A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement.

BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.

Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

A polymorphic 3'UTR element in ATP1B1 regulates alternative polyadenylation and is associated with blood pressure.

Although variants in many genes have previously been shown to be associated with blood pressure (BP) levels, the molecular mechanism underlying these associations are mostly unknown. We identified a multi-allelic T-rich sequence (TRS) in the 3'UTR of ATP1B1 that varies in length and sequence composition (T22-27 and T12GT 3GT6). The 3'UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRS is downstream of the proximal polyadenylation site (A2). Therefore, we hypothesized that alleles of this TRS might influence ATP1B1 expression by regulating alternative polyadenylation. In vitro, the T12GT 3GT6 allele increases polyadenylation at the A2 polyadenylation site as compared to the T23 allele. Consistent with our hypothesis, the relative abundance of the A2-polyadenylated ATP1B1 mRNA was higher in human kidneys with at least one copy of the T12GT 3GT6 allele than in those lacking this allele. The T12GT 3GT6 allele is also associated with higher systolic BP (beta = 3.3 mmHg, p = 0.014) and diastolic BP (beta = 2.4 mmHg, p = 0.003) in a European-American population. Therefore, we have identified a novel multi-allelic TRS in the 3'UTR of ATP1B1 that is associated with higher BP and may mediate its effect by regulating the polyadenylation of the ATP1B1 mRNA.

SOX10 directly modulates ERBB3 transcription via an intronic neural crest enhancer.

BACKGROUND: The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them. RESULTS: In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish. CONCLUSIONS: Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.

Peroxisome-proliferator-activated receptor-binding protein (PBP) is essential for the growth of active Notch4-immortalized mammary epithelial cells by activating SOX10 expression.

PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.

Gpnmb is a melanoblast-expressed, MITF-dependent gene.

Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10-deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross-referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB-MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5'-most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes, and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.