Cystathionine γ-lyase: Clinical, metabolic, genetic, and structural studies
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].
Section snippets
Terminology
DNA and protein sequence variants are described as recommended by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/recs-DNA.html and http://www.hgvs.org/mutnomen/recs-prot.html). The accession number for the CTH genomic sequence is AL354872. The single nucleotide polymorphism in CTH, termed in this paper c.1208G>T (p.Ser403Ile), was originally described as c.1364G>T (p.Ser403Ile) [7]. The nucleotide number has been changed here to agree with the current numbering convention.
Patients
Many of the available metabolic and genetic data for the individuals covered in this paper (six with severe hypercystathioninemia and three members of a family with mild elevations of plasma cystathionine and only a heterozygous CTH mutation) are presented in Table 1, Table 2, with the subjects listed in descending order according to their most recent plasma cystathionine concentrations. The numbers designating these individuals are arbitrary, having been assigned by the laboratories in which
Results and discussion
In the following sections we discuss: (a) the identification of novel mutations and deletions in CTH among the patients reported upon in this paper; (b) the effects each of these novel mutations, as well as those previously characterized by Wang and Hegele [5] and Zhu et al. [6] on enzyme activity (Fig. 2) and clinical responsiveness of persons carrying these mutations to dietary B6 supplementation; (c) the crystal structure of hCTH; and (d) the structural changes due to the mutations under
Acknowledgments
The authors thank Dr. William M. Sugden, Wisconsin Alumni Research Foundation for establishing the immortalized line of lymphocytes from patient #3051. The plasmid pET22b+hCTH was a generous gift from Dr. Markus Wahl. We are grateful to Dr. Harvey Levy for help in gathering patient information. This work was supported by grants from the American Heart Association (AHA 2-5-80663), from the NIH (HL065217-06A2 and AG09834) and Jerome Lejeune foundation to J.P.K, by grants from the Grant Agency of
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