other assignments (Anagnou et al., 1984; Funanage et al., 1984). (From new data, HGM10 concluded that DHFR is located in the region 5q11.2-q13.3.) Killary et al. (1986) found that Dhfr maps to mouse chromosome 13, as does another gene on human chromosome 5, namely, hexosaminidase B (Hex2).
DHFR Pseudogenes
Anagnou et al. (1984) assigned pseudogene 4 (DHFRP4) to chromosome 3. Anagnou et al. (1985) mapped DHFR pseudogene-1 to chromosome 18 and pseudogene-2 (DHFRP2) to chromosome 6 by human-rodent somatic cell hybridization. Pseudogene-1 (DHFRP1) shows an absence/presence polymorphism consistent with recent origin (which is also suggested by sequence identity to the coding sequences of the functional gene). The transposition of the 'perfect' pseudogene must have occurred before the development of racial groups: Mediterraneans show the highest (94%) and American blacks the lowest (32%) frequency of the pseudogene (Anagnou et al., 1988). Anagnou et al. (1987) reported that the DHFR pseudogene-1 identifies 3 RFLPs.
McEntee et al. (2011) found that DHFRP4, which they renamed DHFRL1 (616588), is expressed and encodes a functional enzyme in humans.
Tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase (eNOS) (NOS3; 163729), is regenerated from its oxidized form, dihydrobiopterin (BH2), by DHFR. Chalupsky and Cai (2005) found that inhibition of Dhfr in bovine aortic endothelial cells by RNA interference markedly reduced endothelial BH4 and nitric oxide bioavailability. Angiotensin II (106150) also caused an BH4 deficiency by peroxide-dependent downregulation of Dhfr, and this response was associated with a significant increase in superoxide production by eNOS. Overexpression of human DHFR in angiotensin II-stimulated cells restored nitric oxide production and diminished eNOS production of superoxide. Chalupsky and Cai (2005) concluded that DHFR is critical for BH4 and nitric oxide bioavailability in the endothelium.
Martianov et al. (2007) demonstrated that in quiescent cells the mechanism of transcriptional repression of the major promoter of the DHFR gene depends on a noncoding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between noncoding RNA and the major promoter, direct interaction of the noncoding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation, and RNA immunoprecipitation, Martianov et al. (2007) showed that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.
Using purified recombinant enzymes, McEntee et al. (2011) found that both DHFR and DHFRL1 reduced dihydrofolate to tetrahydrofolate in the presence of NADPH. The specific activity of DHFRL1 was much lower than that of DHFR, but affinity for NADPH was not significantly different. EMSA experiments showed that DHFRL1, like DHFR, bound its own mRNA. In addition, both DHFRL1 and DHFR bound the mRNA of the other enzyme, suggesting not only negative-feedback loops, but possibly a complex pattern of cross-regulation. Both DHFR and DHFRL1 complemented loss of dihydrofolate reductase in E. coli and mammalian cells.
Mammalian cells cultured in the presence of methotrexate, a chemotherapeutic agent, develop resistance to the drug. Sometimes this is due to mutations in the DHFR gene, the primary target of methotrexate. Blakley and Sorrentino (1998) stated that it had not been possible, however, to link such polymorphism to resistance of neoplastic disease to therapy with methotrexate.
DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. In most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs). Singer et al. (2000) isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. All but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial homogeneously staining chromosome region (HSR), its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. The studies suggested that both HSRs and DMs are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.
Johnson et al. (2004) genotyped 157 members of multicase spina bifida (SB) families and 219 unrelated controls for a 19-bp deletion in intron 1 of the DHFR gene. They found that homozygosity for the deletion allele was significantly more frequent in SB mothers (p = 0.049), but not in SB fathers or patients, compared with controls, and was associated with a significantly increased odds ratio (OR, 2.035) of being an SB mother compared with other genotypes. Johnson et al. (2004) suggested that reduced folates might be preferable for supplements during pregnancy to prevent spina bifida.
