A New SLC10A7 Homozygous Missense Mutation Responsible for a Milder Phenotype of Skeletal Dysplasia With Amelogenesis Imperfecta

Abstract

Amelogenesis imperfecta (AI) is a heterogeneous group of rare inherited diseases presenting with enamel defects. More than 30 genes have been reported to be involved in syndromic or non-syndromic AI and new genes are continuously discovered (Smith et al., 2017). Whole-exome sequencing was performed in a consanguineous family. The affected daughter presented with intra-uterine and postnatal growth retardation, skeletal dysplasia, macrocephaly, blue sclerae, and hypoplastic AI. We identified a homozygous missense mutation in exon 11 of SLC10A7 (NM_001300842.2: c.908C>T; p.Pro303Leu) segregating with the disease phenotype. We found that Slc10a7 transcripts were expressed in the epithelium of the developing mouse tooth, bones undergoing ossification, and in vertebrae. Our results revealed that SLC10A7 is overexpressed in patient fibroblasts. Patient cells display altered intracellular calcium localization suggesting that SLC10A7 regulates calcium trafficking. Mutations in this gene were previously reported to cause a similar syndromic phenotype, but with more severe skeletal defects (Ashikov et al., 2018;Dubail et al., 2018). Therefore, phenotypes resulting from a mutation in SLC10A7 can vary in severity. However, AI is the key feature indicative of SLC10A7 mutations in patients with skeletal dysplasia. Identifying this important phenotype will improve clinical diagnosis and patient management.

Keywords: NGS (next generation sequencing); amelogenesis imperfecta; human; rare diseases; skeletal dysplasia.

Figures

Figure 1

(A) X-ray images of the skeletal anomalies at 3 months old (a,d,f,j,l,n), 4 years old (b,e,g,i,k,m,o) and 9 years old (c,h,p). Radiographs of the vertebral column showed the progressive scoliosis (a–c) and the ballooning of the vertebrae (d,e). Radiographs of hands and feet (f–h,j,k) displayed a progressive carpal and tarsal ossification. The patient also presented with short long bones (i,l,m), a genu valgus (l), and a horizontal acetabulum (n–p). (B) Clinical and radiological images of the facial and dental anomalies at 2 years old (a,d,f) and 9 years old (b,c,e,g–i). AI is visible in the clinical intraoral photographs affecting both the primary (f) and permanent dentition (g,h). The panoramic radiograph showed no radio-opacity contrast between dentine and enamel (i). (C) Optical numeric microscope and scanning electronic microscopy (SEM) of primary teeth. Numeric optical view of a sagittal section of a primary tooth. Dentine appears brighter than enamel. Lower central primary incisor (a). SEM micrograph of the thin enamel layer covering the vestibular side of the tooth. A thick layer of calcified calculus is present (b). Higher magnification of the thin enamel layer. Pseudo-prismatic structures are evident (arrows) (c). Micrograph of the thin enamel showing an outer aprismatic layer (between red arrows) exhibiting incremental lines (white arrow) (d). Enamel-dentinal junction showing typical scallops and unusual non-mineralized collagen fibers at the interface (e). Higher magnification of the calculus material capping the tooth. Many entangled calcified filamentous bacteria are observed (f).

Figure 2

(A) Analysis of the SLC10A7 mutation: Pedigree of the AI patient and DNA sequencing chromatograms of the whole family. Parents and other children were heterozygous and the patient was homozygous for the mutation. An arrow points to the mutation. (B) Multiple sequence alignment of SLC10A7 last transmembrane domain (TM10). The largely conserved sequence of the proteins last transmembrane domain is represented by the dark bar. The amino-acid affected by the missense mutation in the patient (red square) is conserved from human to yeast. (C) Human SLC10A7 mutations in the literature. The SLC10A7 gene contains 12 exons. The mutations described in Dubail et al. (2018) are represented by blue arrows and those reported in Ashikov et al., 2018 by red arrows. Our mutation is the only mutation located at the end of the gene (exon 11) and is represented by a green arrow.

Figure 3

(A) Analysis of mouse Slc10a7 transcripts distribution by in situ hybridization. Sections illustrate Slc10a7 expression in the developing bone, vertebrae, molars, and incisors. Developmental stages and section planes are: E14.5 frontal (a,b), E16.5 sagittal (c–h); E18.5 sagittal (i–l) sections. Am, ameloblasts; DP, dental papila; EL, epithelial loop; GUB, gubernaculum; IDE, inner dental epithelium; LI, lower incisor; Od, odontoblasts; ODE, outer dental epithelium; SR, stellate reticulum; Ve, vertebrae. Scale bars: 10 μm (b); 40 μm (a,c,g,h); 30 μm (d,l); 60 μm (i,j); 80 μm (e,f); 150 μm (k). (B) SLC10A7 expression in control and patient skin fibroblasts as detected by western blot. The stain-free membrane, displaying the total amount of protein loaded, was used for quantification. The western blot shown corresponds to one significant experiment (n = 3). The data plotted were the mean of three independent experiments. Statistical analysis was done with the t-test and the p was determined *p < 0.05. (C) Calcium localization in control and patient fibroblasts. The cells were stained for 15 min or 30 min with 4 μm of Fluo4, then washed for 15 or 30 min, respectively and observed by fluorescent microscopy. Images were taken with a 400× magnification, scale bar 10 μm.