Clinical, genetic, epidemiologic, evolutionary, and functional delineation of TSPEAR-related autosomal recessive ectodermal dysplasia 14

Abstract

TSPEAR variants cause autosomal recessive ectodermal dysplasia (ARED) 14. The function of TSPEAR is unknown. The clinical features, the mutation spectrum, and the underlying mechanisms of ARED14 are poorly understood. Combining data from new and previously published individuals established that ARED14 is primarily characterized by dental anomalies such as conical tooth cusps and hypodontia, like those seen in individuals with WNT10A-related odontoonychodermal dysplasia. AlphaFold-predicted structure-based analysis showed that most of the pathogenic TSPEAR missense variants likely destabilize the β-propeller of the protein. Analysis of 100000 Genomes Project (100KGP) data revealed multiple founder TSPEAR variants across different populations. Mutational and recombination clock analyses demonstrated that non-Finnish European founder variants likely originated around the end of the last ice age, a period of major climatic transition. Analysis of gnomAD data showed that the non-Finnish European population TSPEAR gene-carrier rate is ∼1/140, making it one of the commonest AREDs. Phylogenetic and AlphaFold structural analyses showed that TSPEAR is an ortholog of drosophila Closca, an extracellular matrix-dependent signaling regulator. We, therefore, hypothesized that TSPEAR could have a role in enamel knot, a structure that coordinates patterning of developing tooth cusps. Analysis of mouse single-cell RNA sequencing (scRNA-seq) data revealed highly restricted expression of Tspear in clusters representing enamel knots. A tspeara ^-/-;tspearb ^-/- double-knockout zebrafish model recapitulated the clinical features of ARED14 and fin regeneration abnormalities of wnt10a knockout fish, thus suggesting interaction between tspear and wnt10a. In summary, we provide insights into the role of TSPEAR in ectodermal development and the evolutionary history, epidemiology, mechanisms, and consequences of its loss of function variants.

Keywords: Autosomal recessive ectodermal dysplasia type 14; Closca; Conical teeth; Ectodermal dysplasia; Enamel knot; Extracellular matrix dependant signalling; Hypodontia; TSPEAR; WNT10A; zebrafish fin regeneration.

Figure 1

Genotype and phenotype of TSPEAR-related autosomal recessive ectodermal dysplasia (A) Gene diagram showing all protein-coding variants identified in this study (top) and previously reported (bottom), mapped onto the known protein domains. (B) Phenotype heatmap among newly and previously described cases showing a predominant dental phenotype. (C) Images of individuals 2, 3, 5, 6, 9, and 11. Note conical-shaped and widely spaced teeth.

Figure 2

In silico protein modeling of pathogenic/likely pathogenic non-truncating TSPEAR variants (A) Left shows the AlphaFold-predicted structure of TSPEAR with all missense/in-frame indels plotted; note the propensity for these to affect the β-propeller. Right shows the surface of the predicted TSPEAR structure showing overall charge. Note the pocket of negative charge (red) within the inner surface of the β-propeller. Conservation scores are shown below mapped to the predicted structure surface showing conservation of the residues (purple) within the inner surface of the β-propeller. (B) Molecular models for all missense/in-frame indels in this study produced by Modeller 9.24. Steric clashes are shown by red discs. Yellow dotted lines indicated interacting residues.

Figure 3

Population and evolutionary genetics of TSPEAR variants (A) Screenshot from the UCSC browser (GRCh38) showing minimal haplotypes for all founder variants in this study. Regions of homozygosity (ROHs) for the five p.D639N homozygous individuals in the 100KGP are also shown on the lower track. (B) A linkage disequilibrium heatmap of the 12 core haplotype SNVs for heterozygous p.D639N variant carriers in 100KGP (n = 449). (C) Bar plot showing the estimated ages of founder variants seen in out TSPEAR cohort. Estimates shown were calculated using the gamma method (recombination clock) after estimating haplotypes using core SNVs for p.D639N and inconsistent homozygous genotypes for p.R197∗, p.S585I, and p.V576Lfs∗38. The mean for all variants falls within the last ice age, with both p.R197∗ and p.V576Lfs∗38 falling within the Last Glacial Maximum. (D) Lolliplot showing pLoF variants in gnomAD for TSPEAR with allele counts in brackets. (E) Dotplot showing gene-carrier rate for both ClinVar likely pathogenic (LP) and pathogenic (P) alelles (triangles) and all predicted deleterious alleles (circles) in gnomADv.2.1.1. We include p.D639N as a ClinVar LP/P allele given the data we present in this study upgrade this variant to LP based on ACMG criteria (PS4, PM3 and PP3).

Figure 4

Prediction of TSPEAR function (A) Heatmap of pairwise sequence identities using cDNA sequences for 45 diverse species. Note the broad conservation in vertebrates and distinct triangles for mammals, birds, and fish. (B) AlphaFold-predicted structures for both Drosophila Closca and TSPEAR; note a predicted β-propeller in both. (C) Schematic of the known function of Closca in both Torso and Toll receptor activation and terminal and dorsoventral patterning, respectively. Closca, along with Nusrat and Polehole, sequester Torso-like (tsl) and also Nudel at the vitelline membrane (VM), allowing for their precise spatial and temporal accumulation and release, which transfers spatial information to the developing embryo. PM, plasma membrane; PVS, perivitelline space. (D) scRNA-seq data in the murine developing molar showing restricted expression of Tspear to the enamel knot, an important signaling center.

Figure 5

Zebrafish tspear double-knockout model (A) A schematic figure showing ventral view of larval zebrafish with ventral cartilages (blue) and teeth (red). Alizarin red S staining of pharyngeal teeth shows that a double homozygous mutant (tspeara^−/−;tspearb^−/−) displayed thinner and aberrant mineralized teeth (arrowhead) at 12 days post-fertilization (dpf). (B) Schematic figure for pharyngeal dentition of adult zebrafish shown in ventral view (top panel) and lateral view (bottom panel). Arrow indicates missing tooth. A, anterior; D, dorsal; MD, mediodorsal; V, ventral. (C) Quantification of the number of teeth in the ventral, mediodorsal, and dorsal rows. Each dot represents one animal. Control = 30 animals, mutant = 32 animals. Error bars: mean ± SD. Difference was tested using two-tailed unpaired t test with Mann-Whitney test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (D) Adult tail fins were amputated from wild-type control (n = 4) and double homozygous mutants (n = 4). Live fins were imaged at 0, 5, and 12 days post-amputation (dpa). Dashed line indicates the position of amputation.