In a study of 283 Irish neural tube defect (NTD) cases and their parents and 256 controls, Parle-McDermott et al. (2007) found that the 19-bp deletion in intron 1 of the DHFR gene, in contrast to the previous association with spina bifida reported by Johnson et al. (2004), appeared to protect against risk of having an NTD-affected pregnancy when present in 1 or 2 copies (p = 0.01 for both).
Mishra et al. (2007) identified an 829C-T SNP in the 3-prime untranslated region of the DHFR cDNA that is 14 nucleotides downstream of a putative miR24 (see MIRN24-1; 609705)-binding site. The more common 829C allele is conserved in humans and rodents. Overexpression and inhibitor studies showed that miR24 downregulated DHFR protein expression when the DHFR transcript contained the 829C allele but not the 829T allele. When expressed in cultured cells, the 829T allele caused a 2-fold increased DHFR transcript half-life, and due to elevated DHFR protein levels, caused a 4-fold resistance to methotrexate compared with cells expressing the 829C allele. Mishra et al. (2007) concluded that the 829T allele interferes with DHFR downregulation by miR24, resulting in enzyme overproduction and drug resistance.
Megaloblastic Anemia Due to Dihydrofolate Reductase Deficiency
In 6 patients with megaloblastic anemia and DHFR deficiency (613839), Banka et al. (2011) and Cario et al. (2011) simultaneously and independently identified homozygous mutations in the DHFR gene (L80F; 126060.0001 and D153V; 126060.0002, respectively). The phenotypes were different: the 3 patients reported by Banka et al. (2011) had severely delayed psychomotor development, generalized seizures, and cerebral and cerebellar atrophy, whereas the 3 sibs reported by Cario et al. (2011) were either asymptomatic or had childhood absence epilepsy with eyelid myoclonus and mild learning disabilities. Treatment with folinic acid ameliorated the hematologic and seizure phenotypes. The phenotype was caused by decreased cerebral levels of methyltetrahydrofolate.
In a study of 1,751 knockout alleles created by the International Mouse Phenotyping Consortium (IMPC), Dickinson et al. (2016) found that knockout of the mouse homolog of human DHFR is homozygous-lethal (defined as absence of homozygous mice after screening of at least 28 pups before weaning).
In 2 affected sibs, born of first-cousin British Pakistani parents, with megaloblastic anemia due to dihydrofolate reductase deficiency (613839), Banka et al. (2011) identified a homozygous 238C-T transition in exon 3 of the DHFR gene, resulting in a leu80-to-phe (L80F) substitution in a highly conserved residue. A child with the disorder from another consanguineous Pakistani family was also found to be homozygous for the L80F mutation. The mutation was not found in 292 ethnically matched Pakistani control chromosomes. Studies of patient-derived lymphoblastoid cells showed undetectable DHFR protein levels, and severely decreased enzyme activity. Similar studies in the heterozygous parents showed intermediate levels of protein and activity compared to wildtype. There was also an accumulation of folic acid and dihydrofolate in patient erythrocytes, indicating loss of enzyme activity. Molecular modeling of the L80F mutation predicted a significant steric clash and disrupted cofactor binding. Dynamic simulation indicated that the L80F mutation could result in potential destabilization of the DHFR protein or disruption in NADPH binding. The patients had a severe phenotype, with onset in infancy of delayed development, refractory seizures, and cerebral and cerebellar atrophy.
In 3 affected sibs, born of distantly related parents of European descent, with megaloblastic anemia due to dihydrofolate reductase deficiency (613839), Cario et al. (2011) identified a homozygous 458A-T transversion in exon 5 of the DHFR gene, resulting in an asp153-to-val (D153V) substitution. Both parents were heterozygous for the D153V mutation, which was not found in 120 control samples. The mutation was predicted to interrupt hydrogen bonding, affecting fold and conformational stability, resulting in decreased enzyme activity. Studies of patient lymphoblastoid cells showed that DHFR activity was reduced to about 10% of control levels. DHFR expression was similar to controls, but protein levels were severely decreased. Although 1 sib was essentially unaffected except for macrocytosis, the other 2 sibs developed childhood absence epilepsy with eyelid myoclonia in childhood. One had learning disabilities. Levels of 5-methyltetrahydrofolate (5-MTHF) in the CSF were low, but improved with folinic acid treatment.
