The data presented is the result of sustained applications of substitution mapping for over a decade and constitutes the first report of a BP QTL within a very short congenic segment, containing variants within a single rat gene, Adamts16. Similar associations of variants within human ADAMTS16 that were prevalent in two independent populations provide the rationale for further functional analysis of ADAMTS16 in the context of BP regulation and EH. Additional replications would be required to confirm these associations in humans.
Previously we inferred that QTL2 was localized to a 2.73 Mb region on the basis of the differential segment between two congenic strains, one with a BP effect and the other without (16
). By capturing the QTL in its entirety in two congenic substrains, which can be considered as replicated experiments, this QTL was shown to exert a phenotypic effect independently, i.e. without genetic interaction from introgressed LEW alleles of the previously reported strain with the BP effect, S.LEW (D1Mco4x1x3A) (16
Data presented in Figure allowed us to compare the results of substitution mapping to the evidence obtained by genetic linkage analysis of the same region. It is interesting to note that the location of the QTL gene under study here is at some distance from the confidence intervals of LOD peaks for linkage to BP in the F2 (S × LEW) population. This phenomenon is known from theoretical work on simulations of linkage analysis involving two QTLs on the same chromosome and the creation of a ‘ghost’ LOD peak between them (29
). This artifact occurs when two QTLs are ~40 cM apart and when the plus alleles at both QTLs are on the same chromosome, and the contrasting minus alleles are on the other chromosome. In the present case, the two QTLs on rat Chromosome 1 [QTL2 under consideration here and another adjacent QTL called QTL1 (32
)] are configured such that the LOD peak is generated between them, rather than directly over either of them. Other segregating populations, in which QTL1 is presumably not polymorphic, show excellent alignment of QTL2 with the LOD peak (Fig. ). The above situation reiterates the difficulty of mapping QTL genes solely based on linkage analysis as was often done in humans and exemplifies the power of substitution mapping in animal models to resolve QTLs by isolating them as ‘monogenic’ QTL effectors. The advantage of this approach is shown by the data presented here, because the fine-mapped BP QTL did not contain any gene with previously known function related to BP.
The accuracy of mapping QTLs and detecting underlying genetic determinants depends heavily on the quality of assembly of genomes and gene predictions. Early impediments to the positional cloning effort described in this report included the incorrect assignment of the QTL region to rat Chromosome 17 and multiple, discordant gene predictions of Adamts16. The data presented in Figure and Table resolved the error in sequence assembly by correctly placing the region spanned by markers D1Rat211 and D1Rat12 on Chromosome 1. Similarly, the biological data that we have obtained allowed for solving the discrepancies among the available predictions for gene and transcript sequences of rat Adamts16. The most recent version of the predicted Adamts16 gene at the NCBI rat genome resources website (Build 4.1) concurs with the evidence that we have provided in the present study.
It is interesting to note that in addition to other tissues, Adamts16
is expressed in the kidney, which is a major organ involved in BP homeostasis. The function of Adamts16 is unknown (http://rgd.mcw.edu/tools/genes/genes_view.cgi?id=1310046
). Based on the roles of the ADAMTS family members and based on the presence of a metalloproteinase domain and intact active site sequence HESGH+HD within Adamts16, it is likely that Adamts16 functions as a metalloproteinase in vivo
. The non-synonymous SNPs reported in this study do not reside within the metalloproteinase domain of Adamts16. However, they are located in potentially critical regions of the protease, since furin cleavage is known to relieve enzyme latency of other members of the Adamts family of proteins (33
), and the thrombospondin repeat domain is known to influence substrate binding and proteolysis. As the SNPs within the nucleotides coding for the furin cleavage site did not account for changes in efficiency of cleavage by furin, it is possible that the full-length protein may be required for eliciting a differential functionality of Adamts16
variants. For example, a signal peptide mutation in ADAMTS10 causing Weill–Marchesani syndrome was found to have different effects on ADAMTS10 Pro-Cat and full-length ADAMTS10 (39
). Alternately, the ADAMTS16 thrombospondin-motifs, one of which is affected in the second non-synonymous variant (+3508), reported in our study could have key biological activities. Since a substrate is not known for Adamts16, these possibilities are currently unexplored and beyond the scope of the present work. Regardless of the functional alterations, we hypothesized that alterations of the renal transcriptome as a consequence of altered functions of Adamts16 could be important in understanding the potential link between the mechanism of action of this gene product and the physiology of BP regulation. Because the renal transcriptome of the congenic strain may not be the exclusive representation of the downstream effectors regulated by Adamts16
, but may rather represent the net effect of the introgressed congenic segment, we compared the results of our study to the ‘biological signature’ obtained by knock-down of Adamts16
. Overlapping, multiple networks related to BP and organogenesis were perturbed in both cases. The precise mechanism through which such changes in expression lead to the alteration in BP remains to be determined in this model. The results suggested interesting gene targets for BP regulation such as histone deacetylases Hdac5
, genes involved in epigenetic regulation. Taken together, the results are highly supportive of the kidney as one of the sites of action through which variants of Adamts16
participate in the regulation of BP.
Considering the difficulty of defining what constitutes a good standard for identifying a gene underlying a QTL, Glazier et al
. have suggested some criteria (40
). In a recent review on the genetic dissection of hypertension (41
), Cowley indicated that the gold standard for validation of a potential candidate gene for hypertension is that the rescue of the phenotype must be done in the same species and with the same genetic background that was used to identify the QTL. Our study almost meets this ‘gold standard’ because (i) The QTL is fine-mapped to a small region solely based on natural recombination events, thus closely mimicking the performance of a natural allele in the rat as opposed to artificially genetically manipulated systems such as transgenics and knock-ins; and (ii) we are able to map the QTL to a gene-sparse region as confirmed by comparative mapping in rats, mice and humans (16
), thus allowing for testing of the two expressed candidate transcripts, only one of which had coding sequence variants. One could argue that in addition to the variants within Adamts16
, there may be other variants within inter-genic or intra-genic regions of the critical QTL interval that contribute to the QTL effect. Like in any positional cloning study, regardless of any criteria, such possibilities cannot be ruled out. However, with the view of improving the clinical management strategies for high BP through the identification of novel and effective drug targets, the current study in rats as model organisms with follow-up analyses in independent association studies in humans from two unrelated populations, provide the rationale for prioritization of functional studies of ADAMTS16
as a candidate for the BP QTL on human Chromosome 5.