As shown in and , a cohort of nine patients/families diagnosed with either DGI or DD were sequenced for DSPP mutations. Two probands, Patients DGI-7 and DGI-1, had mutations identified in the nonrepeat portion of DSPP (). Patient DGI-7 was found to be heterozygous for a c.13511G4T alteration that affects the splice donor site of intron 3. Interestingly, a different mutation of this same nucleotide, c.13511G4A, has been reported in a family with DGI-II []. Proband DGI-7 (of the long-studied Brandywine cohort) was found to be heterozygous for a c.49C4T transition that is predicted to cause a p.P17S substitution. The identical mutation was recently reported in a Chinese DGI-II family [] and a c.49C4A transition (p.P17 T) was previously reported in a different Chinese DGI-II family []. One affected member of the same Brandywine cohort was reported to have had a 36-bp in-frame deletion followed by an 18-bp inframe insertion within the 30 end of the repeat domain of DSPP []. We confirmed this pair of indels in Proband DGI-1 (DSPP HVRR Hap 38, Genbank accession no. EU278677) but contend this is likely a normal variant and that the c.49C4T alteration is likely the causative event (see below).

The remaining seven new probands were found to have frameshift mutations in the 2-kb repeat portion of DSPP exon 5. All seven showed a net −1 frameshift (i.e., a loss of 1 or 4 bp resulting in the same, new, −1 open reading frame) in the first half of the repeat (). The locations of these four mutations are reported with respect to the current DSPP reference sequence (NM_014208.3), numbering from the start codon (A is nucleotide 1). Because of the high degree of sequence variability among the normal DSPP haplotypes (see below), the values are not the exact numerical location of the mutation within the specific patient’s own DNA. The exact sequence for each unique frameshift is available in GenBank (accession no. EU284750–EU284753) and an alignment is available in ). Two different frameshift events were found in the two DD families. One DD family (Family DD-2) had a −4-bp (loss of TCAG) frameshift (c.1870_1873delTCAG; GenBank accession no. EU284750) while the other DD family (Family DD-3) had a similar 4-bp deletion 50 bp 30 (c.1918_1921delTCAG; GenBank accession no. EU284751). The two frameshifts were verified to be separate events when aligned with each other and to a normal control haplotype, due to the presence of identifiable sequences between the two mutation sites (; ). Interestingly, the TCAG lost in both DD families are two of the three times this precise sequence is found in the entire DSPP repeat, and these four bases may represent currently unexplained mutational hotspots in humans.

Similar to the findings in human mitochondrial DNA and Y chromosome genes, common haplotypes of autosomal DSPP HVRR were present in all the studied ethnic groups from Coriell Cell Repositories [; ; ]. Other haplotypes appeared to be more limited to peoples sharing hypothesized human migration routes often associated with out-of-Africa theories (). For example, haplotype 21 was very frequent in the Asian-descended groups (21% of the 100 chromosomes obtained from Chinese, Japanese, Taiwanese, and their migrant relatives, the Mexican and Andes groups) but was absent in the 88 chromosomes of African (African South Sahara and African American) and European (Caucasian and Northern European) descent (). The cluster including haplotypes 27, 30, 31, 32, and 36 was only observed in our African populations. While analysis of the HVRR is the most simple and data-rich approach, additional information can be obtained by analyzing the entire DSPP repeat. Many of the most common, shared DSPP HVRR haplotypes could be further separated into one or more related but distinct haplotypes based on indels and SNPs that occurred 50 to the HVRR (DPP; GenBank accession no. EU278619–EU278639). For example, with the addition of the 50 SNPs/indels, two of the most frequent and otherwise shared HVRR haplotypes (1 and 17) separated along ethnic group lines: the Chinese and Japanese-associating alleles separating from the Caucasian, Northern European, and Amerindian groups for both of these haplotypes (GenBank accession no. EU278619–EU278624 for Hap 1 and EU278630–EU278631 for Hap 17).

As a corollary, we further propose that the conservation of the chemical properties of the first three amino acids on the carboxyterminal side of the SPP cleavage site may also be critical for the efficient processing of the DSPP’s signal peptide (as is the case for many other proteases in biology). The first two amino acids of the mature protein (immediately after the SPP cleavage site) are normally encoded by the last six bases of exon 2 and the third by the first three bases of exon 3. These three amino acids in DSPP are Ile Pro Val (IPV), with the unique and structurally confined proline being flanked by two hydrophobic amino acids. With the exception of a chemically similar Val substituting for the Ile in the elephant, this tripeptide sequence is, to our knowledge, invariant within all animal species sequenced to date (University of California, Santa Clara (UCSC) Genome Bioinformatics; http://genome.ucsc.edu). The chemically similar motif is also found at the SPP cleavage site in several of the other major human proteins secreted during tooth matrix assembly, including: DMP1 (LeuProVal), OPN (LeuProVal), ameloblastin (ValProPhe), and amelogenin (MetProLeu). This completely conserved Pro in DSPP was shown to be mutated to a serine (p.P17S) in our single representative of the Brandywine DGI family (Family DGI-1), a result identical to that recently reported for a different kindred by , and similar to the p.P17 T first noted by . These changes may cause signal peptide processing errors by the odontoblast SPP and result in dominant dentin diseases similar to that seen in direct changes in the signal peptide. At first, the change of the mature protein’s third position, Val, in the p.V18F (c.52G4T) event [; ; ; ] looks to be just a chemically conserved missense mutation unlikely to cause significant changes in the processing of DSPP. However, we propose that this represents the first in a series of splice-site mutations involving exon 3. The consensus sequence derived for the underlying DNA of the mRNA’s splice donors and acceptors is shown in . The GT and AG ends of the intronic sequence are nearly invariant, although GT and AG are common dinucleotide sequences. Therefore, the likelihood of a splice event occurring at any particular GT (GU in the RNA transcript itself) or AG increases as the sequences better fit the consensus. Using the SplicePort program (http://spliceport.cs.umd.edu), the p.V18F mutation changes the functional (10.91) splice acceptor site: CAG^GTT(Val) into a sequence predicted to be ineffective, CAG^TTT(Phe) (−0.02; see ) []. Due to the lack of a strong alternative splice acceptor site within 50 bp of the damaged one [], the loss of this spice junction would likely cause exon 3 to be skipped, bringing exon 4 (with its normal splice acceptor, CAG^GAT) in-frame to exon 2. This would make the carboxy-terminal side of the SPP cleavage site become Ile Pro Asp, thereby replacing the highly conserved hydrophobic Val with a very hydrophilic amino acid, aspartic acid. Our splice-site mutation in Patient DGI-7, as well as all of the previously published splice junction mutations (), will result in this same loss of exon 3 and consequent replacement of the hydrophobic Val with a hydrophilic Asp. Even the published nonsense mutation pQ45X [; ] can logically be hypothesized to frequently result in the loss of exon 3. The normal splice donor site for exon 3 (including the CAG codon for glutamine, which is conserved in all known species) is CAG^GT, but the transition event makes it TAG^GT, thereby changing a comparatively weak donor site (10.94) into a predicted less functional site (10.39; see ) that may cause exon 3 to be skipped at least some of the time. Using neural network–based splice-site recognition programs in the study of human disease-causing splice junction mutations, noted that many disease-causing mutations were the result of changes in donor splice-sites that were already suboptimal. Thus, all of the early exon missense, nonsense, and splice junction mutations can be hypothesized to be dominant due to errors of signal peptide (or subsequent IPV-dependent) processing events that may, in turn, result in the normal DSPP protein, collagen, or other critical proteins not being properly processed for the rapidly accumulating dentin matrix.