Anagnou, N. P., Antonarakis, S. E., O'Brien, S. J., Modi, W. S., Nienhuis, A. W. Chromosomal localization and racial distribution of the polymorphic human dihydrofolate reductase pseudogene (DHFRPI). Am. J. Hum. Genet. 42: 345-352, 1988. [PubMed: 3341383]
Anagnou, N. P., Antonarakis, S. E., O'Brien, S. J., Nienhuis, A. W. Chromosomal localization and racial distribution of the polymorphic hDHFR-psi-1 pseudogene. (Abstract) Clin. Res. 33: 328A only, 1985.
Anagnou, N. P., Antonarakis, S. E., O'Brien, S. J., Nienhuis, A. W. A novel form of human polymorphism involving the hDHFR-psi-1 pseudogene identifies three RFLPs. Nucleic Acids Res. 15: 5501 only, 1987. [PubMed: 2885811] [Full Text: https://doi.org/10.1093/nar/15.13.5501]
Anagnou, N. P., O'Brien, S. J., Shimada, T., Nash, W. G., Chen, M.-J., Nienhuis, A. W. Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification and a novel form of polymorphism. Proc. Nat. Acad. Sci. 81: 5170-5174, 1984. [PubMed: 6089182] [Full Text: https://doi.org/10.1073/pnas.81.16.5170]
Banka, S., Blom, H. J., Walter, J., Aziz, M., Urquhart, J., Clouthier, C. M., Rice, G. I., de Brouwer, A. P. M., Hilton, E., Vassallo, G., Will, A., Smith, D. E. C., Smulders, Y. M., Wevers, R. A., Steinfeld, R., Heales, S., Crow, Y. J., Pelletier, J. N., Jones, S., Newman, W. G. Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency. Am. J. Hum. Genet. 88: 216-225, 2011. [PubMed: 21310276] [Full Text: https://doi.org/10.1016/j.ajhg.2011.01.004]
Blakley, R. L., Sorrentino, B. P. In vitro mutations in dihydrofolate reductase that confer resistance to methotrexate: potential for clinical application. Hum. Mutat. 11: 259-263, 1998. [PubMed: 9554740] [Full Text: https://doi.org/10.1002/(SICI)1098-1004(1998)11:4<259::AID-HUMU1>3.0.CO;2-W]
Cario, H., Smith, D. E. C., Blom, H., Blau, N., Bode, H., Holzmann, K., Pannicke, U., Hopfner, K.-P., Rump, E.-M., Ayric, Z., Kohne, E., Debatin, K.-M., Smulders, Y., Schwarz, K. Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease. Am. J. Hum. Genet. 88: 226-231, 2011. [PubMed: 21310277] [Full Text: https://doi.org/10.1016/j.ajhg.2011.01.007]
Chalupsky, K., Cai, H. Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. Proc. Nat. Acad. Sci. 102: 9056-9061, 2005. [PubMed: 15941833] [Full Text: https://doi.org/10.1073/pnas.0409594102]
Chen, M. J., Shimada, T., Moulton, A. D., Cline, A., Humphries, R. K., Maizel, J., Nienhuis, A. W. The functional human dihydrofolate reductase gene. J. Biol. Chem. 259: 3933-3943, 1984. [PubMed: 6323448]
Chen, M.-J., Shimada, T., Moulton, A. D., Harrison, M., Nienhuis, A. W. Intronless human dihydrofolate reductase genes are derived from processed RNA molecules. Proc. Nat. Acad. Sci. 79: 7435-7439, 1982. [PubMed: 6961421] [Full Text: https://doi.org/10.1073/pnas.79.23.7435]
Craik, C. S., Rutter, W. J., Fletterick, R. Splice junctions: association with variation in protein structure. Science 220: 1125-1129, 1983. [PubMed: 6344214] [Full Text: https://doi.org/10.1126/science.6344214]
