Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for 
homocysteine remethylation to methionine. Severe MTHFR deficiency is associated 
with hyperhomocysteinemia and homocystinuria. These patients show a wide variety 
of neurological and vascular symptoms, with variable age of onset. Residual 
enzyme activity is usually less than 20% of control values, and correlates 
reasonably well with age of onset of symptoms. A milder deficiency of MTHFR, 
with 30%-50% residual enzyme activity and increased enzyme thermolability, has 
been described as a risk factor for vascular disease and for neural tube 
defects. In earlier work, we isolated the human cDNA for MTHFR, and reported 14 
mutations in severe MTHFR deficiency, as well as a common 677C-->T missense 
mutation (Ala-->Val) that encodes the thermolabile MTHFR. This variant has also 
been observed in some patients with severe MTHFR deficiency, in cis with their 
severe mutations. We report here the in vitro expression of seven severe MTHFR 
mutations in a bacterial expression system; six of these were expressed in cis 
with the Val allele to mimic the situation in the patients. We show that three 
of these constructs have significantly reduced enzyme activity (<10% of 
control); the presence of the thermolabile variant in these patients in cis is 
unlikely to affect enzyme function since activity is already low. One mutation 
causes a dramatic increase in activity when it is expressed in cis with the Ala 
allele, but is associated with extreme lability when in cis with the Val allele. 
Three mutations cause moderate decreases in enzyme activity, with a further 
decrease in activity when they are in cis with the Val allele. We hypothesize 
that deleterious mutations which alter stability may be compromised to a greater 
degree when the thermolabile variant is present on the same allele.