DHFR (dihydrofolate reductase)

2015-12-01 Maja Krajinovic , Rachid Abaji , Bahram Sharif-Askari Affiliation

Identity

HGNC

DHFR

LOCATION

5q14.1

LOCUSID

1719

ALIAS

DHFRP1,DYR

FUSION GENES

Show Gene Fusions

Abstract

Dihydrofolate reductase (DHFR) is a member of the reductase enzyme family, which is ubiquitously expressed in all organisms. Levels of this enzyme peak at the G1\/S cell cycle boundary. Autoregulation, through DHFR-RNA interactions, has also been reported. DHFR catalyzes the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) needed for several one-carbon transfer reactions in purine and pyrimidine synthesis (Jensen et al 1997, Klon et al 2002). It is also the only enzyme that reduces folic acid, a synthetic vitamin not found in nature, to dihydrofolate (Banka et al. 2011). Reduction of DHFR enzymatic activity diminishes the THF pool inside the cell which slows DNA synthesis and cell proliferation eventually leading to cell death (Assaraf et al 2007, Klon et al 2002, Morales et al 2009). DHFR inhibition is essential to the action of antifolate medications used to treat cancer and some inflammatory diseases. Changes in DHFR expression can affect susceptibility to a variety of diseases dependent on folate status such as spina bifida and cancer. Likewise, human DHFR (hDHFR) has become a major drug target in anticancer therapy (Klon et al 2002, Sharif-Askari et al 2010).

DNA/RNA

Description

DHFR encoded on chromosome 5 and has 6 exons which are separated by 5 introns.
Chen et al (1984) determined that the DHFR gene is about 30 kb long and consists of 6 exons separated by 5 introns. The full length transcript is 28756 bp long and length of 3-UTR of some human DHFR mRNA molecules was found to be 2900 nucleotides (Chen et al 1984).

Transcription

The human DHFR contains two promoters, the minor transcript which represents only 1% of DHFR mRNA molecules and the major transcript which codes for 99% DHFR mRNA. Independent regulation, low prevalence, nuclear enrichment and low translational efficiency suggest that the DHFR minor transcript may function in vivo to regulate the transcriptional activity of the major promoter (Blume et al 2003 and Martianov et al 2007).

Pseudogene

The DHFR gene family includes the functional DHFR gene and four other intronless pseudogenes, dihydrofolate reductase pseudogene (DHFRP1-4), based on human-rodent somatic cell hybridization. Pseudogene-4 (DHFRP4) is assigned to chromosome 3, pseudogene-1 to chromosome 18 with two transcripts and pseudogene-2 (DHFRP2) to chromosome 6 with one transcript (Anagnou et al. 1984 and Anagnou et al. 1985). Interestingly, according to Anagnou et al. 1988 report, pseudogene-1 (DHFRP1) was found to be present in some individuals while completely absent in others with an interethnic variation in frequency which might implicate a recent origin in the evolutionary process (Anagnou et al., 1988).
Recent studies suggest that DHFRP4, now known as dihydrofolate reductase-like 1 (DHFRL1) is expressed giving a functional protein product which shows a similar but less specific activity to that of DHFR enzyme (McEntee et al 2011).

Proteins

Description

Sequence length: 187 AA. Craik et al. (1983) in DHFR protein study reported that altered surface structures can account for functional differences among the members of a family. Also they pointed out that sliding of the intron-exon junctions may design a mechanism for generating length polymorphisms and divergent sequences.

Expression

DHFR is extensively expressed in the fetal and adult tissues such as heart, liver, skeletal muscle, thymus, kidney, brains and whole blood with higher expression in adult brain in compare to fetal brain (Banka et al 2011).

Localisation

While the dihydrofolate reductase enzyme is thought to be present in multiple cellular compartments, it is most particularly localized in the cytosol and the nucleoplasm.

Function

DHFR is a key enzyme in folate metabolism as it is involved in 5,10-methylene tetra hydro folate (THF) generation from 7,8-dihydrofolate (DHF). The generated 5,10-methylene THF is used for the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) in a reaction catalyzed by thymidylate synthase (TS). The regenerated THF starts subsequent rounds of thymidylate biosynthesis. Moreover DHFR contributes to the de novo mitochondrial thymidylate biosynthesis pathway and catalyzes de novo glycine and purine synthesis as well as DNA precursor synthesis (Anderson et al 2011 and Assaraf et al 2007).

