Future Studies of Low-Activity PON1 Phenotype Subjects May Reveal How PON1 Protects Against Cardiovascular Disease

Human PON1 Polymorphisms and Point Mutations

Our knowledge of the polymorphic sites for human PON1 has recently been greatly expanded. A major contribution has been the complete sequencing of the PON1 gene from 23 individuals (46 chromosomes) from the genetic collection of the Centre d’Etude du Polymorphisme Humaine (CEPH) (Jarvik et al²). These data, with other recent additions, represent a repository (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusid=5445), which now includes nearly 200 polymorphic sites in the PON1 gene sequence. Of these, 7 are in the 5′-untranslated region, 171 are in intronic regions, and 15 are in the 3′-untranslated region. In addition to the 2 common exonic polymorphic sites (Leu55Met) in exon 3 and (Arg192Gln) in exon 6, there are at least 3 structural variants within the coding region. The latter mutations are: (Trp194stop) in exon 6,² (Arg160Gly) in exon 5 found in the Chinese Han population (Wang et al³), and the Finnish mutant (Ile102Val) in exon 4 (Marchesani et al⁴), which appears to be associated with low PON1 activity (see Tables 1 and 2⇓). Of course, the number of individual PON1 genes sequenced, to date, is still very small, and as the number grows, more examples of PON1 variants will certainly be discovered. We can also expect to find that some of the “polymorphisms” will have to be reclassified as rare mutations, as additional sequencing information becomes available.

Fortunately, PON1 activity can conveniently be directly measured in the serum or plasma. Also, the enzyme is very stable in serum properly frozen for several years. The enzyme is well preserved in heparinized plasma, though less so when citrate is used as the anticoagulant, but it is quickly and irreversibly inactivated by EDTA. Opinions differ today, however, about which PON1 substrates should be used in correlative studies with CVD. Arylesterase activity with phenyl acetate gives about equal weight to both the Q and R isozymes, because both have about the same specific activity. Diazoxon hydrolysis (DZOase) favors somewhat the Q isozyme, and paraoxon hydrolysis (POase) considerably favors the R isozyme; the degree of this bias depends on the pH selected for the assay, as well as the salt concentration. Since it is still not known what substrate best represents the “protective” role of PON1 in connection with cardiovascular disease, this important issue remains unresolved (see Navab et al⁵ and Mackness et al⁶ in the Costa and Furlong book⁷). Jarvik et al¹ used all three substrates and found the correlation with CVD to be best with DZOase activity. However, it is not clear whether this means that the Q isozyme is intrinsically more protective than the R isozyme, or perhaps the Q isozyme is a little more stable during its protective reaction.

Future Directions

The conclusion in the article by Jarvik et al¹ that the level of PON1 activity is the best predictor of vascular disease should encourage other workers in this field to continue in this direction, rather than search for key SNPs to advance the field, at this time. In the past analysis of pharmacogenetic and hereditary metabolic conditions, it generally has been found most profitable to first make use of patient phenotypes, use these to identify the critical genotypes, and then determine the molecular basis of the association (rather than expect to reach this goal directly by genotypic surveys with SNPs.). The importance of focusing on phenotypes in such clinical investigations has recently been well defended by Weiss and Buchanan.⁸

We anticipate that there are a number of critical mutations and some polymorphic sites that exert major influences on the level and quality of PON1 enzymatic activity. The Jarvik group selected several people who have relatively low arylesterase or DZOase activity and found them to be heterozygous for a defective PON1 variant.² If the relationship between the level of PON1 activity and cardiovascular pathology holds up, people with relatively low PON1 enzymatic activity will be found in higher numbers among the CVD patients. Our laboratory has found one such older male cardiovascular disease patient with no detectable serum paraoxonase or arylesterase activity, so we can be sure that a genetic defect resulting in a complete lack of PON1 enzymatic activity can be tolerated in man, just as has been observed for the PON1 knockout mice.⁹

Our surrogate substrates for PON1 activity should still be useful in identifying low-activity phenotypes so these can be characterized, and family studies be undertaken to look for the rarer homozygous or compound heterozygous individuals with essentially no PON1 enzymatic activity. Eventually, we will need new substrates more indicative of the protective role that PON1 seems to play in protecting against oxidative damage in vivo, and switch to these assays later, rather than rely on the standard ones for PON1 hydrolytic activity. The standard hydrolytic assays will serve, for now, to identify gross reductions in enzymatic protein, even though they cannot be expected to measure the special qualitative properties of the enzyme required for protection against oxidative damage in cardiovascular tissues.