A novel WNT10A variant impairs the homeostasis of alveolar bone mesenchymal stem cells.

OBJECTIVES: To explore the influence of a novel WNT10A variant on bone mineral density, proliferation, and osteogenic differentiation capacities of alveolar bone mesenchymal stem cells in humans. SUBJECTS AND METHODS: Whole-exome sequencing and Sanger sequencing were utilized to detect gene variants in a family with non-syndromic tooth agenesis (NSTA). The panoramic mandibular index was calculated on the proband with WNT10A variant and normal controls to evaluate bone mineral density. Alveolar bone mesenchymal stem cells from the proband with a novel WNT10A variant and normal controls were isolated and cultured, then proliferation and osteogenic differentiation capacities were evaluated and compared. RESULTS: We identified a novel WNT10A pathogenic missense variant (c.353A > G/p. Tyr118Cys) in a family with NSTA. The panoramic mandibular index of the proband implied a reduction in bone mineral density. Moreover, the proliferation and osteogenic differentiation capacities of alveolar bone mesenchymal stem cells from the proband with WNT10A Tyr118Cys variant were significantly decreased. CONCLUSIONS: Our findings broaden the spectrum of WNT10A variants in patients with non-syndromic oligodontia, suggest an association between WNT10A and the proliferation and osteogenic differentiation of alveolar bone mesenchymal stem cells, and demonstrate that WNT10A is involved in maintaining jaw bone homeostasis.

BMPR2 Variants Underlie Nonsyndromic Oligodontia.

Oligodontia manifests as a congenital reduction in the number of permanent teeth. Despite the major efforts that have been made, the genetic etiology of oligodontia remains largely unknown. Bone morphogenetic protein receptor type 2 (BMPR2) variants have been associated with pulmonary arterial hypertension (PAH). However, the genetic significance of BMPR2 in oligodontia has not been previously reported. In the present study, we identified a novel heterozygous variant (c.814C > T; p.Arg272Cys) of BMPR2 in a family with nonsyndromic oligodontia by performing whole-exome sequencing. In addition, we identified two additional heterozygous variants (c.1042G > A; p.Val348Ile and c.1429A > G; p.Lys477Glu) among a cohort of 130 unrelated individuals with nonsyndromic oligodontia by performing Sanger sequencing. Functional analysis demonstrated that the activities of phospho-SMAD1/5/8 were significantly inhibited in BMPR2-knockout 293T cells transfected with variant-expressing plasmids, and were significantly lower in BMPR2 heterozygosity simulation groups than in the wild-type group, indicating that haploinsufficiency may represent the genetic mechanism. RNAscope in situ hybridization revealed that BMPR2 transcripts were highly expressed in the dental papilla and adjacent inner enamel epithelium in mice tooth germs, suggesting that BMPR2 may play important roles in tooth development. Our findings broaden the genetic spectrum of oligodontia and provide clinical and genetic evidence supporting the importance of BMPR2 in nonsyndromic oligodontia.

A Novel CDH1 Variant Identified in a Chinese Family with Blepharocheilodontic Syndrome.

The goal of the current study was to identify the pathogenic gene variant in a Chinese family with Blepharocheilodontic (BCD) syndrome. Whole-exome sequencing (WES) and Sanger sequencing were used to identify the pathogenic gene variant. The harmfulness of the variant was predicted by bioinformatics. We identified a novel heterozygous missense variant c.1198G>A (p.Asp400Asn) in the CDH1 gene in the proband and his mother with BCD syndrome. The sequencing results of three healthy individuals in this family are wild type. This result is consistent with familial co-segregation. According to ReVe, REVEL, CADD, gnomAD, dbSNP, and the classification of pathogenic variants with the standards of the 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG), c.1198G>A (p.Asp400Asn) is predicted to be a likely pathogenic. We observed that variant c.1198G>A (p.Asp400Asn) was located in the extracellular cadherin-type repeats in CDH1. Amino acid sequence alignment of the CDH1 protein among multiple species showed that Asp400 was highly evolutionarily conserved. The conformational analysis showed that this variant might cause structural damage to the CDH1 protein. Phenotypic analysis revealed unique dental phenotypes in patients with BCD syndrome, such as oligodontia, conical-shaped teeth, and notching of the incisal edges. Our results broaden the variation spectrum of BCD syndrome and phenotype spectrum of CDH1, which can help with the clinical diagnosis, treatment, and genetic counseling in relation to BCD syndrome.