Homology

Funanage et al (1984) assigned the DHFR gene to chromosome 5 and further narrowed the assignment to 5q11-q22. Based on their evidences, there is a homology between both the short and long arm of hamster chromosome 2 and human chromosome 5. Also, the sequences of the human and mouse DHFR proteins studied by Chen et al was shown to differ in only 21 of 186 amino acids, which reflects an 89% homology in the DNA coding sequences of the genes (Funanage et al 1984 and Chen et al 1984).

Mutations

Germinal

Germline polymorphisms
1. Location: Intron 1
Polymorphism: 19-bp insertion /deletion (rs70991108
Impact: Low-serum folate/ high homocysteine, change in mRNA levels
Related disorders: Neural tube defects and breast cancer
Note: 19 base pair deletion in intron 1 which elevated risk of developing breast cancer, neural-tube defects (NTD) and may be a risk factor for low birth weight and preterm delivery (Xu et al 2007, Vander linden 2006 and Johnson et al 2005). In contrast, Parle-McDermott et al (2007), demonstrated that 19-bp deletion allele (D) may be a protective genetic factor against NTD by increasing DHFR mRNA levels in pregnant women. Moreover, a study by Rafighdoost also suggests that DHFR 19-bp D/D genotype reduce the risk of Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) in Iranian subjects (Rafighdoost et al. 2015).
Ongaro et al (2009) and Vagace et al (2011) reported that homozygosity for DHFR 19 bp deleted allele polymorphism has been associated with increased hepatotoxicity in leukemia patients treated with MTX.

2. Location: 3-UTR
Polymorphism: C829T, A721T, A1171T
Impact: MTX resistance
Related disorder: NTD and Rheumatoid Arthritis
Note: C829T: Goto et al., (2001), by analyzing 3 untranslated region (UTR) of the human DHFR gene transcript discovered C829T substitution located 223 base pairs downstream from the stop codon and positioned between the first and second polyadenylation site. It interferes with miR-24 function leading to higher DHFR mRNA and protein levels. Present in 14.2% of the Japanese populations, the 829T/T mRNA expression level was found to be higher than 829C/C due to the higher stability of the T/T mRNA (Goto et al, 2001).
A721T did not have and significant association with NTD, but was found to be in complete linkage disequilibrium (LD) with the 19-bp indel polymorphism. (Parle-McDermott et al 2007).
Sharma et al (2009) demonstrated that DHFR A1171T (rs7387) polymorphism located in 3UTR is considered as putative predictor for MTX response in rheumatoid arthritis patients.

3. Location: Downstream to 3UTR
Polymorphism: A35289G (rs1232027)
Related disorder: MTX efficacy in patients with psoriatic arthritis.
Note: Chandran et al (2010) found an association of the A allele of A35289G polymorphism with MTX efficacy in patients with psoriatic arthritis.

4. Location: Minor promoter
Polymorphism: C-1610G or T (rs1650694) and A-317/G (rs408626)
Impact: Higher DHFR expression
Related disorder: Higher risk of relapse in ALL
Note: Three polymorphisms in DHFR promoter in the 2 kb region upstream of the first or minor transcription of DHFR gene, (C-1610G/T, C-680A, and A-317G) were found associated with treatment responses in children with acute lymphoblastic leukemia (ALL). Haplotype 1 contains both the A-317 and C-1610 alleles and conferred higher transcriptional activity, as shown by reporter gene assay and quantitative mRNA analysis, likely explaining a worse prognosis in patients carrying this haplotype. The ALL patients who were carriers of this haplotype had reduced event free survival (EFS) (Dulucq et al 2008).

5. Location: Major promoter
Polymorphism: G308A (rs1105525), C35T (rs1650697), Length polymorphism 63/91: 9-bp insertion deletion/ 9-bp repeat (rs3045983/ - )
Impact: Higher DHFR expression
Related disorder: Higher risk of relapse in ALL
Note: Six polymorphisms including five SNPs, C35T, C304T, G308A, G319A, and A413G substitutions, along with one length polymorphism composed of two sequence motifs (i.e. insertion/deletion at position 63 and variable number of 9-bp elements at position 91), were identified in the major promoter of DHFR which participate in regulation as both a major promoter and a noncoding minor transcript. Haplotype 1b was identified as a haplotype responsible for the lower relapse-free survival observed in ALL patients. This haplotype is defined by C-1610, C-680, A-317 in the minor promoter and three alleles (T35, A308 and compound length polymorphisms composed of 9-base pair (bp) insertion at position 63 and triple 9bp element at position 91) in the major promoter (Dulucq et al 2008 and Al-Shakfa, et al 2009).

