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. 2018 Aug 6;9(1):3087.
doi: 10.1038/s41467-018-05191-8.

SLC10A7 Mutations Cause a Skeletal Dysplasia With Amelogenesis Imperfecta Mediated by GAG Biosynthesis Defects

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Free PMC article

SLC10A7 Mutations Cause a Skeletal Dysplasia With Amelogenesis Imperfecta Mediated by GAG Biosynthesis Defects

Johanne Dubail et al. Nat Commun. .
Free PMC article

Abstract

Skeletal dysplasia with multiple dislocations are severe disorders characterized by dislocations of large joints and short stature. The majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases, sulfotransferases or epimerases required for glycosaminoglycan synthesis. Using exome sequencing, we identify homozygous mutations in SLC10A7 in six individuals with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta. SLC10A7 encodes a 10-transmembrane-domain transporter located at the plasma membrane. Functional studies in vitro demonstrate that SLC10A7 mutations reduce SLC10A7 protein expression. We generate a Slc10a7-/- mouse model, which displays shortened long bones, growth plate disorganization and tooth enamel anomalies, recapitulating the human phenotype. Furthermore, we identify decreased heparan sulfate levels in Slc10a7-/- mouse cartilage and patient fibroblasts. Finally, we find an abnormal N-glycoprotein electrophoretic profile in patient blood samples. Together, our findings support the involvement of SLC10A7 in glycosaminoglycan synthesis and specifically in skeletal development.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Morphological and skeletal features of SLC10A7-deficient patients. a Skull X-ray at 6 years of age (Patient 5). Note the retrognathia (arrow). b Hip at one year of age (Patient 1). Note the Swedish key appearance of the proximal femur (medial beak and exaggerated trochanters, arrow). c Hand X-rays at 6 months of age (Patient 2). d Hand X-rays at 3 years of age (Patient 3). Note in (c) and (d, the advanced carpal ossification (presence of three ossified carpal centres at 6 months and seven ossified carpal centres at 3 years instead of one and three, respectively, see arrows). e Knee at 3 years 9 months of age (Patient 3). Note the genu valgum (angled in knee) and flat epiphyses (arrow). f Spine X-rays at 1 month of age (Patient 1). Note the coronal clefts and irregular shape of vertebrae (arrow). g Spine and hip X-rays at 9 years of age (Patient 4). Note the kyphoscoliosis. Informed consent for publication of images was obtained from all individuals or the legal guardians of minors. h Localization of the five SLC10A7 mutations relative to the SLC10A7 gene organization (striped rectangles indicate the 5′ and 3′-UTRs)
Fig. 2
Fig. 2
SLC10A7 mutation consequences and Slc10a7 tissular expression. a, b Characterization of wild-type and mutant SLC10A7 proteins. HEK293F cells were transfected with plasmids encoding c-myc tagged wild-type SLC10A7 proteins or c-myc-tagged mutant SLC10A7 proteins from two different patients (Patient 1 and Patient 3). a Cells were immunostained with anti-c-Myc antibody (red) and nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. The images are representative of three independent experiments. b Total cell lysates were analysed by western blotting using c-Myc antibody. Anti-actin was used as a loading control. The western blot images are cropped from gels that were provided for review as Supplementary Fig. 1d. c In situ hybridization analysis of Slc10a7 mRNA expression in E14.5 mouse embryos and P10 mouse tissues. The blue staining indicates sites of RNA hybridization. At E14.5, empty arrows indicate specific staining in cartilaginous tissues: Meckel cartilage (left panel) in the mandible and phalanges in the digits (central panel) and vertebrae (right panel). Note the positive staining in the lung on the right panel. At P10, filled arrows indicate specific staining in the hypertrophic zone of the growth plate in the digits (left panel), in the tarsals (central panel) and in the epiphysis of the humerus (right panel). Scale bars = 250 μm
Fig. 3
Fig. 3
Slc10a7−/− mice display skeletal dysplasia with skull anomalies. a, b Measurement of body weight and naso-occipital length (body length) and radiographic assessment of Slc10a7+/+, Slc10a7+/− and Slc10a7−/− mice at birth (a) or at 8 weeks (b), demonstrating that Slc10a7−/− mice exhibited a skeletal dysplasia with a more rounded skull. c Evolution of body weight demonstrating the growth delay in Slc10a7−/− mice compared with wild-type littermates. d Three-dimensional reconstruction of 8-week-old mouse skulls by μCT analysis and skull length and width measurements, demonstrating that Slc10a7−/− skulls are less elongated than wild-type skulls. Scale bars = 5 mm. Data are expressed as mean ± SD. NS, nonsignificant; ***p ≤ 0.001; ****p ≤ 0.0001 (two-tailed t-test). n = 13 (Slc10a7+/+), n = 35 (Slc10a7+/−) and n = 19 (Slc10a7−/−) at birth; n = 7 (Slc10a7+/+), n = 7 (Slc10a7+/−) and n = 6 (Slc10a7−/−) at 8 weeks
