1. health and fitness
2. disease

CHAPTER 4
Abstract
S
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
Mary Ann Thomas and David S. Rosenblatt
ce
Severe Methylenetetrahydrofolate Reductase
Deficiency
evere methylenetetrahydrofolate reductase (MTHFR) deficiency is an inborn error of
folate metabolism that is associated with elevated levels of homocysteine and decreased
levels of methionine and S-adenosylmethionine. The clinical spectrum of severe MTHFR
deficiency ranges from the neonatal onset of significant neurological problems to milder adult
onset cases. There have also been several asymptomatic adult cases reported. The majority of
patients present in the first few years of life with developmental delay and other neurological
problems, such as seizures. Although treatment is difficult, the addition of betaine has improved neurological development in some patients and halted the deterioration in others. This
chapter is a summary of the clinical presentation, pathophysiology, laboratory investigations,
prenatal diagnosis, treatment and current knowledge of genotype-phenotype correlations in
severe MTHFR deficiency.
Introduction
Eu
The enzyme 5,10-methylenetetrahydrofolate reductase (methylene-H4Folate reductase,
MTHFR) plays a key regulatory role in folate metabolism. MTHFR reduces
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-methyltetrahydrofolate is involved in the remethylation of homocysteine to methionine, which is then converted to
S-adenosylmethionine, the predominant methyl donor in man. In cases of severe MTHFR
deficiency, the decreased levels of methionine and S-adenosylmethionine adversely affect myelination and are thought to be an important cause of the neurological problems. There is also
increased homocysteine, which accounts for thrombosis being a feature in some of the patients.
Alterations in the gene for MTHFR have been associated with two broad categories of
medical conditions. In the first category, common polymorphisms in MTHFR, such as 677C→T
and 1298A→C, are associated with an increased predisposition to several medical problems.
Although less prevalent in Africans and Asians, homozygosity for the polymorphism 677C→T
is present in 5-18% of many European and North American populations.1 The residual enzyme activity in homozygotes for this polymorphism is 35-50% that of controls,2 which is
sufficient to increase plasma homocysteine if folate intake is inadequate. Homozygosity for
677C→T has been postulated to increase the risk of developing cardiovascular disease and of
women having children with neural tube defects.3 This is explained in more detail in other
chapters of this book. This chapter will concentrate on the second type of medical condition
that is caused by mutations that decrease the specific activity of MTHFR to less than 20% of
controls.4 Individuals with these mutations often have a severe clinical phenotype, including
developmental delay, motor and gait abnormalities, seizures and psychiatric features.
MTHFR Polymorphisms and Disease, edited by Per Magne Ueland and Rima Rozen.
©2004 Eurekah.com.
MTHFR Polymorphisms and Disease
42
Clinical Presentation
Eu
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
A review of at least 85 published cases with severe MTHFR deficiency1,5-58 indicates that the
clinical phenotype varies greatly from individual to individual. This disease can be broadly classified into neonatal onset, late infancy/early childhood onset and late childhood/adult forms.40
The more severe cases typically present at an earlier age with neurological deterioration.
In the neonatal form, the pregnancy and delivery are usually uneventful. Patients present
with decreased muscle tone, drowsiness, poor feeding, apnea, seizures and even coma. This is
occasionally preceded by an infection.44 Brain imaging typically shows brain atrophy and white
matter disease (demyelination). Electroencephalograms (EEG) generally demonstrate abnormal background activity or seizure activity if the patient has clinical seizures. There is no specific seizure type in these patients. In cases tested, visual evoked responses are abnormal and
reflect demyelination. Prior to the inclusion of betaine in the treatment regimen, affected individuals often died within the first year of life, mostly from respiratory failure secondary to
central (CNS) causes or aspiration pneumonia. Some cases treated with betaine caught up with
growth and psychomotor development, although long term neurological outcome of treatment is not known.45,52
Presentation in infancy/early childhood ranges from the age of 3 months to 10 years. Typically, the developmental delay is not as striking in the first few months of life. Patients present
to medical attention when developmental milestones, such as learning to sit or walk, are not
met. Patients may show developmental regression after an infection.44 Some affected children
present later with seizures and mental retardation of unknown cause. A number of children in
this group have microcephaly,23,28,30,33,46 although this is not a consistent finding.10 Neurological symptoms differ among cases and can even be conflicting at times. These include hypotonia, 30,46,48 hypertonia,10,33,38 spastic paraparesis,10 weakness,47 upper motor neuron signs
(brisk deep tendon reflexes and an upgoing Babinsky reflex), pyramidal tract involvement,37
extrapyramidal movements,33 ataxic gait,10,33 lack of coordinated eye movements30 and peripheral neuropathy.38 The majority of patients have seizures and all have developmental delay,
usually severe. One ten year-old boy exhibited a history of developmental delay and physical
signs of Angelman syndrome.57 Brain imaging typically shows abnormalities. Before MRI was
available, CT scans were performed and often demonstrated dilated ventricles10,33,48 and cortical atrophy.23,48 When MRI became available, white matter abnormalities, such as demyelination, were more evident.46 As in earlier onset cases, EEG abnormalities reflect the patient’s clinical
seizure. Studies of visual evoked responses or auditory evoked responses demonstrate abnormalities consistent with abnormal myelination. Treatment regimens that include betaine often improve levels of homocysteine and clinical symptoms. Many cases that did not receive betaine as
part of their treatment died several years after diagnosis, often from respiratory failure.
