Genetics of MTHFR: Gene, Mutations, Variants, Inheritance and Testing  – Genetic Education
Genetics of MTHFR: Gene, Mutations, Variants, Inheritance and Testing 

Genetics of MTHFR: Gene, Mutations, Variants, Inheritance and Testing 

“The MTHFR gene encodes an enzyme known as methylenetetrahydrofolate reductase that breaks down homocysteine amino acid. C677T and A1298C are two common mutations of the MTHFR gene.” 

Enzymes are proteins and encoded by genes. Enzymes are important elements that catalyze many reactions in a cell including replication, transcription and translation.

Mutations in a gene are alterations in a DNA sequence, either pathogenic or not. 

MTHFR gene mutations are responsible for many disorders and pathways, such as neural tube defects, mental retardation, mental disorders, colon cancer, leukemia, infertility and recurrent abortion. 

Therefore it is important to study the MTHFR gene and its related mutations. 

In the present article, we are going to discuss the MTHFR gene, its related mutations, how to diagnose it and related topics here. 

I will also explain to you some of the common mutations of the MTHFR gene. But before that let us briefly understand what MTHFR is and it’s symptoms. 

Abbreviations used: 

Abbreviation Name 
MTHFR methylenetetrahydrofolate reductase
PCR  Polymerase chain reaction 
DNA Deoxyribose nucleic acid 
RFLP Restriction fragment length polymorphism 
RT-PCR  Reverse transcriptase PCR 
ARMS- PCR Amplified refractory mutation system
SSCP Single strand conformation polymorphism 

 Genetics of MTHFR:

The MTHFR (methylenetetrahydrofolate reductase) is an enzyme required to synthesize folic acid or folate or vitamin B9.

Mutations in the MTHFR gene can either increase the homocysteine level in blood or decrease folate. Recent findings suggest that the mutant variants of the MTHFR are associated with a wide variety of symptoms and diseases. 

Starting from mental conditions to the reproduction, mutations in it are sometimes very serious for some while a little for others. Some of the symptoms of it are:


  • Depression 
  • Mental retardation, bipolar disorders
  • Anxiety 
  • Schizophrenia
  • Cancer commonly the colon cancer 
  • Chronic pain 
  • Leukemia 
  • Infertility in male 
  • Recurrent or abnormal miscarriages in female 
  • Neural tube defects 
  • Nerve pain 
  • Migraine and headache

It is also involved in conditions like homocystinuria, alopecia areata, spina bifida, anencephaly, etc. Note that the research on the MTHFR gene mutation profile and its prevalence in different ethnic populations are still ongoing. 

Moreover, the signs and symptoms of the present condition vary from person to person and population to population.

 MTHFR gene: 

The MTHFR gene is located on chromosome 1 at 1p36.22. It is located on the short p arm of chromosome 1 between 11,785,723 to 11,806,103. 

Location of MTHFR gene and mutations C677T and A1298C on chromosome 1
Location of MTHFR gene and mutations C677T and A1298C on chromosome 1.

Functionally the gene MTHFR encodes an enzyme MTHFR that is helpful in the processing of amino acid especially it converts the 5, 10- methylenetetrahydrofolate into 5- methyltetrahydrofolate. This complex converts the amino acid homocysteine present into the blood to methionine. 

As we know that amino acids are the building blocks of different proteins, if not manufactured correctly, correct protein can’t be formed. The rarest form of the inborn error of metabolism- homocystinuria occurs due to the MTHFR gene mutation was reported by Mudd et al firstly. 

Name  MTHFR 
Full name  methylenetetrahydrofolate reductase
Cytological location 1p36.22
Genomic coordinates (GRCh38) 1: 11,785,722-11,806,102
Total exons  11
Size  2.2kb

The identical mouse MTHFR gene is located on chromosome 4, unlike the human MTHFR. Noteworthy, studies guided that due to the retrotransposition, a pseudogene of MTHFR is also present on chromosome 4 in mice which is similar to the MTHFR gene. It is an inactive mis-spliced transcript that can’t form any protein. No such pseudogene is reported in the human genome.  

MTHFR gene mutations and variants:

A change in nucleotide sequence is known as a mutation, and that arisen different alleles or variants of a gene. To date, nearly 34 different MTHFR gene mutations are reported related to some disease conditions. 