6. Location: Intron 3
Polymorphism: A10372C (rs1677693) and A8890G (rs1643659)
Related disorder: Colorectal cancer (Levine AJ et al 2010).

Polymorphism: 79940143T>C (rs1643650)
Related disorder: Rheumatoid Arthritis
Note: According to Salazar et al, this polymorphism was significantly associated with response to MTX in rheumatoid arthritis patients; patients with C/C and C/T genotypes showed a better response to treatment that those with T/T (Salazar et al 2014).

Mutations
Location: 458A>T (Asp153Val)
Related disorder: Megaloblastic Anemia.
Note: Cario et al., 2011 reported a homozygous DHFR mutation, 458A>T (Asp153Val) that leads to DHFR deficiency which in turn results in a complex hematological and neurological disease that can be successfully resolved with folic acid or folinic acid replacement (Banka et al 2011 and Cario et al., 2011).

Implicated in

Entity name

Megaloblastic anemia

Note

Dihydrofolate reductase deficiency is an autosomal recessive metabolic disorder characterized by the hematologic findings of megaloblastic anemia and variable neurologic symptoms.
A germline missense mutation in DHFR was identified causing subsequent extensive enzyme deficiency and resulting in an inborn error of metabolism which is characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency (Banka et al 2011).

Entity name

Neural tube defects (NTD)

Note

Neural tube closure occurs during a period of rapid cellular proliferation and DHFR activity may be a crucial factor in maintaining optimal DNA synthesis during this time. Changes in the activity of the folate cycle enzymes may affect the folate levels and affect NTD development. The most extensively studied DHFR polymorphism is a 19bp insertion to deletion in the first intron and two polymorphisms within the 3 untranslated region (721A>T and 829C>T) of the DHFR gene (Parle-McDermott et al 2007).

Entity name

Rheumatoid Arthritis (RA)

Note

Response to treatment with Methotrexate in RA treatment was found to be influenced by the genotypes of the DHFR polymorphisms rs7387 (Sharma et al 2009) and rs1643650 (Salazar et al 2014).

Entity name

Breast cancer

Note

The DHFR 19-bp deletion polymorphism affects the transcription of DHFR gene in humans which can modify the risk of breast cancer in multivitamin supplement users. A multivitamin supplement has adverse effects in patients carrying the 19-bp insertion allele (Xu et al 2007).

Entity name

Colorectal cancer (CRC)

Note

Levine et al, 2010 demonstrated significant associations between two DHFR tagSNPs (rs1677693 and rs1643659, located on the third intron of the gene) and CRC risk only in individuals not using multivitamin supplements.

Entity name

Retinoblastoma

Note

Risk of retinoblastoma was significantly elevated among children of mothers homozygous for the 19bp deletion allele taking prenatal synthetic folic acid supplements (Orjuela, et al 2012).

Entity name

Nasopharyngeal carcinoma (NPC)

Note

DHFR has a significantly higher expression in NPC and is involved in NPC progression through the nucleotide biosynthetic process (Lee et al 2013).

Entity name

Acute lymphoblastic leukemia (ALL)

Note

MTX is an important component of maintenance therapy in ALL, it exerts its cytotoxicity function by depletion of reduced folates due to interfering with folate metabolism. Changes in DHFR expression level has been found to correlate with MTX efficacy in ALL (Ongaro et al 2009). Polymorphisms in DHFR gene may affect therapeutic responses to antifolates, leading to lower treatment efficacy or higher adverse drug event frequency. Particular haplotype (1b) increases mRNA levels of DHFR and was associated with a higher risk of ALL relapse.