The later childhood or adulthood form of the disease can present with some of the same
features as the early childhood cases. These include similar neurological features, mental retardation and seizures. Others have prominent peripheral neuropathy,38,47 ataxia, arterial thrombosis10,54 and/or psychiatric problems.6,58 There are adults who are asymptomatic and are diagnosed because of a more severely affected sibling. In one family, a younger brother developed limb
weakness, incoordination, paresthesias, and memory lapses at age 15 years and was
wheelchair-bound by his early 20s, whereas his older brother was asymptomatic at age 37 years.38
Pathophysiology
Several patients had an autopsy that confirmed the brain abnormalities seen on imaging,
notably a small brain, cerebral atrophy, enlarged ventricles and demyelination. Macrophage infiltration and gliosis have also been noted.33,36,37 It has been suggested that decreased methionine
and S-adenosylmethionine cause demyelination.37 Monkeys exposed to nitrous oxide, which blocks
the activity of methionine synthase, were found to have decreased methionine formation and
demethylation. These monkeys became ataxic and, at autopsy, the spinal cord and peripheral
nerves demonstrated changes of subacute combined degeneration. Methionine administration to
Severe Methylenetetrahydrofolate Reductase Deficiency
43
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
monkeys exposed to nitrous oxide decreased the severity of the associated demethylation. This
group of monkeys did not develop ataxia during the time they were followed and had little or no
changes of subacute combined degeneration.59
There is at least one known child who had severe worsening of his disease with exposure to
nitrous oxide. Prior to the diagnosis of severe MTHFR deficiency, this 3-month-old boy received nitrous oxide, administered during the surgical removal of a fibrosarcoma. Seventeen
days after the surgery, he was readmitted to hospital with seizures, apneic episodes, severe
hypotonia and absent reflexes. He died at around 4.5 months of age from a respiratory arrest.
The autopsy demonstrated asymmetric cerebral atrophy and severe demyelination with
astrogliosis and oligodendroglial cell depletion in the mid-brain, medulla and cerebellum.60
Several other patients with MTHFR deficiency presented with subacute combined degeneration of the cord, similar to that observed in patients with untreated cobalamin deficiency.21,33
Individuals given betaine have improved CSF levels of S-adenosylmethionine, although treatment with betaine does not always increase CSF methionine levels. This suggests that decreased S-adenosylmethionine levels, more than decreased methionine levels, are responsible
for the demyelination.
Several individuals had thromboses of arteries and cerebral veins, which appear to have
been the cause of their death.10 However, cerebral thromboses do not appear to be the cause of
the neurological symptoms in the majority of patients. It has been suggested that the combination of mutations in MTHFR and factor V Leiden can contribute to the vascular pathology in
some patients.61
There are other proposed explanations for the neurological symptoms in these patients.
One is impaired purine and pyrimidine synthesis in the brain. This has been proposed because
in some cases, there have been neurological symptoms despite normal CSF methionine levels.
Several authors have suggested that the only natural folate that can cross the blood brain barrier
is methyltetrahydrofolate, the product of the MTHFR reaction.16,62 Deficiency of MTHFR
may result in functionally low folate levels in the brain. With the current level of understanding
of this disease, it is unclear what the relative contributions of low folate, low methionine and
low neurotransmitter levels are in the CNS pathology of severe MTHFR deficiency.44
Prenatal Diagnosis
Eu
There are at least 5 published reports of prenatal testing for severe MTHFR deficiency using
enzymatic studies.26,32,48,51,63 One case demonstrated low enzymatic activity in amniocytes and
the pregnancy was continued for religious reasons. The first urine contained homocystine and
cord blood showed low MTHFR activity, confirming an affected newborn.26 Another case had
testing in both chorionic villi (11 weeks) and amniocytes (18 weeks). Both samples showed
decreased enzymatic activity in the heterozygous range. This was confirmed 9 months post-natally.
In our laboratory, using the specific activity of MTHFR in confluent amniocytes, we have excluded the diagnosis of severe MTHFR deficiency in 9 cases and diagnosed one affected fetus.
However, a recent publication demonstrated the potential difficulty in interpreting biochemical
results since enzymatic activity was in the heterozygous range in the prenatal studies, but the
child’s enzymatic activity was very low after birth.51 Another family exemplified the complex
relationship between residual enzyme activity and clinical findings. The mother had severely low
enzyme function, in the range of her severely affected child, but had no clinical symptoms.48
Given the complicated association between residual enzyme activity and clinical severity, molecular testing is the ultimate prenatal test. This would consist of testing for known causal mutations or performing linkage analysis, rather than assessing enzymatic activity.
Since MTHFR polymorphisms, such as 677C→T and 1298A→C, are relatively common,
prenatal diagnosis may be possible in some families using linkage analysis, even when the mutations are not known. Because identification of mutations can be time-consuming, linkage
analysis may actually be the preferred molecular test. A sample from the affected proband is
necessary to track the mutant allele in the family, and parents need to be heterozygous for at
MTHFR Polymorphisms and Disease
44
Laboratory Findings
ce
least one variant in the MTHFR gene. However, since a number of other SNPs (single nucleotide polymorphisms), in addition to 677C→T and 1298A→C, are known in the MTHFR
gene (Table 5, chapter 2), linkage analysis should be possible in most families. Prenatal diagnosis for severe MTHFR deficiency has been performed by linkage analysis for 3 families in our
institution (R. Rozen, personal communication); in the one family that requested amniocentesis, instead of chorionic villus sampling, enzymatic analysis in our laboratory confirmed the
prenatal result obtained by DNA testing (unpublished data).
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
The major biochemical findings include moderate homocystinuria and hyperhomocysteinemia
with low or low-normal levels of plasma methionine. Whereas patients reported in the early
papers had free plasma homocystine measured, current publications usually include total plasma
homocysteine values. In Erbe’s clinical review in 1986,64 homocystinuria was present in all
patients, with a reported range of 15 to 667 µmol/24 h and a mean value of 130 µmol/24 h.
Homocystine, not normally detected in urine or free in plasma, was found in the plasma: mean
value 57 µM (range, 12 to 233 µM). Recent data on total plasma or serum homocysteine
(tHcy) reveal levels (before treatment) of 42-220 µM (controls 4-20 µM).38,46,48,50,51,56,65,66
Plasma methionine levels were low in all patients, ranging from 0 to 18 µM, with a mean of 12
µM; normal is 23-35 µM,64 although values vary among laboratories.