Dose Dependence Effect in Biallelic WNT10A Variant-Associated Tooth Agenesis Phenotype.

The goal of this study was to identify the pathogenic gene variants in patients with odonto-onycho-dermal dysplasia syndrome (OODD) or nonsyndromic tooth agenesis. Four unrelated individuals with tooth agenesis and their available family members were recruited. Peripheral blood was collected from four probands and five family members. Whole-exome sequencing (WES) and Sanger sequencing were used to identify the pathogenic gene variants. The harmfulness of these variations was predicted by bioinformatics. We identified four biallelic variants of the WNT10A gene in four patients, respectively: the proband#660: c.1176C > A (p.Cys392*) and c.812G > A (p.Cys271Tyr); the proband#681: c.637G > A (p.Gly213Ser) and c.985C > T (p.Arg329*); the proband#829: c.511C > T (p.Arg171Cys) and c.637G > A (p.Gly213Ser); and the proband#338: c.926A> G (p.Gln309Arg) and c.511C > T (p.Arg171Cys). Among them, two variants (c.812G > A; p.Cys271Tyr and c.985C > T; p.Arg329*) were previously unreported. Bioinformatics analysis showed that the pathogenicity of these six variants was different. Tertiary structure analysis showed that these variants were predicted to cause structural damage to the WNT10A protein. Genotype−phenotype analysis showed that the biallelic variants with more harmful effects, such as nonsense variants, caused OODD syndrome (#660 Ⅱ-1) or severe nonsyndromic tooth agenesis (NSTA) (#681 Ⅱ-1); the biallelic variants with less harmful effects, such as missense variants, caused a mild form of NSTA (#829 Ⅱ-2 and #338 Ⅱ-1). Individuals with a heterozygous variant presented a mild form of NSTA or a normal state. Our results further suggest the existence of the dose dependence of WNT10A pathogenicity on the tooth agenesis pattern, which broadens the variation spectrum and phenotype spectrum of WNT10A and could help with clinical diagnosis, treatment, and genetic counseling.

Rare X-Linked Hypohidrotic Ectodermal Dysplasia in Females Associated with Ectodysplasin-A Variants and the X-Chromosome Inactivation Pattern.

The goal of this study was to identify the pathogenic gene variants in female patients with severe X-linked hypohidrotic ectodermal dysplasia (XLHED). Whole-exome sequencing (WES) and Sanger sequencing were used to screen for the pathogenic gene variants. The harmfulness of these variations was predicted by bioinformatics. Then, skewed X-chromosome inactivation (XCI) was measured by PCR analysis of the CAG repeat region in the human androgen receptor (AR) gene in peripheral blood cells. Two novel Ectodysplasin-A (EDA) heterozygous variants (c.588_606del19bp and c.837G>A) and one heterozygous variant (c.1045G>A, rs132630317) were identified in the three female XLHED patients. The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these variants could cause structural damage to EDA proteins. Analysis of the skewed X-chromosome inactivation revealed that extreme skewed X-chromosome inactivation was found in patient #35 (98:2), whereas it was comparatively moderate in patients #347 and #204 (21:79 and 30:70). Our results broaden the variation spectrum of EDA and the phenotype spectrum of XLHED, which could help with clinical diagnosis, treatment, and genetic counseling.

KDF1 Novel Variant Causes Unique Dental and Oral Epithelial Defects.

Keratinocyte differentiation factor 1 (KDF1) is a recently identified and rare candidate gene for human tooth agenesis; however, KDF1-related morphological characteristics and pathological changes in dental tissue and the oral epithelium remain largely unknown. Here, we employed whole-exome sequencing (WES) and Sanger sequencing to screen for the suspected variants in a cohort of 151 tooth agenesis patients, and we segregated a novel KDF1 heterozygous missense variation, c.920G>C (p.R307P), in a non-syndromic tooth agenesis family. Essential bioinformatics analyses and tertiary structural predictions were performed to analyze the structural changes and functional impacts of the novel KDF1 variant. The subsequent functional assessment using a TOP-flash/FOP-flash luciferase reporter system demonstrated that KDF1 variants suppressed the activation of canonical Wnt signaling in 293T cells. To comprehensively investigate the KDF1-related oral morphological anomalies, we performed scanning electron microscopy and ground section of the lower right lateral deciduous incisor extracted from #285 proband, and histopathological assessment of the gingiva. The phenotypic analyses revealed a series of tooth morphological anomalies related to the KDF1 variant R307P, including a shovel-shaped lingual surface of incisors and cornicione-shaped marginal ridges with anomalous morphological occlusal grooves of premolars and molars. Notably, keratinized gingival epithelium abnormalities were revealed in the proband and characterized by epithelial dyskeratosis with residual nuclei, indistinct stratum granulosum, epithelial hyperproliferation, and impaired epithelial differentiation. Our findings revealed new developmental anomalies in the tooth and gingival epithelium of a non-syndromic tooth agenesis individual with a novel pathogenic KDF1 variant, broadening the phenotypic spectrum of KDF1-related disorders and providing new evidence for the crucial role of KDF1 in regulating human dental and oral epithelial development.