Variants are the different alleles for one particular gene. Various alleles for an MTHFR gene originate due to mutations, for example, C677T and A1298C are the two most common variants of the MTHFR gene. 

Two copies of genes or we can say alleles are present on two different chromosome number-1. Hence some people have two different variants one for normal and one for mutant- the heterozygous, some have two same types of variants present in their genome- the homozygous one while some have two normal variants- homozygous normal

Symptoms in homozygous normal conditions are less likely to occur. In the heterozygous conditions fewer symptoms and in homozygous (mutant) conditions, more symptoms of the mutation are seen in the patient. 

Therefore, conditions with two mutant alleles become more worsen for a person, though again it varies from person to person. Some of the variants of mutations of the MTHFR gene are enlisted here: 

Type of mutation  Variants of MTHFR gene 
Missense mutations 1141CT (C1141T), 1172GA, 1274GC, 1615CT, 1727CT, 1755GA, 1768GA, 468GT, 692CT, 764CT, 983AG, 985CT, 1010TC, 1027TG, 167GC, 980TC, 1015CT, 1025TC.
Nonsense mutations 28AT, 559CT, 1084CT, 1134CG, 1274GA, 1420GT, 1711CT and 1762AT
Splice site mutation 249-1GT and 792+1GA.
Other mutations

Among all these mutations C677T and A1298C are the two most candidate mutations associated with a wide spectrum of abnormal conditions in patients. 

C677T variant: 

The present mutation is one of the most common MTHFR gene mutations so far and reported in many different conditions. It is present in nearly half of the American population (25 to 40%), though it is not so lethal. 

C677T is an SNP in which the C nucleotide is replaced by the T at position 677 on the MTHFR gene. This causes a change in amino acid from alanine to valine (A222V).

Studies suggest that the present MTHFR gene mutation is widely associated with the congenital neural tube defect and venous thromboembolism in females. Related article: Top 10 Congenital Disorders and How it occurs? 

Venous thromboembolism is the risk of blood clotting due to high homocysteine levels in the blood. The C667T or often denoted as 677C -T is commonly present in hyperhomocysteinemia.

The thermolabile form of the enzyme occurs due to the present mutation. Because the mutation disrupts the homocysteine pathway it is the most common risk factor for so many multifactorial and complex disorders. 

 “90% of the amino acids of MTHFR protein of mouse and human are identical”


The second candidate single nucleotide polymorphism is the A1298C which results in glutamate to alanine substitution (E429A). 

Studies suggest that the presence of CC homozygous genotype in the patient though not affect the MTHFR protein directly but abrupt the conversion of MTHFR to tetrahydrobiopterin which is a cofactor of neurotransmitters. 

Moreover, the variant is also associated with depression and other mental conditions as well as a chance of fetal down syndrome in pregnant females. 

Noteworthy less published data are available to investigate the role of A1298C variant mutation in complex disorders. 

Inheritance pattern: 

The inheritance pattern of MTHFR gene mutations is unclear though it follows either autosomal dominant or autosomal recessive mode of inheritance. Let me explain what it is!

One set of the haploid genome is inherited from the mother and another from the father. Therefore 23 chromosomes from each parent come to the fetus. Genes also inherited from parents to their offspring as it is located on chromosomes, obviously, one copy from father and one from mother. 

Suppose one parent has a normal allele for MTHFR, for instance, C at 677 while the other has T instead of C at 677, two different alleles inherited into the fetus, C677C (normal) and C677T (mutant). 

The fetus encounters a condition known as heterozygous having two different alleles. Some signs and symptoms are shown in the heterozygous conditions. 

In another condition, if both parents transmit mutant alleles to the fetus, the fetus encounters a condition known as a homozygous mutant. The present conditions are more serious than the heterozygous one!

The MTHFR condition is a complex one, different mutations have different inheritance patterns therefore, we can’t predict the clear reason and inheritance pattern.

MTHFR gene, pregnancy and infertility

Many reasons are reported for recurrent pregnancy loss, MTHFR gene mutations are one of them. The C677T candidate mutation potentially causes recurrent pregnancy loss. This happens due to abnormal levels of folate. 

Moreover, women having a mutant variant of MTHFR (either single or both) have a higher risk of pregnancy loss, spina bifida, neural tube defect and birth defects in a baby. 

Folic acid is much needed for getting pregnant as well as the healthy development of the fetus. Henceforth, MTHFR gene mutation not only creates problems in getting pregnant but also causes congenital inborn errors. 