Although homocystinuria was consistently seen in all patients, and indeed is the clinical
sign by which the diagnosis of MTHFR deficiency is made, the excretion of homocystine in
urine is much less than that found in homocystinuria due to cystathionine synthase deficiency.
Indeed, it may not be detected on spot testing, which should not be used in isolation to diagnose severe MTHFR deficiency.67 Methionine levels in MTHFR deficiency are usually low.
This again distinguishes these patients from those with cystathionine synthase deficiency, who
generally have high levels of methionine. Although serum folate levels were not always low,
many of the patients with MTHFR deficiency had serum folate levels that were low on at least
one determination. In contrast, serum cobalamin levels were almost always normal. Although
the levels of neurotransmitters in the cerebrospinal fluid have been measured in only a minority of patients, they have usually been low.47,64
Another group of inborn errors of metabolism that can have homocystinuria are the cobalamin (vitamin B12) abnormalities. These patients are functionally deficient in methionine biosynthesis because of abnormalities in methylcobalamin formation (complementation groups
cblC, cblD, cblE (methionine synthase reductase deficiency), cblF, and cblG (methionine synthase deficiency)), and differ from patients with MTHFR deficiency by having megaloblastic
anemia. In addition, in contrast to patients with the cblC, cblD, and cblF disorders, patients
with MTHFR deficiency have no methylmalonic aciduria. Tests to assess for megaloblastic
anemia and methylmalonic aciduria should be performed to distinguish cobalamin abnormalities from MTHFR deficiency.
Studies on Cultured Cells
Eu
A deficiency of MTHFR has been confirmed in studies of liver, leukocytes, cultured fibroblasts and lymphoblasts. The MTHFR reaction is irreversible in vivo, but the enzyme activity
can be measured in the reverse (nonphysiological) direction in vitro. Traditionally, this is the
enzyme assay used to measure MTHFR activity and uses radioactive methyltetrahydrofolate as
a substrate and menadione as the electron acceptor. This is the conventional assay for practical
reasons, including lack of commercial availability of radiolabelled 5,10-methylenetetrahydrofolate.
Enzyme activity is extremely sensitive to the stage of the culture cycle of fibroblasts, with the
specific activity in control fibroblast cells being highest in confluent cultures.68 This variability
is sufficiently great to allow for the misclassification of controls and heterozygotes if the stage of
the culture cycle is not taken into account. In general, there is a rough correlation between
residual enzyme activity and the clinical severity (Table 1).
ce
0
1762AT/1762AT
0
1553delAG/1420GT
0
1084CT/1084CT
0
559CT/559CT
0
1755GA/ Presumed
heterozygote
0.2
1027TG/1027TG
1
559CT/559CT
1.6
2
164GC/249-1GT
980TC/1141CT
N/A
1027TG/1027TG
N/A
1010TC/1010TC
©2
ka 00
h/ 4
Do La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
Mutation
Clinical Presentation
Diagnosis between 0-3 months
Pakistani male presented in the first month of life with neurological symptoms and
failure to thrive. Responded to betaine and folate.
Caucasian male presented at 2 weeks of life with vomiting. Over the next few weeks,
developed stridor, hypotonia and head lag.
Turkish male, presented at 1 month of age with psychomotor delay and severe
hypotonia. He was not treated. At 10 months, there was severe psychomotor delay,
severe hypotonia and no social interaction. Betaine was started. He died at 7 years
from hyperpyrexia and had severe mental retardation.
1 month-old Native American (Hopi) male with hypotonia. Developed seizures and
corneal clouding.
Caucasian male diagnosed at 3 months with an infantile fibrosarcoma. He was
administered nitrous oxide during surgery. Returned to hospital with marked
hypotonia and apneas, and died at 4 months.
Turkish male referred at 5 weeks with hypotonia, developmental delay, apnea
and poor suck.
1 month-old Native American (Choctaw) presented with apnea, failure to thrive,
unresponsiveness, seizures and anemia.
2 week-old Caucasian male presented with failure to thrive and irregular breathing
African American/Caucasian female with lethargy and failure to thrive at 1 month
of age. At 3 months, had seizures, apnea and hypotonia
Turkish male, treated with betaine, starting at 6 days of life (poor compliance),
because of a positive family history. At 4 years, had severe mental retardation
and cerebral demyelination.
4 week-old with severe muscular hypotonia, died at 4 months. Severe cerebral
demyelination.
Patient
Reference
1794
83
1569
84
K
67
1554
80
1084
60
2231
84
1627
80
1772
1767
82, 83
82
UB
67
2
43
continued on next page
45
ure
MTHFR Activity
(% Control)
Severe Methylenetetrahydrofolate Reductase Deficiency
Table 1.
0
983AG/ 983AG
2
692CT/692CT
3
764CT/764CT
4
5.3
458GT/458GT
1727CT/1025TC
7.8
28AT/1615CT
8
28AT/1615CT
N/A
1420GT/1274GC
N/A
1711CT/1711CT
Clinical Presentation
Diagnosis between 3 months-10 years
Greek female, presented at 2 years with psychomotor retardation, microcephaly,
hypotonia, restlessness and inability to sit unsupported. At 17 years, had severe
mental retardation.
African Indian female presented at 7 months with microcephaly, progressive
deterioration of mental development, apnea and coma.
Japanese female with delayed walking and speech at 2 years, seizures at 6 years and
gait disturbance with peripheral neuropathy at 16.
Japanese female with developmental delay and seizures who died at 9 months of age.
Caucasian male presented in the first year of life with developmental delay and
seizures. At age 4 years, had gait problems and hyperactivity.
Female who presented at 11.5 years with moderate delay (sister of II3). At 2 years,
she had speech delay, attention deficit and hyperactivity and, at 3 years, was
overweight (+4SD). She had no seizures.
Male diagnosed at 3 years (brother of II1) when his sister was diagnosed. At the time,
he had a short concentration span and speech delay.
Presented at 5 years with psychomotor retardation, epilepsy and hyperkinetic
movements. Improved on betaine and folate.
