BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis.

BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation (Bmp7R-GFlag) that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While Bmp7 null homozygotes are live born, Bmp7R-GFlag homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the Bmp7R-G allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either Bmp4 or Bmp7 lead to a similar spectrum of congenital defects in humans.

Next-Generation Sequencing in Salivary Gland Basal Cell Adenocarcinoma and Basal Cell Adenoma.

Basal cell adenoma and basal cell adenocarcinoma represent basaloid salivary gland neoplasms that show cyto-morphologic similarity but differ at the histologic level by their invasive qualities, as adenocarcinomas show invasion beyond their capsule, a finding not seen in the adenomas. Due to the low incidence of these tumors, the molecular mechanism underlying their pathogenesis is poorly understood. We sought to further delineate these neoplasms through mutation profiling by targeted next-generation sequencing (NGS). Twenty cases (basal cell adenocarcinoma = 10; basal cell adenoma = 10) were retrospectively selected from a previous analysis. NGS was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA). The data was analyzed using the Ion Torrent Suite Software (Life Technologies) followed by a laboratory-developed pipeline. One of eight cases of basal cell adenocarcinoma had a mutation, which was an activating mutation in PIK3CA (c.3140A>G, p.H1047R). No mutations were detected in the remaining basal cell adenocarcinomas. In the basal cell adenomas, the CTNNB1 p.I35T mutation was identified in three of nine (3/9) cases. A missense mutation in the ATM gene (c.2572T>C, p.F858L) was seen in a basal cell adenoma with an allele frequency of 53 %, raising the possibility of a germline mutation. The overall findings suggest that although there is cytomorphologic similarity, differences exist between these two tumors at the histologic and genetic level. Although the numbers of cases are limited, the aberrations in genes affecting different signaling pathways in the basal cell adenocarcinoma versus the basal cell adenomas suggest that basal cell adenocarcinoma likely arises de novo and not from basal cell adenoma.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.

Polymorphism Arg72Pro of p53 confers susceptibility to squamous cell carcinoma of lungs in a North Indian population.

The causes of lung cancer might be many, but genetic variation in the genes of carcinogen-metabolizing enzymes, tumor suppressor proteins, and/or DNA-repairing enzymes can also play a significant role in lung cancer susceptibility. The tumor suppressor protein p53 functions to induce cell cycle arrest, DNA repair, or apoptosis. Polymorphism in its gene can, therefore, play a significant role in cancer susceptibility. Present report evaluated the association of polymorphism in exon 4 Arg72Pro (G>C) of the p53 gene with lung cancer susceptibility using 175 cancer cases and 202 controls from the North Indian population. Binary logistic regression analysis revealed that the Pro72Pro genotype was significantly associated with increasing risk for lung cancer in younger age patients (≤55 years) (adjusted odds ratio [OR]=2.72, 95% confidence intervals [95% CI] 0.99-7.85, p<0.05). Histological stratification of lung cancer revealed that the Pro72Pro genotype was associated with higher risk for squamous cell carcinoma (OR=3.05, 95% CI 1.07-8.87, p<0.05). Genetic variation Arg72Pro of the p53 gene may contribute to higher risk of SCC of lung in the North Indian population.

Association of functionally important polymorphism of microsomal epoxide hydrolase gene (EPHX1) with lung cancer susceptibility.

Distribution and gene-environment interaction of EPHX1 polymorphism was evaluated in 175 lung cancer patients and 322 controls from north India. Two novel non-synonymous, Lys117Arg and Leu263Phe, and twelve single nucleotide polymorphisms were identified in the present study. Binary logistic regression analysis showed association of polymorphism Tyr113His with increased risk of lung cancer (OR = 2.2, 95% CI = 1.2-4.0, p < .05). Gene-environment interaction revealed that patients with His113His and smoking habit had significantly greater risk of lung cancer (OR = 4.52, 95% CI = 0.93-43.05, p < .05). Present study provided evidence that EPHX1 polymorphism is associated with lung cancer susceptibility in Indian population.

Microsomal epoxide hydrolase (EPHX1), slow (exon 3, 113His) and fast (exon 4, 139Arg) alleles confer susceptibility to squamous cell esophageal cancer.

Genetic polymorphisms in xenobiotic metabolizing enzymes may alter risk of various cancers. Present case-control study evaluated the influence of EPHX1 genetic variations on squamous cell esophageal cancer (ESCC) susceptibility in 107 patients and 320 controls. EPHX1 polymorphic alleles were genotyped by direct sequencing (exon 3, Tyr113His) or PCR-RFLP (exon 4, His139Arg). Patients with exon 3 genotypes (Tyr113His, His113His) and 113His allele were at risk of ESCC (OR(Tyr113His) 2.0, 95% CI=1.2-3.4, p=0.007; OR(His113His) 2.3 95% CI=1.0-5.2, p=0.03 and OR(His) 1.5, 95% CI=1.0-2.1, p=0.01). In contrast, individuals with exon 4, 139Arg allele were at low risk of cancer (OR 0.34, 95% CI=0.20-0.56, p=0.001). However, none of haplotype combinations of exon 3 (Tyr113His) and exon 4 (His139Arg) polymorphisms showed modulation of risk for ESCC. Sub-grouping of patients based on anatomical location of tumor predicted that patients with exon 3, His113His and Tyr113His genotypes were at higher risk for developing ESCC tumor at upper and middle third locations (OR 4.4, 95% CI=1.0-18.5, p=0.04; OR 2.5, 95% CI=1.3-5.0, p=0.005 respectively). The frequency of exon 4, His139Arg genotype was significantly lower in ESCC patients with lower third tumor location as compared to controls (14.8% vs. 36.3%, p=0.02). In case-only study, gene-environment interaction of EPHX1 genotypes with tobacco, alcohol and occupational exposures did not appear to modulate the cancer susceptibility. In conclusion, exon 3, Tyr113His genotype was associated with higher risk of ESCC particularly at upper and middle-third anatomical locations of tumor. However, His139Arg genotype of exon 4, exhibited low risk for ESCC as well as its clinical characteristics.