Genotype-phenotype analysis and functional study of three novel LRP6 variants in non-syndromic oligodontia

Abstract

Introduction: Tooth agenesis (TA) is a common craniofacial malformation in humans, characterized by the absence of one or more permanent teeth. Recent studies have identified the low-density lipoprotein receptor-related protein 6 (LRP6) gene as an autosomal dominant contributor to TA. Herein we aimed to identify novel LRP6 variants in patients with non-syndromic oligodontia (NSO) and perform functional analyses of these variants.

Methods: Whole-exome sequencing (WES) was conducted on probands and their first-degree relatives to identify potential pathogenic variants. Identified LRP6 variants underwent computational pathogenicity prediction using integrated bioinformatics tools. Subcellular localization patterns were analyzed via immunofluorescence microscopy. Functional characterization of WNT/β-catenin signaling alterations was achieved through Western blot analysis and dual-luciferase reporter assays (TOP-Flash/FOP-Flash systems). Finally, genotype-phenotype correlations in LRP6-associated non-syndromic oligodontia (NSO) were systematically investigated.

Results: We identified three novel LRP6 variations (NM_002336): a truncating variant [c.2182C>T (p.Arg728*)] and two missense variants [c.3773C>T (p.Thr1258Met) and c.1441C>T (p.Arg481Cys)]. Immunofluorescence characterization revealed that the missense variants exhibited subcellular localization patterns comparable to wild-type LRP6, with predominant distribution in the plasma membrane and cytoplasmic compartments. Western blot analysis revealed impaired β-catenin expression in cells harboring the LRP6 missense variants, suggesting compromised canonical WNT signaling pathway activity. Functional assessment using the TOP/FOP-Flash luciferase reporter system demonstrated significantly reduced TCF/LEF transcriptional activity associated with these variants, though statistical significance was exclusively observed for the Arg481Cys variant (P < 0.05). Literature review identified 39 LRP6 variants associated with 52 NSO patients, revealing that mandibular second premolars were the most frequently affected teeth, while maxillary first molars were least likely to be affected.

Discussion: We identified three novel LRP6 variants in patients with NSO from three Chinese families. Furthermore, we have confirmed through in vitro experiments that these novel LRP6 missense variants lead to impaired activation of the WNT signalling pathway. Finally, we summarized the genotype-phenotype correlation for LRP6-related NSO, finding that LRP6 variants are most likely to affect the mandibular second premolars.

Keywords: LRP6; genotype-phenotype; non-syndromic oligodontia; tooth agenesis; β-catenin.

FIGURE 1

Dental characteristics of three probands with non-syndromic oligodontia. (A) Facial photographs, intraoral images, panoramic radiographs, and schematic of missing teeth for proband 1, (B) proband 2, and (C) proband 3. White arrows indicate missing teeth; white triangles represent unseparated tooth roots; asterisks indicate taurodontism; crosses indicate microdontia. Max, maxillary; Mand, mandibular.

FIGURE 2

Conserved sequence analysis and structural modeling of three novel LRP6 variants. (A) A new LRP6 truncation variant [c.2182C>T (p.Arg728*)] identified in proband II:1 and her father (I:1) in family 1. (B,C) Arg728 is highly conserved across all six species. (D) Structural modeling of wild-type Arg728 and (D′) p. Arg728*. (E) A new LRP6 missense variant [c.3773C>T (p.Thr1258Met)] identified in proband II:1 and her mother (I:2) in family 2. (F–G) Thr1258 is highly conserved across all six species. (H) Structural modeling of wild-type Thr1258 and (H′) p. Thr1258Met. (I) Hydrophobicity analysis of wild-type Thr1258 and (I′) p. Thr1258Met. (J) A new LRP6 missense variant [c.1441C>T (p.Arg481Cys)] identified in proband II:1 and her father (I) 1 in family 3. (K–L) Arg481 was highly conserved across all six species. (M) Structural modeling of wild-type Arg481 and (M′) p. Arg481Cys. (N) Electrostatic potential surface of wild-type Arg481 and (N′) p. Arg481Cys. The mutant allele is marked in a red box. * indicates a completely conserved column of amino acids.

FIGURE 3

In vitro data. (A) Subcellular localization of LRP6 before and after variant. (B,C) β-catenin expression in LRP6 mutants, determined by Western blotting. Quantitative data are presented. (D) Activity of the WNT signaling pathway in HEK-293T cells, measured using TOP-Flash/FOP-Flash luciferase reporter assay. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4

Pattern of LRP6-related non-syndromic tooth agenesis. (A) Schematic diagram of wild-type LRP6, indicating the distribution of all LRP6 variants in patients with tooth agenesis. Red arrows indicate variants identified in this study, and blue arrows indicate LRP6-related syndromic phenotype variant sites. (B) Proportions of LRP6 variant types. (C) Analysis of missing teeth in 52 NSO patients with LRP6 variants, showing tooth loss at each permanent dentition position (excluding third molars). (D) Tooth loss rate in NSO patients with LRP6 variants (excluding third molars). Mo, molar; PM, premolar; Ca, canine; LI, lateral incisor; CI, central incisor; Max, maxillary; Mand, mandibular. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.