Turkish female, presented at 10 months with psychomotor delay, severe
microcephaly. Received betaine, and, at 4 years, she had severe mental retardation.
ure
Mutation
Patient
Reference
CM
67
735
81
1807
81
670
1951
82
84
II1
1
II3
1
1
43
U
67
continued on next page
MTHFR Polymorphisms and Disease
MTHFR Activity
(% Control)
©2
ka 00
h/ 4
Do La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
46
Table 1. Continued
ce
5.5
1025TC/1141CT
7
8
482GA/1711CT
482GA/1711CT
8.2
1172GA/1768GA
10
13
167GA/1015CT
1274GA/471CG
14
792+1GA/?
14
167GA/1081CT
14.2
482GA/1727CT
19
792+1GA/?
20
985CT/985CT
29.1
358GA/1134CG
©2
ka 00
h/ 4
Do La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
Mutation
Clinical Presentation
Diagnosis after 10 years
Caucasian male referred at 13 years with developmental delay, noted during the first
year of life, with a history of seizures, excessive growth, and immature behavior.
37 year-old asymptomatic French Canadian male (brother of 1779).
French Canadian male (brother of 1834) presented at age 15 with weakness,
incoordination, paresthesiae and memory lapses. He was wheelchair bound by
his twenties.
14 year-old Caucasian female presenting with 4-year history of dementia and 3-year
history of dysthymia.
Caucasian male diagnosed at 12 years with ataxia and marginal school performance.
Saudi Arabian female had school difficulties at age 12, a stroke at 15 years and
spastic paraplegia and seizures at 16.
African American female (sister of 354) presented at 15 years with anorexia, tremor,
hallucinations and progressive withdrawal.
Caucasian female presented at 14 years with ataxia, foot drop, and inability to walk.
During childhood, she was clumsy and had global developmental delay. She
developed deep vein thrombosis and bilateral pulmonary emboli.
21 year-old Caucasian male, presenting with gait abnormalities of 2-3 years,
and found to have spastic paraparesis.
African American female (sister of 355) diagnosed at 13 years with mild
mental retardation.
Italian male presented at 16 years with muscle weakness, abnormal gait,
and flinging movements of the upper extremities.
Caucasian presented at 16 years with slow neurological deterioration,
including changes in mental ability and difficulty walking,
Patient
Reference
2351
84
1834
1779
80, 83
80, 83
2006
83
458
2255
81
84
355
81
1396
81
1863
80, 83
354
81
356
81
2184
83
47
ure
MTHFR Activity
(% Control)
Severe Methylenetetrahydrofolate Reductase Deficiency
Table 1. Continued
MTHFR Polymorphisms and Disease
48
Treatment
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
There are several reported problems with the reverse direction assay. The assay uses organic
solvent extraction with incomplete recovery and less than optimal specificity. The blank values
can be variable and even high because of impurities in the substrate and dependence on protein
concentration. Recently, a sensitive assay in the physiological direction has been reported.69
MTHFR activity is measured by assessing the conversion of 5,10-methylenetetrahydrofolate,
with NADPH, to 5-methyltetrahydrofolate. This is accomplished with HPLC and fluorescence detection. The mean activity with the physiological assay was 2.5- 3 fold higher than the
reverse assay and can be used to detect residual activities as low as 2.6%.
Other measures to assess the function of MTHFR include: (1) The synthesis of methionine
from homocysteine using labeled formate.22 Methionine synthesis, in the presence of normal
methionine synthase activity, is a function of MTHFR activity. The goal is to measure the
appearance of label in methionine; (2) Assessing the proportion of folate present in cultured
cells as methyltetrahydrofolate, which correlates with clinical severity. Studies in cultured fibroblasts8,15 and liver21,30 have determined the levels and distribution of folate derivatives. In
both control and mutant fibroblasts, most of the folates present were polyglutamates, and the
proportion of polyglutamates relative to folate monoglutamates was similar. In cultured fibroblasts, a decrease in the proportion of cellular methyltetrahydrofolate (as a fraction of total
folate) is correlated with worse clinical symptoms and decreased residual activity. This indicates
that the distribution of the different folates may be an important control of intracellular folate
metabolism;4,15 (3) Cultured fibroblasts from patients with severe MTHFR deficiency do not
grow in tissue culture medium lacking methionine, an essential amino acid for these cells. This
is in contrast to control fibroblasts which can grow when homocysteine, along with folate and
cobalamin, is substituted in the culture medium for methionine;9,70 and (4) A differential
microbiologic assay, which makes use of the fact that Lactobacillus casei can utilize
methyltetrahydrofolate for growth but Pediococcus acidilactici (previously known as Pediococcus
cerevisiae) cannot. This is a useful screening test for methylenetetrahydrofolate reductase deficiency since analysis only requires small numbers of cultured fibroblasts.8
Eu
Interestingly, therapy with methionine alone or with methyltetrahydrofolate has not been
particularly effective in most cases, even though S-adenosylmethionine deficiency in the central nervous system appears to play a major role in the pathogenesis of this disease.44 Individuals with MTHFR deficiency have been treated with a variety of agents including folates, methionine, pyridoxine, cobalamin, carnitine, betaine, and riboflavin, either alone or in
combination. The rationale for therapy has included: (1) folates, such as folic acid or folinic
acid, in an attempt to maximize any residual enzyme activity; (2) methyltetrahydrofolate to
replace the missing product; (3) methionine to correct the cellular methionine deficiency; (4)
pyridoxine to lower homocysteine levels, because of its role as a cofactor for cystathionine
synthase (enhancing the transsulfuration pathway);40 (5) cobalamin, because of its role as a
cofactor for methionine synthase and at least one case who developed subacute combined
degeneration of the cord when treated with methyltetrahydrofolate alone;33,71 (6) carnitine,
since deficiency can occur because its synthesis requires S-adenosylmethionine; (7) betaine,20
because it is a substrate for betaine:homocysteine methyltransferase,72 a liver-specific enzyme
which converts homocysteine to methionine; and (8) riboflavin, because of the flavin requirement of MTHFR.