Dickinson, M. E., Flenniken, A. M., Ji, X., Teboul, L., Wong, M. D., White, J. K., Meehan, T. F., Weninger, W. J., Westerberg, H., Adissu, H., Baker, C. N., Bower, L., and 73 others. High-throughput discovery of novel developmental phenotypes. Nature 537: 508-514, 2016. Note: Erratum: Nature 551: 398 only, 2017. [PubMed: 27626380] [Full Text: https://doi.org/10.1038/nature19356]
Funanage, V. L., Myoda, T. T., Moses, P. A., Cowell, H. R. Assignment of the human dihydrofolate reductase gene to the q11-q22 region of chromosome 5. Molec. Cell. Biol. 4: 2010-2016, 1984. [PubMed: 6504041] [Full Text: https://doi.org/10.1128/mcb.4.10.2010-2016.1984]
Johnson, W. G., Stenroos, E. S., Spychala, J. R., Chatkupt, S., Ming, S. X., Buyske, S. New 19 bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR): a risk factor for spina bifida acting in mothers during pregnancy? Am. J. Med. Genet. 124A: 339-345, 2004. [PubMed: 14735580] [Full Text: https://doi.org/10.1002/ajmg.a.20505]
Killary, A. M., Leach, R. J., Moran, R. G., Fournier, R. E. K. Assignment of genes encoding dihydrofolate reductase and hexosaminidase B to Mus musculus chromosome 13. Somat. Cell Molec. Genet. 12: 641-648, 1986. [PubMed: 2947337] [Full Text: https://doi.org/10.1007/BF01671950]
Martianov, I., Ramadass, A., Barros, A. S., Chow, N., Akoulitchev, A. Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript. Nature 445: 666-670, 2007. [PubMed: 17237763] [Full Text: https://doi.org/10.1038/nature05519]
Maurer, B. J., Barker, P. E., Masters, J. N., Ruddle, F. H., Attardi, G. Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes. Proc. Nat. Acad. Sci. 81: 1484-1488, 1984. [PubMed: 6584893] [Full Text: https://doi.org/10.1073/pnas.81.5.1484]
Maurer, B. J., Carlock, L., Wasmuth, J., Attardi, G. Assignment of human dihydrofolate reductase gene to band q23 of chromosome 5 and of related pseudogene psiHD1 to chromosome 3. Somat. Cell Molec. Genet. 11: 79-85, 1985. [PubMed: 3856332] [Full Text: https://doi.org/10.1007/BF01534737]
McEntee, G., Minguzzi, S., O'Brien, K., Larbi, N. B., Loscher, C., O'Fagain, C., Parle-McDermott, A. The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional. Proc. Nat. Acad. Sci. 108: 15157-15162, 2011. [PubMed: 21876184] [Full Text: https://doi.org/10.1073/pnas.1103605108]
Mishra, P. J., Humeniuk, R., Mishra, P. J., Longo-Sorbello, G. S. A., Banerjee, D., Bertino, J. R. A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc. Nat. Acad. Sci. 104: 13513-13518, 2007. [PubMed: 17686970] [Full Text: https://doi.org/10.1073/pnas.0706217104]
Myoda, T. T., Funanage, V. L. Personal Communication. Wilmington, Del. 10/7/1983.
Parle-McDermott, A., Pangilinan, F., Mills, J. L., Kirke, P. N., Gibney, E. R., Troendle, J., O'Leary, V. B., Molloy, A. M., Conley, M., Scott, J. M., Brody, L. C. The 19-bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR) may decrease rather than increase risk for spina bifida in the Irish population. Am. J. Med. Genet. 143A: 1174-1180, 2007. [PubMed: 17486595] [Full Text: https://doi.org/10.1002/ajmg.a.31725]
Singer, M. J., Mesner, L. D., Friedman, C. L., Trask, B. J., Hamlin, J. L. Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks. Proc. Nat. Acad. Sci. 97: 7921-7926, 2000. [PubMed: 10859355] [Full Text: https://doi.org/10.1073/pnas.130194897]