Bibliography

Pubmed ID	Last Year	Title	Authors
19861437	2009	DNA variants in region for noncoding interfering transcript of dihydrofolate reductase gene and outcome in childhood acute lymphoblastic leukemia.	Al-Shakfa F et al
2885811	1987	A novel form of human polymorphism involving the hDHFR-psi 1 pseudogene identifies three RFLPs.	Anagnou NP et al
21876188	2011	Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria.	Anderson DD et al
21629435	2010	Dihydrofolate reductase gene variations in susceptibility to disease and treatment outcomes.	Askari BS et al
17333344	2007	Molecular basis of antifolate resistance.	Assaraf YG et al
21310276	2011	Identification and characterization of an inborn error of metabolism caused by dihydrofolate reductase deficiency.	Banka S et al
12461786	2003	The 5'-untranslated RNA of the human dhfr minor transcript alters transcription pre-initiation complex assembly at the major (core) promoter.	Blume SW et al
21310277	2011	Dihydrofolate reductase deficiency due to a homozygous DHFR mutation causes megaloblastic anemia and cerebral folate deficiency leading to severe neurologic disease.	Cario H et al
20472929	2010	Folate pathway enzyme gene polymorphisms and the efficacy and toxicity of methotrexate in psoriatic arthritis.	Chandran V et al
6323448	1984	The functional human dihydrofolate reductase gene.	Chen MJ et al
6344214	1983	Splice junctions: association with variation in protein structure.	Craik CS et al
6504041	1984	Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.	Funanage VL et al
11448909	2001	A novel single-nucleotide polymorphism in the 3'-untranslated region of the human dihydrofolate reductase gene with enhanced expression.	Goto Y et al
9328836	1997	Distinct roles for Sp1 and E2F sites in the growth/cell cycle regulation of the DHFR promoter.	Jensen DE et al
15755837	2005	Common dihydrofolate reductase 19-base pair deletion allele: a novel risk factor for preterm delivery.	Johnson WG et al
12096917	2002	Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.	Klon AE et al
23726796	2013	Overexpression of thymidylate synthetase confers an independent prognostic indicator in nasopharyngeal carcinoma.	Lee SW et al
20615890	2010	A candidate gene study of folate-associated one carbon metabolism genes and colorectal cancer risk.	Levine AJ et al
17237763	2007	Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript.	Martianov I et al
21876184	2011	The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional.	McEntee G et al
19190117	2009	Dihydrofolate reductase amplification and sensitization to methotrexate of methotrexate-resistant colon cancer cells.	Morales C et al
19648163	2009	Gene polymorphisms in folate metabolizing enzymes in adult acute lymphoblastic leukemia: effects on methotrexate-related toxicity and survival.	Ongaro A et al
22648968	2012	Risk of retinoblastoma is associated with a maternal polymorphism in dihydrofolatereductase (DHFR) and prenatal folic acid intake.	Orjuela MA et al
17486595	2007	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.	Parle-McDermott A et al
26221921	2015	The 19-bp deletion polymorphism of dihydrofolate reductase (DHFR) and nonsyndromic cleft lip with or without cleft palate: evidence for a protective role.	Rafighdoost F et al
25084201	2014	Polymorphisms in genes involved in the mechanism of action of methotrexate: are they associated with outcome in rheumatoid arthritis patients?	Salazar J et al
19902562	2009	Purine biosynthetic pathway genes and methotrexate response in rheumatoid arthritis patients among north Indians.	Sharma S et al
21064136	2011	Methotrexate-induced subacute neurotoxicity in a child with acute lymphoblastic leukemia carrying genetic polymorphisms related to folate homeostasis.	Vagace JM et al
17413111	2007	A functional 19-base pair deletion polymorphism of dihydrofolate reductase (DHFR) and risk of breast cancer in multivitamin users.	Xu X et al
16672082	2006	Genetic variation in genes of folate metabolism and neural-tube defect risk.	van der Linden IJ et al

Other Information

Locus ID:

NCBI: 1719
MIM: 126060
HGNC: 2861
Ensembl: ENSG00000228716

Variants:

dbSNP: 1719
ClinVar: 1719
TCGA: ENSG00000228716
COSMIC: DHFR

RNA/Proteins

Gene ID	Transcript ID	Uniprot
ENSG00000228716	ENST00000439211	P00374
ENSG00000228716	ENST00000439211	B0YJ76
ENSG00000228716	ENST00000504396	P00374
ENSG00000228716	ENST00000505337	P00374
ENSG00000228716	ENST00000505337	B0YJ76
ENSG00000228716	ENST00000511032	B4DM58

Expression (GTEx)