Treatment is considered successful if there is reduction of the plasma homocysteine levels,
elevation of plasma methionine levels to normal and improvement in the clinical picture.64
In most cases, several of the agents mentioned above have been used in combination, and it
is somewhat difficult to assess the efficacy of a single one. Prior to the addition of betaine to
the treatment regimen, most cases were very resistant to treatment.39,40,64,71,73 There are
exceptions to this, including a 7 2 month-old who showed rapid improvement on methionine, pyridoxine, folinic acid, and cobalamin.23 Another patient responded to high doses of
Severe Methylenetetrahydrofolate Reductase Deficiency
49
Genetics
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
folic acid (400mg/day) with the disappearance of homocystine in the urine and increased
methionine in the plasma.74 The majority of cases, however, did not improve.10,30,45,47,48,75
One patient’s clinical deterioration was attributed to pyridoxine.28 When betaine is added to
the treatment, there is often decreased homocystine levels, elevated methionine levels and a
variable degree of clinical improvement.44-47,51,52,54-56
Thus, betaine25,34,45,50 appears to be the most promising agent for therapy of MTHFR
deficiency, although some of the other therapies have been partially successful. There is not a
great deal of data on the optimum dose of betaine in these patients because of limited experience. Ronge and Kjellman suggested a dose of 6 g/day (2 x 3 g). Ten g/day of betaine was tested
and was not found to further improve the clinical or biochemical features of this disease.50
Ogier de Baulny and colleagues suggested a dose of 2-3 g/day in young infants and 6-9 g/day in
children and adults.40 Sakura and colleagues studied the relationship of serum total homocysteine and betaine levels during treatment of a patient with oral betaine in doses of between 20
and 120 mg/kg/day.66 They found that serum levels of total homocysteine decreased proportionately until betaine levels reached 400 µM. They suggested that this was the therapeutic
threshold for serum betaine.
Many authors41,50,64,75 have stressed the importance of early diagnosis and therapy because
of the poor prognosis in this disorder once there is evidence of neurologic involvement. Even
with early diagnosis, it is not clear that any of the therapeutic regimens are universally successful, and it is possible that genetic heterogeneity in the disease itself is responsible for some of
the variability in clinical response to therapy.
Autosomal recessive inheritance of MTHFR deficiency has been assumed based on clinical
information. Consanguinity has been reported.13,64 The disease has occurred in siblings in
several families, both males and females have been affected and there is decreased activity of the
enzyme in the fibroblasts 9 and lymphocytes 11 of obligate heterozygotes. The clinical suspicion
was confirmed following cloning of the gene, which is on chromosome 1p36.3 and has eleven
exons.76 Most mutations are missense, although nonsense and splice site mutations have been
reported in patients with MTHFR deficiency. Each mutation has been reported in only one or
two families.1,43,65,77-81 Thirty-four different mutations causing severe disease are known, in
addition to polymorphisms which may contribute to disease in the general population. Chapter 2 contains a list of all known mutations as well as information on functional impact of some
of these sequence changes.
Genotype-Phenotype Correlations
Eu
Genetic heterogeneity in the severe form of this disorder was suggested by the fact that
fibroblast extracts from two of the original families showed differential heat inactivation at 55
degrees.9 Although several of the later-onset patients had a thermolabile reductase under these
conditions, thermolability was also found in patients with early-onset disease.82 In some patients, this has been shown to be due to the presence of severe MTHFR mutations in combination with the common 677C→T polymorphism, which is responsible for the majority of enzyme thermolability in the general population.79,83 Recent evidence has shown that having a
severe mutation in cis with the 677C→T polymorphism produced lower enzyme activity than
the severe mutation alone.2 The presence of the 677C→T polymorphism, in combination
with a severe mutation, resulted in an additional decrease of 50%.81
Although there is a correlation between residual enzyme activity and clinical severity, it is
still difficult to make genotype-phenotype correlations. There are many different mutations in
the 32 cases with identified mutations (Table 1). In addition, many of these patients are compound heterozygotes. This makes it difficult to associate a particular mutation with a specific
amount of residual enzyme activity. There can also be clinical variability among family members harboring the same mutations. In the patient described with an adverse reaction to nitrous
MTHFR Polymorphisms and Disease
50
ce
oxide exposure, only a single mutation was found in combination with the two common
MTHFR polymorphisms.60 In the 32 cases with identified mutations, the range of residual
enzyme activity correlates directly with the age of onset of symptoms. The 9 cases with onset
between 0-3 months had a range of enzyme activity that was 0-2%, average 0.5%. The range in
the 7 cases with onset between 3 months and 10 years was 0-8%, average 3%. In the group over
10 years, the range was 6-23%, average 13%. One case was asymptomatic and had 7% residual
enzyme activity. Four cases (2 in the first and 2 in the second groups) had unknown residual
enzyme activity. Therefore, in general, individuals with severely decreased enzyme activity present
at a younger age with a more severe phenotype.
Conclusion
References
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
MTHFR deficiency is an inborn error of folate metabolism that is associated with decreased
methionine and S-adenosylmethionine, and elevated homocysteine levels. There are numerous
mutations in MTHFR that cause a severe reduction in the enzyme activity. In general, the
more severely reduced the enzyme activity is, the more severe is the phenotype. Clinical presentation can occur anytime from the neonatal period to adulthood. There are often neurological
abnormalities associated with abnormal brain pathology, occasional thromboses and, rarely,
psychiatric symptoms. There are also cases that are asymptomatic. This is a very difficult disease to treat. The addition of betaine in the treatment regimen has halted the neurological
deterioration in many patients and has even improved the development in others.
Eu
1. Tonetti C, Amiel J, Munnich A et al. Impact of new mutations in the methylenetetrahydrofolate
reductase gene assessed on biochemical phenotypes: A familial study. J Inherit Metab Dis 2001;
24:833-842.
2. Goyette P, Rozen R. The thermolabile variant 677C->T can further reduce activity when expressed
in cis with severe mutations for human methylenetetrahydrofolate reductase. Hum Mutat 2000;
16:132-138.