Fig. 4
Fig. 4
SLC10A7 deficiency leads to enamel anomalies in human and in mice. a Intra-oral photography of Patient 4 at 9 years of age showing hypomineralized amelogenesis imperfecta (left panel). X-ray panoramic of Patient 5 at 6 years of age showing absence of enamel radiolucency corresponding to amelogenesis imperfecta associated with severe oligodontia (right panel). b Three-dimensional reconstruction of mandibles from μCT analysis of 8-week-old mouse skulls and volume measurement of mandibles, lower incisors and lower molars at 8 weeks. Scale bars = 1 mm. Data are expressed as mean ± SD. NS, nonsignificant; ****p ≤ 0.0001 (two-tailed t-test). n = 7 (Slc10a7+/+), n = 7 (Slc10a7+/−) and n = 6 (Slc10a7−/−). c Scanning electron microscopy of mandible incisor from Slc10a7+/+ and Slc10a7−/− mice. Low magnification (left panels) shows conservation of enamel morphology but decreased thickness in Slc10a7−/− mice. The boxed areas in the left panels are shown at higher magnification (middle and right panels). In Slc10a7−/− mouse enamel, the aprismatic layer was absent and the external prismatic layer was altered giving a rough aspect to the enamel surface (middle panels: arrows indicate hole in the external prismatic layer; a = aprismatic enamel layer, ep = external prismatic layer, ip = internal prismatic layer). High magnification of internal prismatic enamel shows absence of a well-defined prismatic pattern in Slc10a7−/− mice, with fused rods and inter-rod structures (right panels; r = rod, ir = inter-rod). Scale bars = 20 μm. These images represent three incisors analysed
Fig. 5
Fig. 5
Slc10a7−/− mice exhibit long-bone macro- and microstructure defects. a Alizarin red/Alcian blue staining of newborn femurs and measurement of newborn femur length and femur length/width ratio. Scale bars = 1 mm. n = 8 (Slc10a7+/+), n = 9 (Slc10a7+/−) and n = 10 (Slc10a7−/−). b Three-dimensional reconstruction of μCT analysis of 8-week-old mouse femurs and measurement of 8-week-old femur length and femur length/width ratio. Scale bars = 1 mm. Panels (a) and (b) demonstrate that Slc10a7−/− femurs, both at birth and at 8 weeks, are shorter and thicker, and exhibit morphological defects. c Three-dimensional μCT of sections of 8-week-old distal femur metaphyses from Slc10a7+/+ and Slc10a7−/− mice. Scale bars = 1 mm. Graphs show trabecular and cortical bone volume (BV/TV) and bone mineral density (BMD). n = 7 (Slc10a7+/+), n = 7 (Slc10a7+/−) and n = 6 Slc10a7−/−). d Safranin O staining of the distal femur epiphysis of newborn Slc10a7+/+ and Slc10a7−/− mice. Right panels show higher magnification of the growth plate. Scale bars = 250 μm. e Masson’s Trichome staining of distal femur epiphysis of newborn Slc10a7+/+ and Slc10a7−/− mice. Scale bars = 250 μm. HZ, hypertrophic zone; PHZ, prehypertrophic zone; PZ, proliferative zone; RZ, resting zone. Images are representative of n = 10 and n = 5 per group for Safranin O and Masson’s Trichome staining, respectively. Data are expressed as mean ± SD. NS, nonsignificant; *p ≤ 0.05; **p ≤ 0.0.1; ****p ≤ 0.0001 (two-tailed t-test)
Fig. 6
Fig. 6
SLC10A7 deficiency leads to defective GAG and enhanced Ca2+ intake. a, b Total sulfated GAGs and heparan sulfates (HS) were quantified according to the DMMB procedure in extracts of SLC10A7-deficient patient fibroblasts and control fibroblasts (n = 3) (a) or in cartilage extracts from 10-day-old Slc10a7−/− or Slc10a7+/+ mice (n = 5) (b). Proportions of HS are expressed as a percentage of total sulfated GAGs (% HS). c Immunohistofluorescence for HS (red) or CS (red) counterstained with DAPI (blue) on distal femurs of newborn Slc10a7+/+ and Slc10a7−/− mice (n = 5 mice). Arrows indicate more intense CS staining at the close proximity of chondrocytes. Scale bars = 100 μm. Graphs show red fluorescent signal intensity in the growth plate for each marker. a.u., arbitrary unit. Data are expressed as mean ± SD. NS, nonsignificant; **p ≤ 0.01 (two-tailed t-test). d A representative recording of intracellular free Ca2+ in SLC10A7-deficient patients fibroblasts and control fibroblasts (n = 3). Fibroblasts were loaded with Fluo-4-AM and preincubated in calcium-free buffer for 30 min before addition of 20 μM CaCl2. Data are presented as mean ± SEM, *p ≤ 0.05; **p ≤ 0.0.1 (two-tailed t-test)

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