Adipose - Subcutaneous

Adipose - Visceral

Adrenal Gland

Artery - Aorta

Artery - Tibial

Bladder

Brain - Amygdala

Brain - Anterior cingulate cortex

Brain - Caudate

Brain - Cerebellar Hemisphere

Brain - Cerebellum

Brain - Cortex

Brain - Frontal Cortex

Brain - Hippocampus

Brain - Hypothalamus

Brain - Nucleus accumbens

Brain - Putamen

Brain - Spinal cord

Brain - Substantia nigra

Breast - Mammary Tissue

Cells - Cultured fibroblasts

Cells - EBV - transformed lymphocytes

Cervix - Ectocervix

Cervix - Endocervix

Colon - Sigmoid

Colon - Transverse

Esophagus - Gastroesophageal Junction

Esophagus - Mucosa

Esophagus - Muscularis

Fallopian Tube

Heart - Atrial Appendage

Heart - Left Ventricle

Kidney - Cortex

Kidney - Medulla

Liver

Lung

Minor Salivary Gland

Muscle - Skeletal

Nerve - Tibial

Ovary

Pancreas

Pituitary

Prostate

Skin - Not Sun Exposed

Skin - Sun Exposed

Small Intestine - Terminal Ileum

Spleen

Stomach

Testis

Thyroid

Uterus

Vagina

Whole Blood

Pathways

Pathway	Source	External ID
One carbon pool by folate	KEGG	ko00670
Folate biosynthesis	KEGG	ko00790
One carbon pool by folate	KEGG	hsa00670
Folate biosynthesis	KEGG	hsa00790
Metabolic pathways	KEGG	hsa01100
Cell Cycle	REACTOME	R-HSA-1640170
Cell Cycle, Mitotic	REACTOME	R-HSA-69278
Mitotic G1-G1/S phases	REACTOME	R-HSA-453279
G1/S Transition	REACTOME	R-HSA-69206
G1/S-Specific Transcription	REACTOME	R-HSA-69205
E2F mediated regulation of DNA replication	REACTOME	R-HSA-113510
Metabolism	REACTOME	R-HSA-1430728
Metabolism of nitric oxide	REACTOME	R-HSA-202131
eNOS activation and regulation	REACTOME	R-HSA-203765
Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation	REACTOME	R-HSA-1474151
Metabolism of vitamins and cofactors	REACTOME	R-HSA-196854
Metabolism of water-soluble vitamins and cofactors	REACTOME	R-HSA-196849
Metabolism of folate and pterines	REACTOME	R-HSA-196757
Antifolate resistance	KEGG	ko01523
Antifolate resistance	KEGG	hsa01523

Protein levels (Protein atlas)

Not detected

Low

Medium

High

PharmGKB

Entity ID	Name	Type	Evidence	Association	PD	PMIDs
PA10810	pemetrexed	Chemical	ClinicalAnnotation	associated	PD	23709418, 24732178
PA443434	Arthritis, Rheumatoid	Disease	ClinicalAnnotation, VariantAnnotation	associated	PD	19902562, 25084201
PA443622	Carcinoma, Non-Small-Cell Lung	Disease	ClinicalAnnotation	associated	PD	23709418, 24732178
PA444937	Mesothelioma	Disease	ClinicalAnnotation	associated	PD	24732178
PA445601	Osteosarcoma	Disease	ClinicalAnnotation	associated	PD	25778468
PA446155	Precursor Cell Lymphoblastic Leukemia-Lymphoma	Disease	ClinicalAnnotation	associated	PD	19648163, 19861437, 21747412, 26335211
PA450428	methotrexate	Chemical	ClinicalAnnotation, VariantAnnotation	associated	PD	19648163, 19861437, 19902562, 21747412, 25084201, 25778468, 26335211

References

Pubmed ID	Year	Title	Citations
17237763	2007	Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript.	282
19913121	2009	Gene-centric association signals for lipids and apolipoproteins identified via the HumanCVD BeadChip.	85
19898482	2009	Genetic variants in TPMT and COMT are associated with hearing loss in children receiving cisplatin chemotherapy.	84
19706381	2009	The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake.	66
16735474	2006	Understanding bistability in complex enzyme-driven reaction networks.	62
19493349	2009	118 SNPs of folate-related genes and risks of spina bifida and conotruncal heart defects.	62
12084458	2002	Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase.	53
11964483	2002	Divergent regulation of dihydrofolate reductase between malaria parasite and human host.	49
12612070	2003	RNA polymerase II accumulation in the promoter-proximal region of the dihydrofolate reductase and gamma-actin genes.	47
17449906	2007	Genetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of diet.	46

Citation

Maja Krajinovic ; Rachid Abaji ; Bahram Sharif-Askari

DHFR (dihydrofolate reductase)

Atlas Genet Cytogenet Oncol Haematol. 2015-12-01

Online version: http://atlasgeneticsoncology.org/gene/40303/dhfr