3. Botto LD, Yang Q. 5,10-methylenetetrahydrofolate reductase gene variants and congenital anomalies: A HuGE review. Am J Epidemiol 2000; 151:862-877.
4. Rosenblatt D, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism.
In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW et al, eds. The Metabolic &
Molecular Bases of Inherited Disease. 8th ed. New York: McGraw-Hill, 2001:3897-3933.
5. Freeman JM, Finkelstein JD, Mudd SH et al. Homocystinuria presenting as reversible “schizophrenia”: A new defect in methionine metabolism with reduced 5,10-methylenetetrahydrofolate reductase activity. Pediatr Res 1972; 6:423.
6. Freeman JM, Finkelstein JD, Mudd SH. Folate-responsive homocystinuria and “schizophrenia”: A
defect in methylation due to deficient 5,10-methylenetetrahydrofolate reductase activity. N Engl J
Med 1975; 292:491-496.
7. Kanwar YS, Manaligod JR, Wong PWK. Morphologic studies in a patient with homocystinuria
due to 5,10-methylenetetrahydrofolate reductase deficiency. Pediatr Res 1976; 10:598-609.
8. Cooper BA, Rosenblatt DS. Folate coenzyme forms in fibroblasts from patients deficient in
5,10-methylenetetrahydrofolate reductase. Biochem Soc Trans 1976; 4:921-922.
9. Rosenblatt DS, Erbe RW. Methylenetetrahydrofolate reductase in cultured human cells. II. Studies
of methylenetetrahydrofolate reductase deficiency. Pediatr Res 1977; 11:1141-1143.
10. Wong PWK, Justice P, Hruby M et al. Folic acid nonresponsive homocystinuria due to
methylenetetrahydrofolate reductase deficiency. Pediatrics 1977; 59:749-756.
11. Wong PWK, Justice P, Berlow S. Detection of homozygotes and heterozygotes with
methylenetetrahydrofolate reductase deficiency. J Lab Clin Med 1977; 90:283-288.
12. Baumgartner ER, Schweizer K, Wick H. Different congenital forms of defective remethylation in
homocystinuria. Clinical, biochemical, and morphological studies. Pediatr Res 1977; 11:1015
13. Narisawa K, Wada Y, Saito T et al. Infantile type of homocystinuria with N5,10-methylenetetrahydrofolate
reductase defect. Tohoku J Exp Med 1977; 121:185-194.
14. Rosenblatt DS, Cooper BA. Methylenetetrahydrofolate reductase deficiency: Clinical and biochemical
correlations. In: Botez MI, Reynolds EH, eds. Folic acid in Neurology, Psychiatry, and Internal
Medicine. New York: Raven Press, 1979:385-390.
Severe Methylenetetrahydrofolate Reductase Deficiency
51
Eu
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
15. Rosenblatt DS, Cooper BA, Lue-Shing S et al. Folate distribution in cultured human cells. Studies
on 5,10-CH2- H4PteGlu reductase deficiency. J Clin Invest 1979; 63:1019-1025.
16. Narisawa K. Brain damage in the infantile type of 5,10-methylenetetrahydrofolate reductase deficiency. In: Botez MI, Reynolds EH, eds. Folic acid in Neurology, Psychiatry, and Internal Medicine. New York: Raven Press, 1979:391-400.
17. Singer HS, Butler I, Rothenberg S et al. Interrelationships among serum folate, CSF folate, neurotransmitters, and neuropsychiatric symptoms. Neurology 1980; 30:419
18. Baumgartner R, Wick, Ohnacker H et al. Vascular lesions in two patients with congenital
homocystinuria due to different defects of remethylation. J Inher Met Dis 1980; 3:101-103.
19. Cederbaum SD, Shaw KNF, Cox DR et al. Homocystinuria due to methylenetetrahydrofolate reductase (MTHFR) deficiency: Response to a high protein diet. Pediatr Res 1981; 15:560
20. Allen RJ, Wong PWK, Rothenberg SP et al. Progressive neonatal leukoencephalomyopathy due to
absent methylenetetrahydrofolate reductase, responsive to treatment. Ann Neurol 1980; 8:211
21. Narisawa K. Folate metabolism infantile type of 5,10-methylenetetrahydrofolate reductase deficiency.
Acta Paediatr Jap 1981; 23:82.
22. Boss G, Erbe RW. Decreased rates of methionine synthesis by methylenetetrahydrofolate
reductase-deficient fibroblasts and lymphoblasts. J Clin Invest 1981; 67:1659-1664.
23. Harpey JP, Rosenblatt DS, Cooper BA et al. Homocystinuria caused by 5,10-methylenetetrahydrofolate
reductase deficiency: A case in an infant responding to methionine, folinic acid, pyridoxine, and
vitamin B12 therapy. J Pediatr 1981; 98:275-278.
24. Harpey JP, Lemoel G, Zittoun J. Follow-up in a child with 5,10-methylenetetrahydrofolate reductase deficiency. J Pediatr 1983; 103:1007
25. Wendel U, Bremer HJ. Betaine in the treatment of homocystinuria due to 5,10-methylenetetrahydrofolate
reductase deficiency. Eur J Pediatr 1984; 142:147-150.
26. Christensen E, Brandt NJ. Prenatal diagnosis of 5,10-methylenetetrahydrofolate reductase deficiency.
N Engl J Med 1985; 313:50-51.
27. Nishimura M, Yoshino K, Tomita Y et al. Central and peripheral nervous system pathology of
homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency. Pediatr Neurol 1985;
1:375-378.
28. Haan E, Rogers J, Lewis G et al. 5,10-methylenetetrahydrofolate reductase deficiency: Clinical and
biochemical features of a further case. J Inher Met Dis 1985; 8:53-57.