Note that besides the above-mentioned conditions, the change of getting down syndrome, Autism, postpartum, preeclampsia and delayed development are also inflated. 

Abnormal MTHFR enzymes also impact fertility in both males and females adversely. Female faces problems in getting pregnant while males’ potential to fertilize an egg gets decreased. 

Notwithstanding, the distinct role of MTHFR gene mutations in pregnancy and related problems are still in dilemma. Folate or folic acid rich food, veggies and supplement may help to overcome problems. 

Testing of MTHFR gene: 

Genetic testing is costly medical testing and insurance sometimes also does not cover it! So to test the MTHFR gene is not feasible for all. It is not required in all conditions, indeed. 

So one may wonder in which conditions should I go for MTHFR testing? 

 Well, there are several indications suggesting that you should undergo MTHFR testing. Some are;

  • Previous pregnancy loss without any reason. 
  • History of pregnancy loss or infertility in family
  • History of any genetic problem in the family 
  • Multiple miscarriages 
  • Problems getting pregnant 
  • Genetic complications with pregnancy. 

These are some, if you have any, contact a genetic counselor and ask them for MTHFR testing. 

Process of MTHFR gene testing: 

A testing expert collects blood samples of father and mother or a patient in an EDTA tube. 

Using chemical and physical methods, DNA is isolated from the sample and tested for quality. 

The MTHFR gene is amplified in PCR using the set of primers under strict temperature gradients. 

Any of the below-listed techniques (ARMS- PCR, SSCP, sequencing, microarray or restriction digestion) is performed to get the results. 

In the case of prenatal testing, a fetus sample either amniotic fluid or chorionic villi is taken and processed to encounter mutations. 

Related articles: Cell-Free DNA Test- What is it & How to do it?

Diagnosis of MTHFR gene mutations: 

There are four different approaches, using which the MTHFR gene mutations can be studied and detected. These options are,

  1. SSCP(single-strand conformation polymorphism)
  2. PCR-RFLP (polymerase chain reaction and restriction digestion)
  3. RT-PCR analysis
  4. Triplex tetra-primer ARMS- PCR 
  5. DNA sequencing of MTHFR gene 
  6. MTHFR gene microarray


The broad spectrum of the MTHFR gene mutations including the C677T and A1298C are investigated using the single-strand conformation polymorphism. The present technique is based on the principle of change in the conformation of the single-stranded DNA. 

The confirmation or the structure of the double-stranded DNA remains the same (or unchanged) even after the single base change but that is not the case with the single strand. 

When single base change occurs in a DNA sequence, the conformation or the three-dimensional structure of a single-strand also change which consequence in the different migration pattern in the gel. 

A PCR is performed using the DNA sample and denatured to make it a single strand, afterward the amplified single-stranded DNA is run on the gel under the influence of electrical charge. Different variants or alleles migrate differently. 

Notably, the latest automated version of the present technique is slightly different. Here the fluoro-labeled primer set is used to amplify different variants of MTHFR. Later on, they run on the capillary electrophoresis that differentiates variants based on the conformation changes. 

Related article: PCR primer design guidelines.


The PCR-based restriction fragment length polymorphism technique is widely used to investigate known MTHFR mutations. HinFI and MobII are two restriction enzymes employed to digest the variants of C677T and A1298C, respectively. 

Here in the first step, the DNA is extracted from the sample and PCR is performed by the sequence-specific primers. Two different primer sets for two different variants are amplified in PCR cycles. Denaturation, annealing and extension are performed simultaneously for 94ºC (50s), 55ºC (45s) and 72ºC (45s), respectively. 

Afterward, the amplicons are digested using the two restriction endonucleases given below applying the standard manufacturer’s protocol and run on 3% gel. Based on the digestion pattern, the results are evaluated. The results of restriction digestion are looks like this: 

The hypothetical representation of restriction digestion of C677T with the restriction endonuclease.
The hypothetical representation of restriction digestion of C677T with the restriction endonuclease.

RT-PCR analysis: 

Yet another technique widely employed to study the MTHFR gene mutations is the real-time PCR analysis or through the reverse transcriptase- reverse transcriptase PCR. 

The present technique investigates gene expression for MTHFR and measures the number of different variants present in the sample or population. 

