A missense variant in specificity protein 6 (SP6) is associated with amelogenesis imperfecta

Abstract

Amelogenesis is the process of enamel formation. For amelogenesis to proceed, the cells of the inner enamel epithelium (IEE) must first proliferate and then differentiate into the enamel-producing ameloblasts. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective or absent tooth enamel. We identified a 2 bp variant c.817_818GC>AA in SP6, the gene encoding the SP6 transcription factor, in a Caucasian family with autosomal dominant hypoplastic AI. The resulting missense protein change, p.(Ala273Lys), is predicted to alter a DNA-binding residue in the first of three zinc fingers. SP6 has been shown to be crucial to both proliferation of the IEE and to its differentiation into ameloblasts. SP6 has also been implicated as an AI candidate gene through its study in rodent models. We investigated the effect of the missense variant in SP6 (p.(Ala273Lys)) using surface plasmon resonance protein-DNA binding studies. We identified a potential SP6 binding motif in the AMBN proximal promoter sequence and showed that wild-type (WT) SP6 binds more strongly to it than the mutant protein. We hypothesize that SP6 variants may be a very rare cause of AI due to the critical roles of SP6 in development and that the relatively mild effect of the missense variant identified in this study is sufficient to affect amelogenesis causing AI, but not so severe as to be incompatible with life. We suggest that current AI cohorts, both with autosomal recessive and dominant disease, be screened for SP6 variants.

Figure 1

Family pedigree, dental phenotype, genotyping and conservation. (A) Pedigree of the British family investigated. Affected family members are shaded. WES was carried out on the individuals marked with asterisk. Segregation analysis of the SP6 c.817_818GC>AA for all available family members is also shown. (B) The permanent dentition of the index case, IV:2 (arrow on pedigree) was characterized by generalized hypoplastic AI with an irregular surface involving all teeth. Note: the small soft tissue lesion involving the hard palate (marked with asterisk) is a reactive lesion unrelated to the dentition. (C) Sanger sequencing electropherograms to show the WT SP6 and the SP6 c.817_818GC>AA (NM_199262) variant sequence. (D) Conservation analysis of the p.Ala273 residue in orthologous and paralogous proteins.

Figure 2

Comparison of DNA-binding activity of WT and mutant SP6 proteins using Biacore SPR. Biotinylated oligonucleotides were captured on a streptavidin–derivatized sensor chip surface. WT and mutant SP6 proteins were washed over these surfaces across a range of concentrations. Each 3-min injection was followed by buffer washes to follow dissociation rates of the SP6-DNA complex. Sensorgram results are shown for the AMBN oligonucleotide (sequence in Supplementary Material, Table S8) (A) WT SP6; (B) mutant SP6. DNA-binding by the mutant SP6 protein demonstrates that this variant does not abolish binding, consistent with the mutation affecting only one of the three zinc finger motifs. However, the mutant does show reduced binding compared to the WT, with the dissociation rate for the mutant SP6 protein being faster than for the WT. This is clear on the AMBN promoter sequence surface with the signal dropping to lower values than for the WT protein.