29. Hyland K, Smith I, Howells DW et al. The determination of pterins, biogenic amino metabolites,
and aromatic amino acids in cerebrospinal fluid using isocratic reverse phase liquid chromatography within series dual cell coulometric electrochemical and fluorescence determinations - Use in
the study of inborn errors of dihydropteridine reductase and 5,10-methylenetetrahydrofolate reductase. In: Wachter H, Curtius H, Pfleiderer W, eds. Biochemical and Clinical Aspects of Pteridines.
Berlin: Walter de Gruyter, 1985:4:85-99.
30. Baumgartner ER, Stokstad ELR, Wick H et al. Comparison of folic acid coenzyme distribution
patterns in patients with methylenetetrahydrofolate reductase and methionine synthetase deficiencies. Pediatr Res 1985; 19:1288-1292.
31. Berlow S. Critical review of cobalamin-folate interrelations (letter). Blood 1986; 67:1526
32. Shin YS, Pilz G, Enders W. Methylenetetrahydrofolate reductase and methylenetetrahydrofolate
methyltransferase in human fetal tissues and chorionic villi. J Inher Med Dis 1986; 9:275-276.
33. Clayton PT, Smith I, Harding B et al. Subacute combined degeneration of the cord, dementia and
Parkinsonian due to an inborn error of folate metabolism. J Neurol Neurosurg Psychiatry 1986;
49:920-927.
34. Brandt NJ, Christensen E, Skovby F et al. Treatment of methylenetetrahydrofolate reductase deficiency from the neonatal period. The Society for the Study of Inborn Errors of Metabolism. In:
Anonymous The Netherlands (Abstract): Amersfoort, 1986:23.
35. Fowler B. Homocystinuria, remethylation defects. Methionine synthesis and cofactor response in
cultured fibroblasts. Society for the Study of Inborn Errors of Metabolism, 24th Annual Symposium, Amersfoort, the Netherlands, Sept 1986; 9-12.
36. Beckman DR, Hoganson G, Berlow S et al. Pathological findings in 5,10-methylenetetrahydrofolate
reductase deficiency. Birth Defects: Original Article Series 1987; 23:47-64.
37. Visy JM, Le Coz P, Chadefaux B et al. Homocystinuria due to 5,10-methylenetetrahydrofolate
reductase deficiency revealed by stroke in adult siblings. Neurology 1991; 41:1313-1315.
38. Haworth JC, Dilling LA, Surtees RAH et al. Symptomatic and asymptomatic methylenetetrahydrofolate
reductase deficiency in two adult brothers. Am J Med Genet 1993; 45:572-576.
39. Fowler B. Genetic defects of folate and cobalamin metabolism. Eur J Pediatr 1998; 157:S60-S66.
40. Ogier de Baulny H, Gerard M, Saudubray JM et al. Remethylation defects: Guidelines for clinical
diagnosis and treatment. Eur J Pediatr 1998; 157:S77-S83.
MTHFR Polymorphisms and Disease
52
Eu
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
41. Abeling NGGM, van Gennip AH, Blom H et al. Rapid diagnosis: Basis for a favourable outcome
in a patient with MTHFR deficiency. J Inher Metab Dis 1998; 21:21 Abstract.
42. Sewell AC, Neirich U, Fowler B. Early infantile methylenetetrahydrofolate reductase deficiency: A
rare cause of progressive brain atrophy. J Inher Metab Dis 1998; 21:22 Abstract.
43. Homberger A, Linnebank M, Sewell A et al. Severe methylenetetrahydrofolate reductase deficiency:
Two novel genotypes with different clinical course. J Inher Metab Dis 2001; 24(suppl 1):50 (abstract)
44. Hyland K, Smith I, Bottiglieri T et al. Demyelination and decreased S-adenosylmethionine in
5,10-methylenetetrahydrofolate reductase deficiency. Neurology 1988; 38:459-462.
45. Holme E, Kjellman B, Ronge E. Betaine for treatment of homocystinuria caused by
methylenetetrahydrofolate reductase deficiency. Arch Dis Child 1989; 64:1061-1064.
46. Engelbrecht V, Rassek M, Huismann J et al. MR and proton MR spectroscopy of the brain in
hyperhomocysteinemia caused by methylenetetrahydrofolate reductase deficiency. AJNR 1997;
18:536-539.
47. Kishi T, Kawamura I, Harada Y et al. Effect of betaine on S-adenosylmethionine levels in the
cerebrospinal fluid in a patient with methylenetetrahydrofolate reductase deficiency and peripheral
neuropathy. J Inher Metab Dis 1994; 17:560-565.
48. Marquet J, Chadefaux B, Bonnefont JP et al. Methylenetetrahydrofolate reductase deficiency prenatal diagnosis and family studies. Prenat Diagn 1994; 14(1):29-33.
49. Walk D, Kang S, Horwitz A. Intermittent encephalopathy, reversible nerve conduction slowing,
and MRI evidence of cerebral white matter disease in methylenetetrahydrofolate reductase deficiency. Neurology 1994; 44 (2):344-347.
50. Ronge E, Kjellman B. Long term treatment with betaine in methylenetetrahydrofolate reductase
deficiency. Arch Dis Child 1996; 74:239-241.
51. Tonetti C, Burtscher A, Bories D et al. Methylenetetrahydrofolate reductase deficiency in four
siblings: A clinical, biochemical, and molecular study of the family. Amer J Hum Genet 2000;
9:363-367.
52. Al Essa M, Sakati NA, Dabbagh O et al. Inborn error of vitamin B12 metabolism: A treatable
cause of childhood dementia/paralysis. J Child Neurology 1999; 13:239-243.
53. Al Tawari AA, Ramadan DG, Neubauer D et al. An early onset form of methylenetetrahydroflate
reductase deficiency a report of a family from Kuwait. Brain Development 2002; 24:304-309.
54. Tonetti C, Ruivard M, Rieu V et al. Severe methylenetetrahydrofolate reductase deficiency revealed by a pulmonary embolism in a young adult. Br J Heamatology 2002; 119:397-399.
55. Fattal-Valevski A, Bassan H, Korman SH et al. Methylenetetrahydrofolate reductase deficiency:
Importance of early diagnosis. J Child Neurol 2000; 15:539-543.