In the present technique, firstly mRNA is isolated and reverse transcribed into the cDNA using the reverse transcriptase enzyme in order to measure the transcript. 

After that, as the variant amplifies the amount of fluorescence emitted, is recorded and measured by the detector. 

The RT-PCR analysis is important to know the number of particular variants in the patient and how it affects different conditions. 

Read related article: Reverse transcriptase PCR.

DNA sequencing of the MTHFR gene: 

MTHFR gene sequencing is an accurate, fast and reliable technique to investigate known and unknown mutations associated with the MTHFR. Here every nucleotide is reported and compared with the reference MTHFR gene sequence to evaluate mutations. 

The sequencing technique can even encounter gene expression as well. It is the topmost technique to study gene mutations. It can distinguish homozygous and heterozygous. This article on DNA sequencing will help you to learn more: What Is DNA Sequencing? A Beginners Guide.

MTHFR gene microarray:

An array is developed to study various MTHFR gene mutations. In gene microarray, known mutations specific probes are immobilized on the glass slide and allowed to hybridize with the sample. 

The hybridization signal indicates the presence of a mutation or mutations in the sample. The technique can also report the expression of genes or variants as well. 

On the downside, the microarray can’t identify new mutations or variants. 

Related article: Genome-On-A-Chip: DNA Microarray.


The amplified refractory mutations system is one of the most versatile conversions of conventional PCR to study mutations. Three different primers for one variant are designed to amplify. 

The isolated DNA is amplified in the PCR using the triplex tetra primers. The primer amplifies only the complementary sequence if present. 

The results are analyzed on the agarose gel electrophoresis. It can distinguish homozygous as well as heterozygous alleles. However, it can neither study gene expression nor new mutations. 

Treatments for MTHFR gene mutation: 

The mutant variants of the MTHFR are not so lethal, unlike other genetic or congenital disorders. Though a person with the confirmed mutant variant should have to take care of their homocysteine level. 

Note that there are other reasons as well that increase the level of homocysteine in the blood, therefore changing the lifestyle can overcome several problems. For instance, by consuming a vitamin B rich diet or supplements or folate-rich diet the conditions can be ruled out. 

In addition to this, quitting smoking, exercise and a healthy diet helps to some extent. 

Beans, peas, veggies, lentils, honeydew, bananas, strawberries, fruits, peanut butter, and sunflower oil are good sources of multivitamins that are highly needed in case of MTHFR gene mutation. 

Moreover in some serious conditions like infertility and recurrent abortion treatments like artificial insemination, in vitro fertilization, IUI or surrogacy is advised. 


Conclusively, we can say that though the variants of MTHFR are not so lethal, it should be ruled out as soon as possible. There are many supplements and medications that are nowadays available to maintain folate in the body. Also, separate assays and biochemical testing are also now commonly available to test MTHFR.


What is MTHFR gene mutation? 

MTHFR gene mutation is a change in the nucleotide sequence (a single base change) that causes serious health issues, congenital conditions and pregnancy-related problems. 

What are the common MTHFR gene mutations? 

C667T and A1298C are two variants or mutations of the MTHFR gene, associated with various developmental, reproductive and mental problems. C667T causes a change in amino acids from alanine to valine while A1298C causes a change in amino acid glutamate to alanine. 

What do MTHFR gene mutations cause? 

The MTHFR gene mutations cause varieties of health problems like depression, bipolar disorders, Alzheimer’s disease, neural tube defect, infertility and recurrent pregnancy loss.

What does a positive MTHFR test mean?

The positive MTHFR test means you have a single or two alleles or variants of the mutant MTHFR gene, you should have to contact your doctor in that case. 

What to do in the case of MTHFR mutation? 

MTHFR gene mutations can’t cause serious problems in most cases. Take folate supplements as per your doctor’s advice or eat a healthy folate-rich diet. 

What are the common symptoms of MTHFR gene mutations? 

Mental illness, depression, bipolar disorders, recurrent abortion, reduced folate level and infertility are common symptoms of it.  

How to overcome the MTHFR problem? 

Eat healthy and folate-rich foods, veggies and fruits. Use medications if required. 

Sources and links:

Leclerc D, Sibani S, Rozen R. Molecular Biology of Methylenetetrahydrofolate Reductase (MTHFR) and Overview of Mutations/Polymorphisms. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

NCBI: MTHFR gene Homo sapiens

dbSNP: MTHFR gene 

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