WNT/β-catenin signaling is initiated by binding of a WNT protein to a Frizzled (FZD) receptor and a co-receptor, low-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5/6). The objective of this study was to find the genetic variants responsible for dental anomalies found in 4 families. Clinical and radiographic examination and whole exome sequencing were performed on 5 patients affected with dental anomalies and the mutant proteins modeled. Five patients were heterozygous for the WNT10A variants, including c.877C>T; p.Arg293Cys, c.874A>G; p.Ser292Gly, c.1042C>T; p.Arg348Cys, and c.1039G>T; p.347GluX. The p.Arg293Cys and p.Ser292Gly mutations are located in the WNT10A N-terminal domain region with binding sites for FZD receptor, porcupine, WNTLESS, and extracellular binding proteins, so they are likely to have adverse effects on binding these proteins. The p.Arg348Cys mutation, which is located in the binding site of LRP5/6 co-receptors, is postulated to result in impaired binding to these co-receptors. The nonsense mutation p.347GluX is predicted to result in the truncation of most of the C-terminal domain, which is likely to disrupt the binding of WNT10A to WNTLESS, the membrane protein that binds lipid-acylated WNT proteins to carry them from the endoplasmic reticulum to the cell surface and FZD. Four novel mutations in WNT10A were identified in patients with isolated tooth agenesis. The mutations in the N-terminal domain and the interface between the N- and C-terminal domains of WNT10A in our patients are likely to disrupt its binding with FZD, LRP5/6, and various other proteins involved in WNT10A processing and transport, impair WNT and SHH signaling, and subsequently result in tooth agenesis, microdontia, and root maldevelopment.

Summary: Background: Canonical and non-canonical WNT signaling are important for odontogenesis. WNT ligand secretion mediator (WLS; MIM611514) is required to transport lipid-modified WNT proteins from the Golgi to the cell membrane, where canonical and non-canonical WNT proteins are released into the extracellular milieu. Biallelic pathogenic variants in WLS are implicated in autosomal recessive Zaki syndrome (ZKS; MIM 619648), the only genetic condition known to be caused by pathogenic variants in WLS. Objective: To investigate molecular etiology of dental anomalies in 250 patients with or without oral exostoses. Patients and methods: Clinical and radiographic examination, and whole exome sequencing, were performed in the case of 250 patients with dental anomalies with or without oral exostoses. Results: Four extremely rare heterozygous missense variants (p.Ile20Thr, p.Met46Leu, p.Ser453Ile and p.Leu516Phe) in WLS were identified in 11 patients with dental anomalies. In five of these patients, a torus palatinus or a torus mandibularis was observed. Conclusion: We report for the first time the heterozygous WLS variants in patients with dental anomalies. Root maldevelopments in patients with WLS variants supports the role of canonical and non-canonical WNT signaling in root development. We also show that variants in WLS were implicated in torus palatinus and torus mandibularis. In addition, this is the first time that heterozygous carriers of WLS variants were found to manifest phenotypes. WLS variants were likely to have adverse effects on the concentration of WNT ligands delivered to the cell membrane, resulting in aberrant canonical and non-canonical WNT signaling, and subsequent phenotypes. Limitations of the study: Patient’s positioning during the acquisition of panoramic radiography might have affected the appearance of the tooth structures. If we had all family members of each patient to study co-segregation between genotype and phenotype, it would have strengthened the association of WLS variants and the phenotypes.

KCTD1 plays crucial roles in regulating both the SHH and WNT/β-catenin signaling pathways, which are essential for tooth development. The objective of this study was to investigate if genetic variants in KCTD1 might also be associated with isolated dental anomalies. We clinically and radiographically investigated 362 patients affected with isolated dental anomalies. Whole exome sequencing identified two unrelated families with rare (p.Arg241Gln) or novel (p.Pro243Ser) variants in KCTD1. The variants segregated with the dental anomalies in all nine patients from the two families. Clinical findings of the patients included taurodontism, unseparated roots, long roots, tooth agenesis, a supernumerary tooth, torus palatinus, and torus mandibularis. The role of Kctd1 in root development is supported by our immunohistochemical study showing high expression of Kctd1 in Hertwig epithelial root sheath. The KCTD1 variants in our patients are the first variants found to be located in the C-terminal domain, which might disrupt protein–protein interactions and/or SUMOylation and subsequently result in aberrant WNT-SHH-BMP signaling and isolated dental anomalies. Functional studies on the p.Arg241Gln variant are consistent with an impact on β-catenin levels and canonical WNT signaling. This is the first report of the association of KCTD1 variants and isolated dental anomalies.

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.

Cholangiocarcinoma (CCA) is an aggressive tumor of the biliary epithelium with poor survival that shows limited response to conventional chemotherapy. Increased expression of glucosylceramide synthase (GCS) contributes to drug resistance and the progression of various cancers; the expression profiles of GCS (UGCG) and the genes for glucocerebrosidases 1, 2, and 3 (GBA1, GBA2, and GBA3) were therefore studied in CCA. The biological functions of GCS for cell proliferation and cisplatin sensitivity in CCA were explored. GCS expression was higher in CCA tumor tissues than that of GBA1, GBA2, and GBA3. Verification of GCS expression in 29 paired frozen CCA tissues showed that 8 of 29 cases (27.6%) had high GCS expression. The expression of GCS and GBA2 was induced in CCA cell lines following low-dose cisplatin treatment. Suppression of GCS by either palmitoylamino-3-morpholino-1-propanol (PPMP), GCS knockdown or a combination of the two resulted in reduced cell proliferation. These treatments enhanced the effect of cisplatin-induced CCA cell death, increased the expression of apoptotic proteins and reduced phosphorylation of ERK upon cisplatin treatment. Taken together, inhibition of the GCS increased cisplatin-induced CCA apoptosis via the inhibition of the ERK signaling pathway. Thus, targeting GCS might be a strategy for CCA treatment.

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. It is continuously formed and degraded in glycosphingolipid metabolism. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2). Deficiencies of GBA and GBA2 affect glycosphingolipid metabolism, resulting in neurological diseases, such as Gaucher Disease and Hereditary Spastic Paraplegia. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/ accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels compared to control and other isoforms. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide. These studies suggest that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell, acting on glucosylceramides with a wide range of lipid components. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.