Homocystinuria: Studies in Tissue Culture

Pediat. Res. 7: 645-658 (1973) Cystathionine synthase fibroblast genetic disease homocystinuria Homocystinuria: Studies in Tissue Culture B. WILLIAM UHLENDORF< 54 J, E. BRINSON GONERLY, AND S. HARVEY MUDD Division of Virology, Bureau of Biologies, and Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland, USA Extract Cystathionine synthase activities are reported for extracts of fibroblasts grown from 39 control subjects, 47 homocystinuric individuals, and 10 parents of cystathionine synthase-deficient patients. Among the group with homocystinuria, fibroblast extracts from 38 had specific activities of cystathionine synthase below the control range. A number of considerations indicate that these 38 patients excrete homocystine because of cystathionine synthase deficiency. Fibroblasts from nine patients with homocystinuria had specific activities of cystathionine synthase within the control range. This group of nine was shown to be comprised of two individuals with cystathionine synthase deficiency, three with deficient activity of methylenetetrahydrofolate reductase, three with deficient activity of iV5-methyltetrahydrofolate-homocysteine methyltransferase, and one in whom homocystine excretion found by others in earlier studies could not be confirmed during the present investigation. The specific activities in fibroblasts of parents of cystathionine synthase deficient patients in most cases fall near the low end of the control range. An assay of increased sensitivity was used to measure the cystathionine synthase activities in extracts of fibroblasts from cystathionine synthase-deficient patients at several concentrations of added pyridoxal phosphate. Of 25 cystathionine synthase-deficient patients judged to be clinically responsive to pyridoxine treatment, 24 had detectable cystathionine synthase activities in fibroblast extracts when the assays were performed without added pyridoxal phosphate. These activities ranged from approximately 0.1 % to 10% of the mean control value, and generally were stimulated no more by the addition of pyridoxal phosphate than were extracts from normal cells. Of 10 cystathionine synthase-deficient patients judged not to be responsive to pyridoxine, 9 had no cystathionine synthase activity in fibroblast extracts detected by the method employed. The cystathionine synthase in the cell extracts of the single nonresponsive patient with significant activity was stimulated much more by in vitro addition of pyridoxal phosphate than was control cystathionine synthase. Genetic heterogeneity in cystathionine synthase-deficient patients and some of the genetic implications of the demonstrated a2/32 subunit structure of mammalian cystathionine synthase are discussed. Speculation Knowledge of enzyme activities, which includes cystathionine synthase, iV5-methyltetrahydrofolate-homocysteine methyltransferase and methylenetetrahydrofolate reductase, in cultured fibroblasts is of diagnostic value in distinguishing between the several genetic abnormalities which may lead to excessive excretion of homocystine in the urine. 645 646 UHLENDORF, CONERLY, AND MUDD Clinical response of cystathionine synthase-deficient patients to pyridoxine therapy is thought to be due to a several-fold increase of residual cystathionine synthase activity. This increase occurs in the patient by an unknown mechanism, but in general is not caused by an unusual stimulation of residual enzyme activity by high concentrations of pyridoxal phosphate. Introduction Patients with a genetically determined deficient activity of cystathionine synthase, the enzyme which catalyzes the condensation of homocysteine and serine to form cystathionine (reaction 4, Fig. 1), excrete excessive amounts of homocystine in their urine [4, 7, 14, 26, 30, 38], The term "homocystinuria" has often been used to designate this genetic disorder, but this restricted use of the term is no longer appropriate because it is now known that excessive homocystine excretion (i.e., homocystinuria) may occur in a number of other situations. For example, homocystinuria accompanies any condition in which the rate of homocysteine methylation catalyzed by iV5-methyltetrahydrofolate-homocysteine methyltransferase (reaction 8, Fig. 1) is sufficiently decreased. There are conditions known to decrease pathologically the rate of this methyl transfer reaction. (1) Failure to form iV5-methyltetrahydrofolate, the methyl donor for the enzyme in question, decreases the rate of transfer. This failure is due to deficient activity of methylenetetrahydrofolate reductase (reaction 18, Fig. 1) [35]. (2) Failure to form methyl-B12, a cofactor required by iV6-methyltetraliydrofolate-homocysteine methyltransferase may also be responsible. This failure may be the result of either B12 deficiency, occurring, for example, in familial gastrointestinal malabsorption of B12 [15, 17], or the result of impaired cellular uptake or metabolism of B12 (deficiency in reaction pathway 16, Fig. 1) [23, 33, 34, 36]. In addition to these genetic conditions, another situation leading to excessive homocystine excretion is the administration of 6-azaurdine triacetate [16]. The mechanism of this drug effect has not been clarified. From the above considerations, and in accord with a suggestion first published in 1964 [4], we prefer to use the term "homocystinuria" not as the name of a particular disease, but rather "to denote excretion of [excessive] homocystine in the urine, without etiologic connotation." The specific diseases which include homocystinuria among their manifestations may then be termed "cystathionine synthase deficiency," "methylenetetrahydrofolate reductase deficiency," and so on. For the sake of gaining further understanding of the various diseases in question, and in the interest of rational patient management [12], it is now necessary to define the etiology of each case of homocystinuria. Of the many lines of evidence which may assist in distinguishing between the various causes of homocystinuria, the least equivocal is a direct demonstration of deficient activity of a specific enzyme. Unfortunately, studies of cystathionine synthase activity in humans have been limited by the restricted distribution of this enzyme, which is found in liver and brain, but not in more accessible tissues [30, 31, 42]. To meet the need for a readily available experimental system, we turned to the use of tissue culture and found that fibroblasts grown from biopsies of normal human skin contain easily detectable concentrations of cystathionine synthase, whereas those grown from several homocystinuric patients had markedly decreased, or absent, activity of this enzyme [42]. This tissue culture system has provided a convenient means to study cystathionine synthase from normal and homocystinuric subjects. We have subsequently investigated lines of fibroblasts from more than 80 individuals, including 47 hom (...truncated)