56. Abeling NGGM, van Gennip AH, Blom H et al. Rapid diagnosis and methionine administration:
Basis for a favourable outcome in a patient with methylene tetrahydrofolate reductase deficiency. J
Inher Metab Dis 1999; 22:240-242.
57. Arn PH, Williams CA, Zori RT et al. Methylenetetrahydrofolate reductase deficiency in a patient
with phenotypic findings of Angelman syndrome. Amer J Med Genet 1998; 77:198-200.
58. Pasquier F, Lebert F, Zittoun J, Marquet J. Methylenetetrahydrofolate reductase deficiency revealed by a neuropathy in a psychotic adult [letter]. J Neurol Neurosurg Psychiatry 1994;
57(6):765-766.
59. Scott JM, Dinn JJ, Wilson P et al. Pathogenesis of subacute combined degeneration: A result of
methyl group deficiency. Lancet 1981; 2:334-337.
60. Selzer RR, Rosenblatt DS, Laxova R et al. Nitrous oxide and 5,10-methylenetetrahydrofolate reductase deficiency. N Engl J Med 2003; 349:49-50.
61. Goyette P, Rosenblatt DS, Rozen R. Homocystinuria (methylenetetrahydrofolate reductase deficiency) and mutations of Factor V gene. J Inher Metab Dis 1998; 21:690-691.
62. Levitt M, Nixon PF, Pincus JH et al. Transport of folates in cerebrospinal fluid:A study using
doubly labelled 5-methyltetrahydrofolate and 5-formyltetrahydrofolate. J Clin Invest 1971; 50:1301
63. Wendel U, Claussen U, Dickmann E. Prenatal diagnosis for methylenetetrahydrofolate reductase
deficiency. J Pediatr 1983; 102:938-940.
64. Erbe RW. Inborn errors of folate metabolism. In: Blakley RL, Whitehead VM, eds. Folates and
Pterins Nutritional, Pharmacological and Physiological Aspects. New York: Wiley, 1986; 3:413-466.
65. Kluijtmans LAJ, Wendel U, Stevens EMB, van den Heuvel LPWJ, Trijbels FJM, Blom HJ. Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency. Eur J
Hum Genet 1998; 6:257-265.
66. Sakura N, Ono H, Nomura H et al. Betaine dose and treatment intervals in therapy for
homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency. J Inher Metab Dis 1998;
21:84-85.
Severe Methylenetetrahydrofolate Reductase Deficiency
53
Eu
rek ©2
ah 004
Do / La Co
No nde pyri
t D s B ght
ist io
rib sc
ute ien
ce
67. Fowler B, Jakobs C. Post- and prenatal diagnostic methods for the homocystinurias. Eur J Pediatr
1998; 157:S88-S93.
68. Rosenblatt DS, Erbe RW. Methylenetetrahydrofolate reductase in cultured human cells. Growth
and metabolic studies. Pediatr Res 1977; 11:1137-1141.
69. Suormala T, Gamse G, Fowler B. 5,10 methylenetetrahydrofolate reductase (MTHFR) assay in the
forward direction: Residual activity in MTHFR deficiency. Clin Chem 2002; 48:835-843.
70. Mudd SH, Uhlendorf BW, Freeman JM et al. Homocystinuria associated with decreased
methylenetetrahydrofolate reductase activity. Biochem Biophys Res Commun 1972; 46:905-912.
71. Cooper BA. Anomalies congenitales du metabolisme des folates. In: Zittoun J, Cooper BA, eds.
Folates et cobalamines. Paris: Doin, 1987:193-208.
72. Gaull GE, Von Berg W, Raiha NCR et al. Development of methyltransferase activities of human
fetal tissues. Pediatr Res 1973; 7:527-533.
73. Zittoun J. Congenital errors of folate metabolism. In: Wickringamasinghe S, ed. Megaloblastic
anaemias. London: Bailière Tindall, 1995:603-616.
74. Takenaka T, Shimomura T, Nakayasu H, Urakami K, Takahashi K. Effect of folic acid for treatment of homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency [Japanese].
Rinsho-Shinkeigaku-Clinical Neurology 1993; 33(11):1140-1145.
75. Erbe RW. Genetic aspects of folate metabolism. Adv Hum Genet 1979; 9:293-354.
76. Goyette P, Pai A, Milos R et al. Gene structure of human and mouse methylenetetrahydrofolate
reductase (MTHFR). Mamm Genome 1998; 9:652-656.
77. Goyette P, Sumner JS, Milos R et al. Human methylenetetrahydrofolate reductase: Isolation of
cDNA, mapping and mutation identification. Nat Genet 1994; 7(2):195-200.
78. Goyette P, Frosst P, Rosenblatt DS et al. Seven novel mutations in the methylenetetrahydrofolate
reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase
deficiency. Amer J Hum Genet 1995; 56:1052-1059.
79. Goyette P, Christensen B, Rosenblatt DS et al. Severe and mild mutations in cis for the
methylenetetrahydrofolate (MTHFR) gene, and description of 5 novel mutations in MTHFR. Amer
J Hum Genet 1996; 59:1268-1275.
80. Sibani S, Christensen B, O‘Ferrall E et al. Characterization of six novel mutations in the
methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria. Hum Mutat
2000; 15(3):280-7.
81. Sibani S, Leclerc D, Weisberg IS et al. Characterization of mutations in severe methylenetetrahydrofolate
reductase deficiency reveals an FAD-responsive mutation. Hum Mutat 2003; 21(5):509-20.
82. Rosenblatt DS, Lue-Shing H, Arzoumanian A et al. Methylenetetrahydrofolate reductase (MR)
deficiency: Thermolability of residual MR activity, methionine synthase activity, and methylcobalamin
levels in cultured fibroblasts. Biochem Med Met Biol 1992; 47(3):221-225.
83. Frosst P, Blom HJ, Milos R et al. A candidate genetic risk factor for vascular disease: A common
mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111-113.