We carried out a GWAS of serum AST activity levels in 866 Amish subjects from the HAPI Heart Study. Characteristics of study participants are summarized in . In the GWAS, the strongest SNP associations with serum AST levels were observed for a group of SNPs located on chromosome 10q24.1. The peak association was with rs17109512 (P-value=2.80E-14), in the vicinity of GOT1 (~1 Mb downstream), the gene encoding cAST (). The frequency of the associated SNP is rare (MAF=0.006), with only 10 heterozygotes for rs17109512 found in the HAPI Heart Study. The 10 heterozygotes had significantly lower AST levels compared with the 856 homozygotes of the wide-type allele (mean±s.d. of AST: 10.0±2.8 versus 18.8±5.2 U l−1, P=2.80E-14).
Clinical characteristics of the HAPI Heart Study participants
Figure 1 Genome-wide association analysis plot for serum AST levels in 866 Old Order Amish subjects. Arrow indicates that SNP rs17109512 on chromosome 10q24.1 is highly associated with serum AST activity (P=2.80E-14). A full color version of this figure is available (more ...)
Given the low frequency of the associated SNP, its proximity to GOT1, and the very large effect size of the association, we hypothesized that rs17109512 might tag a functional SNP, most likely a mutation changing the protein sequence of cAST, in GOT1. We subsequently sequenced the gene and identified an in-frame deletion of three nucleic acids encoding asparagine at position 389 in GOT1 gene (). All of 10 heterozygotes for rs17109512 were heterozygotes for c.1165_1167delAAC. The deletion c.1165_1167delAAC (p.Asn389del) was in complete linkage with rs17109512. By constructing the pedigree, all the deletion carriers can be traced back to one most likely founder who was born in mid-18th century, suggesting a founder effect. Genotyping of 647 non-Amish Caucasians from Baltimore area did not identify carriers of this deletion.
Figure 2 Detection of GOT1 (c.1165_1167delAAC) mutation. (a) Sequencing identified an in-frame deletion of three nucleic acids encoding asparagine at position 389 in GOT1 gene. (b) Asparagine (N, indicated by arrow) is conserved in cAST protein sequences among (more ...)
Glutamine at amino acid at 389 is conserved among known cAST of vertebrate species, including the human, chimpanzee, mouse and zebrafish (). Further genotyping of deletion c.1165_1167delAAC (p.Asn389del) in an additional 1,932 Amish samples identified 20 more carriers (MAF=0.0052), among which 14 had phenotype measurements. No homozygotes for the deletion were found. Phenotype comparison between the 24 (10 from the HAPI Heart Study plus 14 from the additional screening) c.1165_1167delAAC (p.Asn389del) carriers and 856 non-carriers (from the HAPI Heart Study) is shown in . The deletion carriers had serum AST activity levels that were approximately one-half that of normal homozygotes, suggesting that the deletion resulted in a complete loss of enzymatic function. Despite this large difference in AST activity, we did not find any association between the mutation and metabolic traits, including serum fasting glucose or insulin, fasting lipids (). Neither did we find any association with post-meal lipids, inflammatory markers or sub-clinical markers of cardiovascular disease (data not shown).
Clinical characteristics of the GOT1 c.1165_1167delAAC carriers and non-carriers
To further examine the functional consequences of p.Asn389del, we conducted in vitro functional studies by expressing the recombinant wild-type and mutant cAST (p.Asn389del) in the mammalian kidney cell line HEK293 and measuring AST activity. As shown in , the basal activity of the cells transfected with empty vector (lane 1) was barely detectable. AST activity levels were increased by >100 fold in a dose-dependent manner when the cells were expressing wild-type cAST (lanes 2 and 3). By contrast, lysates from cells transfected with mutant cAST (lanes 5 and 6) had almost no AST activity, suggesting that the mutant cAST is enzymatically inactive. Western analysis was conducted to determine the levels of recombinant protein expression in the cells. As shown in , mutant cAST only was barely detectable, compared with the wild type (lanes 5 and 6 versus 2 and 3). Interestingly, the activities from the cells transfected with wild-type and mutant cAST at a 1:1 ratio were less than half of that of wild-type AST (lane 4 versus 3; P<0.001), suggesting a dominant negative effect. Western analysis revealed that less mutant cAST protein was expressed after correction for GAPDH, suggesting a decreased expression, probably due to decreased RNA stability and/or increased protein degradation (). However, even after correcting for AST protein, mutant cAST had markedly decreased activity relative to wild-type cAST (lane 2 versus 5, P<0.001).
Figure 3 Functional study of recombinant mutant and wild-type cAST proteins. (a) AST activities were measured in lysates of HEK293 cells transfected with empty vector (Vector), wild-type (cASTwt), or p.Asn389del mutant (cASTmut) at the indicated amount in a six-well (more ...)