Cystathionine γ-lyase: Clinical, metabolic, genetic, and structural studies

Abstract

We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine γ-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B₆ administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects.

Introduction

Cystathioninuria or hypercystathioninemia may be due to a variety of causes. Less marked elevations of urinary excretion and/or plasma concentrations occur with prematurity, deficiencies of vitamins B₆, B₁₂, or folic acid, neural tumors that produce cystathionine, a renal defect in the transport of this amino acid, or in genetically determined defects in the conversion of homocysteine to methionine. In the latter situation the excessive accumulation of homocysteine leads to an abnormally high rate of production of cystathionine by cystathionine β-synthase. These conditions have been reviewed in more detail elsewhere [1], [2]. More marked cystathioninuria and hypercystathioninemia of genetic origin is caused by mutations that decrease the activity of cystathionine γ-lyase (CTH; EC 4.4.1.1)¹, the enzyme that catalyzes the conversion of cystathionine to cysteine, ammonia, and 2-oxobutyrate. The initial patient in whom the metabolic findings strongly suggested a CTH defect was reported in 1959 by Harris and colleagues [3], and by 1983 a survey of published cases listed briefly the metabolic and clinical findings in a total of 47 such cases with gross cystathioninuria thought to be of genetic origin [4]. However, among these cases the diagnosis had been confirmed in only ten by assay of CTH activity (using extracts of either liver or long-term lymphoid cell lines). Pyridoxal 5′-phosphate (PLP) is a cofactor for CTH. The effect of administration of oral pyridoxine (vitamin B₆) on cystathionine excretion had been tested in 37 of the 47 individuals mentioned above. Most (33/37) showed marked decreases. The CTH activities in tissue extracts from B₆-responsive individuals has been stimulated by the in vitro addition of PLP by between 1.3-fold and as much as 50-fold [4]. It was not until five years ago that the first report of identification of CTH mutations in cystathioninuric individuals was published. In four individuals two frame shift mutations, c.940_941delCT (p.Leu262ThrfsX20), and c.1220delC (p.Thr355IlefsX18) and two missense mutations, c.200C>T (p.Thr67Ile) and c.718C>G (p.Gln240Glu) were found, occurring in either homozygous or compound heterozygous forms [5]. The two missense CTH mutations have very recently been expressed in Escherichia coli and the mutant proteins purified and characterized with regard to their kinetic properties, the amounts of PLP they bind, and the effects of preincubation with PLP upon enzyme activity [6]. In the present paper we report clinical, metabolic, and molecular genetic studies of several additional cystathioninuric persons, among whose CTH genes three novel missense mutations, one of the previously reported alterations, and a novel large deletion have been identified. We have also further characterized in a large group of Czech individuals the effects of a CTH polymorphism previously studied in Canadian individuals and found to cause mild elevations of plasma total homocysteine (tHcy) [7].