Human S-adenosylhomocysteine hydrolase: common gene sequence variation and functional genomic characterization

Abstract

S-Adenosylhomocysteine hydrolase (AHCY) is the only mammalian enzyme known to catalyze the hydrolysis of S-adenosylhomocysteine. We have used a genotype-to-phenotype strategy to study this important enzyme by resequencing AHCY in 240 DNA samples from four ethnic groups. Thirty-nine polymorphisms were identified - 28 of which were novel. Functional genomic studies for wild type AHCY and the three variant allozymes identified showed that two variant allozymes had slight, but significant decreases in enzyme activity, but with no significant differences in levels of immunoreactive protein. Luciferase reporter gene assays for common 5'-flanking region haplotypes revealed that one haplotype with a frequency of approximately 2% in Caucasian-American subjects displayed a decreased ability to drive transcription. The variant nucleotide at 5'-flanking region single nucleotide polymorphism (SNP) (-34) in that haplotype altered the DNA-protein binding pattern during electrophoresis mobility shift assay. Finally, an AHCY genotype-phenotype association study for expression in lymphoblastoid cells identified four SNPs that were associated with decreased expression. For the IVS6 (intervening sequence 6, i.e., intron 6) G56 > C SNP among those four, electrophoresis mobility shift assay showed that a C > G nucleotide change resulted in an additional shifted band. These results represent a step toward understanding the functional consequences of common genetic variation in AHCY for the regulation of neurotransmitter, drug and macromolecule methylation.

Figure 1

The Methionine Cycle. AHCY = S-adenosylhomocysteine hydrolase; AD = adenosine deaminase; BHMT = betaine homocysteine methyltransferase; CBS = cystathione β-synthase; MAT = methionine adenosyltransferase; MTR = 5-methyltetrahydrofolate-homocysteine methyltransferase; MTRR = 5-methyltetrahydrofolate homocysteine methyltransferase reductase; MT = methyltransferase.

Figure 2

Human AHCY gene polymorphisms. The figure shows a schematic representation of the human AHCY gene structure, with arrows indicating the locations of polymorphisms. Black rectangles represent exons encoding the open-reading frame (ORF), open rectangles represent portions of exons encoding untranslated region (UTR) sequence, and the grey rectangle represents coding region when exon 1B is transcribed. “Indel” represents an insertion/deletion event. The colors of arrows indicate minor allele frequencies.

Figure 3

AHCY allozyme functional genomics. (A) AHCY allozyme enzyme activity. AHCY activity was assayed in the hydrolase direction with 50 μM AdoHcy as substrate. Activities are expressed as a percentage of WT. Values shown are mean ± SD for 3 determinations, * p<0.001 by ANOVA. (B) AHCY allozyme immunoreactive protein levels. Protein levels are expressed as a percentage of WT. Values shown are mean ± SD for 3 determinations, p=0.11 by ANOVA. (C) A representative Western blot. Cells transfected with empty vector were assayed as a negative control (NC).

Figure 4

Human AHCY 5′-FR reporter gene and EMS assays. (A) and (B) AHCY 5′-FR reporter gene constructs were used to transfect HEK293T and HepG2 cells. Luciferase activity is expressed as a percentage of the value for WT constructs. Each bar represents the average of 6 independent experiments (mean ± SD), * p<0.001 by ANOVA (C) Human AHCY 5′-FR C(-34)T SNP effect on nuclear protein binding. Nuclear extract from HEK293T and HepG2 cells was incubated with the WT probe containing C(-34), or with the variant probe containing T(-34).

Figure 5

Human AHCY intron 6 reporter gene and EMS assays. (A) Effect of human AHCY intron 6 C(56)G SNP on nuclear protein binding. Nuclear extract from HEK293T and HepG2 cells was incubated with a WT C56 intron 6 probe, or with a variant G56 intron 6 probe. (B) and (C) AHCY intron 6 reporter gene studies. Constructs were used to transfect HEK293T and HepG2 cells. Luciferase activity is expressed as a percentage of the value for the WT construct. Each bar represents the average of 3 independent experiments (mean ± SD).