Concordant genetic and physiological data are required for candidate genes.

Concordant Genetic and Physiological Data Are Required for Candidate Genes To the Editor: In a recent article, Chen et al present additional data on differences in the biochemical and physiological properties of the inducible form of nitric oxide synthase (NOS2) between Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. The data establish that the enzyme responds differently to increasing concentrations of L-arginine with regard to nitrite production, such that the S enzyme is less active at a given substrate concentration than the R enzyme. This makes sense as a potential mechanism for hypertension in S rats, which would be expected to produce less NO. Also, there was a single base change in the coding region of NOS2, resulting in an amino acid substitution and a DNA restriction fragment length polymorphism (RFLP); this RFLP appeared to be unique to S rats among the several strains tested. The question arises as to the causative relationship between the above interesting changes and blood pressure differences between S and R rats. In the last paragraph of their article, Chen et al provide a rather misguided attempt to reconcile their biochemical/physiological data with our genetic data. Chen et al quote our early genetic linkage data using F2 populations derived by crossing S with Wistar-Kyoto rats (WKY) or crossing S with the Milan normotensive strain (MNS) that localizes a blood pressure quantitative trait locus (BP QTL) to a region of rat chromosome 10. This region does include NOS2 and did initially support NOS2 as a locus causing inherited differences in blood pressure. Subsequent data using congenic strains, however, definitively ruled out NOS2 as a locus causing inherited differences in blood pressure between S and MNS. Congenic strains substituting a relatively large region of chromosome including NOS2 from MNS into the S background showed a marked reduction in blood pressure compared with S. However, when a congenic substrain was constructed to include only the MNS-derived region containing NOS2 and small flanking chromosomal segments on either side of NOS2, the blood pressure effect was lost, effectively eliminating NOS2 as a candidate for the QTL between S and MNS and localizing the QTL to an adjacent chromosomal segment. This work was published in April 1997 but was not quoted by Chen et al. Chen et al correctly quoted our result that NOS2 in an F2 population derived from S and R rats does not cosegregate with blood pressure. The most logical reason that NOS2 cosegregates with blood pressure in F2 (S3MNS) and not in F2 (S3R) is that the different normotensive strains introduce different alleles at the BP QTL and/or different genetic backgrounds. If S and R are not polymorphic for the QTL on chromosome 10, it follows that the physiological/biochemical differences observed by Chen et al in NOS2 between S and R have nothing to do with inherited differences in blood pressure. Chen et al reject this obvious and logical explanation in favor of an explanation based on spurious logic. They state that “the unique presence of the NOS2 RFLP in the S strain, but not in other strains of rat including WKY, suggests that the normotensive strain that was used to produce the F2 cross with S rats was not the factor that produced different results with respect to cosegregation of S NOS2 with blood pressure.” Basically, they are stating that strain associations (in this case, the NOS2 RFLP association with blood pressure across several strains) are more powerful than linkage analysis on hundreds of rats. Such strain comparisons for drawing cause and effect conclusions between traits and blood pressure have plagued the hypertension community for decades and were shown to be spurious 15 years ago. Unfortunately, enzymatic differences and an RFLP in NOS2 that make biological sense as regulators of genetic differences in blood pressure by themselves prove absolutely nothing about NOS2 as a BP QTL without genetic evidence to corroborate the idea. So far, the genetic evidence is solidly against that proposition. It may be useful to realize that 1 in every 1500 bases is likely to be different between the inbred S and R strains. Because the mammalian genome is 3310 bases in size, there will be '2310 such differences, some of which are bound to cause interesting biological differences that have nothing to do with blood pressure. Systematic genetic analysis is the only way to sort the wheat from the chaff, and the genetic data cannot be dismissed as was done by Chen et al. In summary, there are nice physiological/biochemical differences between S and R in the NOS2 enzyme, but these are not supported as a causative factor in inherited hypertension by genetic linkage analysis in S and R rats. In a comparison of S and MNS, there is a major BP QTL near NOS2, but NOS2 has been eliminated as the QTL in that strain comparison by use of congenic strains.