Methylenetetrahydrofolate reductase (MTHFR) synthesizes 
5-methyltetrahydrofolate, a major methyl donor for homocysteine remethylation to 
methionine. Severe MTHFR deficiency results in marked hyperhomocysteinemia and 
homocystinuria. Patients display developmental delay and a variety of 
neurological and vascular symptoms. Cloning of the human cDNA and gene has 
enabled the identification of 29 rare mutations in homocystinuric patients and 
two common variants [677C>T (A222V) and 1298A>C (E429A)] with mild enzymatic 
deficiency. Homozygosity for 677C>T or combined heterozygosity for both 
polymorphisms is associated with mild hyperhomocysteinemia. In this 
communication, we describe four novel mutations in patients with homocystinuria: 
two missense mutations (471C>G, I153M; 1025T>C, M338T), a nonsense mutation 
(1274G>A, W421X), and a 2-bp deletion (1553delAG). We expressed the 1025T>C 
mutation as well as two previously reported amino acid substitutions [983A>G 
(N324S) and 1027T>G (W339G)] and observed decreased enzyme activity at 10%, 36%, 
and 21% of control levels, respectively, with little or no effect on affinity 
for 5-methyltetrahydrofolate. One of these mutations, 983A>G (N324S), showed 
flavin adenine dinucleotide (FAD) responsiveness in vitro. Expression of these 
mutations in cis with the 677C>T polymorphism, as observed in the patients, 
resulted in an additional 50% decrease in enzyme activity. This report brings 
the total to 33 severe mutations identified in patients with severe MTHFR 
deficiency.