Four Novel PAX9 Variants and the PAX9-Related Non-Syndromic Tooth Agenesis Patterns.

The purpose of this research was to investigate and identify PAX9 gene variants in four Chinese families with non-syndromic tooth agenesis. We identified pathogenic gene variants by whole-exome sequencing (WES) and Sanger sequencing and then studied the effects of these variants on function by bioinformatics analysis and in vitro experiments. Four novel PAX9 heterozygous variants were identified: two missense variants (c.191G > T (p.G64V) and c.350T > G (p.V117G)) and two frameshift variants (c.352delC (p.S119Pfs*2) and c.648_649insC(p.Y217Lfs*100)). The bioinformatics analysis showed that these variants might be pathogenic. The tertiary structure analysis showed that these four variants could cause structural damage to PAX9 proteins. In vitro functional studies demonstrated that (1) the p.Y217Lfs*100 variant greatly affects mRNA stability, thereby affecting endogenous expression; (2) the p. S119Pfs* 2 variant impairs the subcellular localization of the nuclear expression of the wild-type PAX9 protein; and (3) the four variants (p.G64V, p.V117G, p.S119Pfs*2, and p.Y217Lfs*100) all significantly affect the downstream transcriptional activity of the BMP4 gene. In addition, we summarized and analyzed tooth missing positions caused by PAX9 variants and found that the maxillary second molar (84.11%) and mandibular second molar (84.11%) were the most affected tooth positions by summarizing and analyzing the PAX9-related non-syndromic tooth agenesis positions. Our results broaden the variant spectrum of the PAX9 gene related to non-syndromic tooth agenesis and provide useful information for future genetic counseling.

Novel DLX3 variant identified in a family with tricho-dento-osseous syndrome.

OBJECTIVES: To identify DLX3 variants in a Chinese family with typical clinical manifestations of tricho-dento-osseous syndrome (TDO). DESIGN: Sanger sequencing was performed to detect DLX3 variants in the TDO family. Three-dimensional laser scanning microscopy, bioinformatic and conformational analyses were employed to explore the phenotypic characterization and the functional impact. RESULTS: We identified a novel heterozygous variant in the DLX3 gene (c.534G>C; p.Gln178His). Familial co-segregation verified an autosomal dominant inheritance pattern. Bioinformatic prediction demonstrated the deleterious effects of the variant, and DLX3 structure changes suggested the corresponding functional impairments. CONCLUSIONS: We identified a variant in the DLX3 gene in an integrated family of Han nationality for the first time. This study expands the variant spectrum of DLX3 and phenotype spectrum of TDO syndrome.

Progresses and Perspectives of Anti-PD-1/PD-L1 Antibody Therapy in Head and Neck Cancers.

Head and neck cancer is the 6th most common malignancy worldwide and urgently requires novel therapy methods to change the situation of low 5-years survival rate and poor prognosis. Targeted therapy provides more precision, higher efficiency while lower adverse effects than traditional treatments like surgery, radiotherapy, and chemotherapy. Blockade of PD-1 pathway with antibodies against PD-1 or PD-L1 is such a typical targeted therapy which reconstitutes anti-tumor activity of T cell in treatments of cancers, especially those highly expressing PD-L1, including head and neck cancers. There are many clinical trials all over the world and FDA has approved anti-PD-1/PD-L1 drugs for head and neck cancers. However, with the time going, the dark side of this therapy has emerged, including some serious side effects and drug resistance. Novel materials like nanoparticles and combination therapy have been developed to improve the efficacy. At the same time, standards for evaluation of activity and safety are to be established for this new therapy. Here we provide a systematic review with comprehensive depth on the application of anti-PD1/PD-L1 antibodies in head and neck cancer treatment: mechanism, drugs, clinical studies, influencing factors, adverse effects and managements, and the potential future developments.