Molecular analysis of the cystathionine ß-synthase gene

Abstract

Homocysteine is a sulfur containing amino acid that results from the demethylation of methionine. Metabolic pathways utilising homocysteine include remethylation to methionine and transsulfuration to cysteine. Disruption o f these pathways leads to an abnormal accumulation of homocyst(e)ine in the blood (hyperhomocyst(e)inemia). Severe hyperhomocyst(e)inemia causes excretion o f large amounts o f homocystine into the urine (homocystinuria). Clinical features associated with homocystinuria include mental retardation, dislocation o f the optic lens, osteoporosis, thinning and lengthening o f the long bones, and thrombotic tendencies. Mild hyperhomocyst(e)inemia usually does not cause the clinical symptoms associated with homocystinuria. However, mild hyperhomocyst(e)inemia has been suggested as a risk factor for occlusive vascular disease. Cystathionine s-synthase (CBS) is an enzyme o f the transsulfuration pathway condensing homocysteine with serine to form cystathionine. The CBS enzyme is a homotetramer o f 63 kDa subunits which binds its two substrates, homocysteine and serine, and three additional ligands: pyridoxal 5 '-phosphate (vitamin B6), S-adenosylmethionine, and haem. The gene that encodes CBS maps to human chromosome 21 at position q22.3. This gene contains a protein coding region o f 1653 nucleotides which is translated into the CBS subunit o f 551 amino acids. Partial deficiency of CBS is one of several factors which may cause mild hyperhomocyst(e)inemia. Severe deficiency of CBS is the most common inherited cause of homocystinuria. The incidence of heterozygous CBS deficiency varies geographically and is estimated to be approximately 1 % in the general population. It has been suggested that these individuals may account for a large percentage of those at risk for occlusive vascular disease. However, testing this hypothesis has proven difficult due to the lack of an accurate assay to detect heterozygotes. Treatments for CBS deficiency are aimed at lowering plasma homocyst(e)ine levels. These treatments include simple dietary supplementation with pyridoxine (vitamin B6 precursor) and patients are classed as pyridoxine responsive or non- esponsive. The availability of potentially effective treatments for hyperhomocyst(e)inemia underscores the need for improved methods of detecting individuals who are heterozygous for CBS deficiency. More recently, the application of molecular biology techniques has permitted the identification of CBS mutations in patients with homocystinuria and in their heterozygous family members. Identification of mutations responsible for CBS deficiency offers the potential for therapeutic intervention and the possible prevention of premature vascular disease. This thesis describes the identification of genetic lesions responsible for defective CBS enzyme in five patients with homocystinuria. Messenger RNA was isolated from in vitro cultured fibroblasts and/or lymphoblasts derived from patients. The mRNA was reverse transcribed to cDNA. Direct DNA sequence analysis of PCR amplified CBS cDNA identified the individual mutations. These mutations were all single nucleotide changes which predict an amino acid substitution in the primary sequence of the CBS protein subunit. Four additional patients with intermediate hyperhomocyst(e)inemia and reduced CBS activity in extracts from their cultured fibroblast cells, were also screened for CBS mutations. However no mutations that cause abnormal catalytic function were detected in the CBS coding region. These four patients were also screened for a reported mutation in the gene for methylenetetrahydrofolate reductase (MTHFR). Two of the four patients were homozygous for this known mutation which confers thermal instability to the MTHFR protein (another enzyme involved in the metabolism of homocysteine). Knowledge of the individual CBS mutations was used to accurately identify potential heterozygous family members, at risk of premature vascular disease. Genomic DNA was extracted from blood samples derived from family members. Portions of CBS genomic DNA were amplified by PCR and used to screen for the individual mutations by one or more of the following techniques: direct DNA sequencing, restriction enzyme analysis, dideoxy fingerprinting or a combination of single strand conformation polymorphism and temperature gradient gel electrophoresis. Appropriate segments of patient CBS cDNA, containing the defined mutation, were amplified by PCR and used to replace corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7.7. These recombinant mutant and normal CBS constructs were expressed in E. coli and the catalytic activities of the mutant proteins were compared with normal. Differences in in vitro catalytic capacity confirmed the association between the individual mutations and CBS dysfunction in vivo.