A Fourth KLK4 Mutation Is Associated with Enamel Hypomineralisation and Structural Abnormalities

Abstract

"Amelogenesis imperfecta" (AI) describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4), expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs^*37) (NM_004917.4, NP_004908.4). This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and microhardness testing (MH). Enamel from the affected individual (referred to as KLK4 enamel) was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p < 0.001). However, the hardness of control inner enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4^-/- mouse and suggest that KLK4 is required for the hardening and mineralization of the inner enamel layer but is less essential for hardening and mineralization of the outer enamel layer.

Keywords: KLK4; amelogenesis imperfecta; enamel; mutation; phenotype.

Figure 1

Pedigrees and clinical images of families 1–5. Red labels indicate the individuals for which DNA was subjected to whole exome sequencing for families 1–3 and 5. Asterisks mark individuals in family 1 whose DNA underwent SNP genotyping analysis. Dots indicate individuals that self-reported as affected with AI or were reported by other family members to be affected with AI. However these individuals were not themselves clinically assessed by a dental practitioner. Question marks indicate individuals for which details of their AI phenotype were unavailable. The genotypes of the KLK4 c.632delT variant (NM_004917.4) for each individual for which DNA was available for analysis are marked on each pedigree. Clinical images show hypomaturation AI in families 1–4. Clinical images were unavailable for family 5.

Figure 2

High resolution X-ray CT analysis of WT control and KLK4 teeth from individual V:1 (family 1). (A,B): molar teeth, (C,D): canine teeth, (E,F): incisal tooth slices. Arrows indicate the presence of an inner layer of enamel of lower mineral density not seen in control teeth. Scale bars represent 1 mm.

Figure 3

SEM and microhardness testing of control and KLK4 teeth from individual V:1 (family 1). (A–E): Control tooth; (F–J): KLK4 tooth. (A): Whole tooth longitudinal section. (B): The enamel layer with the enamel surface shown at the top and the enamel-dentine junction at the bottom. White boxes indicate the positions of magnified images (C,D) and (E) respectively (top to bottom). (C–E): outer (C): close to the surface; middle (D) and inner (E): close to the EDJ, enamel. (F): Whole tooth longitudinal section—note the clear demarcation in the enamel layer between the inner and outer enamel on the lingual side of the tooth (arrow). An area of decay is present on the one side of the tooth (star). (G): The enamel layer with surface at the top and the enamel-dentine junction at the bottom. White boxes indicate the positions of magnified images (H–J) respectively (top to bottom). (H–J): outer (H): close to the surface: middle (I) and inner (J): close to the EDJ, enamel. (K): Knoop microhardness testing for control and KLK4 incisor and molar teeth. Error bars represent 2x standard error of the mean. Brackets indicate the conditions for which unpaired, two-tailed T-tests were undertaken. Results are abbreviated as follows: NS; not significant, i.e., p > 0.05; ^*** significant at p < 0.001. (L): Knoop microhardness testing results in table form. (M): Light microscopy image showing two indentations made around 100 μm apart on the KLK4 incisor tooth, one in the outer enamel layer and the other in the middle enamel layer. Scale bars represent (A,F): 1 mm; (B,G): 100 μm; (C–E) and (H–J): 25 μm; (M): 50 μm.

Figure 4

Elemental analysis by energy dispersive X-ray spectroscopy (EDX). Measurements are detailed in Supplementary Table 10. Note that Mg and Na content were also analyzed but are not included here. Measurements were taken from both sides of the tooth: (A): buccal, (B): lingual.