Human DSPP: Novel System for Slip-Replication and Epigenetic-Based Variation Studies

Objectives: Examine dentin's most abundant noncollagenous protein, DSPP's (dentin sialophosphoprotein), extraordinary variation in the ~240 SerSerAsp-encoded repeat within normal humans. Methods: Sequencing of DSPP-repeat domain from 188 human chromosomes of diverse geographical origins. Bisulfite-treated sequencing of embryonic and somatic cell genomic DNA to explore epigenetic explanation of the many single nucleic acid polymorphisms (SNPs) encountered. Results: DSPP-repeat contained a ~1.2kb hypervariable region (DSPP-hvrr) with extraordinary rates of change. There were 37 haplotypes comprised of specific combinations of 20 insertions/deletions (indels) due to slip-replication during meiosis and 37 predominantly C-to-T SNPs, making DSPP more diverse than most genes. Throughout the DSPP-repeat, the most frequent changes from the proposed primordial 9-basepair repeat (AGC-AGC-GAC encoding the SerSerAsp repeat) were at CpG motifs that are generally accepted to be prone to epigenetic methylation and subsequent deamination to TpG in other genes. Interestingly, the second most frequent transitions occurred at CpNpG sites for which methylation/deamination to TpNpG has been described in plants but is not well documented in normal human cells. Bisulfite-sequencing showed that DSPP-hvrr cytosines can be methylated at both CpG and CpNpG (specifically, CAG) sites. Analysis of the 37 DSPP-hvrr haplotypes from 94 geographically diverse people suggests it apparently changes so rapidly that it may be a useful autosomal marker to track ancient human migration. For example, DSPP-hvrr haplotype 21 was frequent in Asians as well as their descendent Amerindians of North and South America, but was absent in the chromosomes of European descent. Conclusions: The normal population was found to have extraordinarily high levels of DSPP indels (comparable to microsatellites) and epigenetic-related SNPs. Indeed, bisulfite-sequencing shows that DSPP is methylated at both the common CpG and, for the first time in normal humans, CpNpG sequences. Furthermore, DSPP-hvrr may be a useful autosomal marker for ethno-geographic human migration studies. (supported by DIR/NIDCR/NIH/DHHS)