Functional Assessment of Clubfoot Associated HOXA9, TPM1, and TPM2 Variants Suggests a Potential Gene Regulation Mechanism.

BACKGROUND: Isolated nonsyndromic clubfoot is a common birth defect affecting 135,000 newborns worldwide each year. Although treatment has improved, substantial long-term morbidity persists. Genetic causes have been implicated in family-based studies but the genetic changes have eluded identification. Previously, using a candidate gene approach in our family-based dataset, we identified associations between clubfoot and four single nucleotide polymorphisms (SNPs) located in potential regulatory regions of genes involved in muscle development and patterning (HOXA9) and muscle function (TPM1 and TPM2) were identified. QUESTIONS/PURPOSES: Four SNPs, rs3801776/HOXA9, rs4075583/TPM1, rs2025126/TPM2, and rs2145925/TPM2, located in potential regulatory regions, were evaluated to determine whether they altered promoter activity. METHODS: Electrophoretic mobility shift assays were performed on these four SNPs to identify allele-specific DNA-protein interactions. SNPs showing differential banding patterns were assessed for effect on promoter activity by luciferase assay. Undifferentiated (for HOXA9) and differentiated (for TPM1 and TPM2) mouse cells were used in functional assays as a proxy for the in vivo developmental stage. RESULTS: Functional analyses showed that the ancestral alleles of rs3801776/HOXA9, rs4075583/TPM1, and rs2025126/TPM2 and the alternate allele of rs2145925/TPM2 created allele-specific nuclear protein interactions and caused higher promoter activity. Interestingly, while rs4075583/TPM1 showed an allele-specific nuclear protein interaction, an effect on promoter activity was observed only when rs4075583/TPM1 was expressed in the 1.7kb haplotype construct. CONCLUSION: Our results show that associated promoter variants in HOXA9, TPM1, and TPM2, alter promoter expression suggesting that they have a functional role. Moreover and importantly, we show that alterations in promoter activity may be observed only in the context of the genomic architecture. Therefore, future studies focusing on proteins binding to these regulatory SNPs may provide important key insights into gene regulation in clubfoot. CLINICAL RELEVANCE: Identifying the genetic risk signature for clubfoot is important to provide accurate genetic counseling for at-risk families, for development of prevention programs and new treatments.

Generation of luciferase-expressing Leishmania infantum chagasi and assessment of miltefosine efficacy in infected hamsters through bioimaging.

BACKGROUND: The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters. METHODOLOGY/PRINCIPAL FINDINGS: A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival. CONCLUSIONS/SIGNIFICANCE: Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.

Assessment of the estrogenicity of isoflavonoids, using MCF-7-ERE-Luc cells.

In the current study, our research focused on the estrogenic activity of isoflavonoids, mainly genistein, biochanin A and daidzein. Genistein enhanced the reporter gene expression of MCF-7-ERE-Luc cells, at a concentration as low as 10 nM, with a concentration of 100 nM the achieved gene expression effects were similar to those of 10 pM 17beta-estradiol, Based on the estrogenic activities of biochanin A and daidzein, hydroxyl groups at the 4' and 5 positions are needed for the maximal effect of the genistein. The estrogenic effects of these isoflavonoids were inhibited by the concomitant treatment with tamoxifen. The data showed that the estrogenic effects of isoflavonoids were mediated through estrogen receptors. When the isoflavonoids were tested as mixtures, the estrogenic effects were lower than the arithmetic sum of those induced by each individual isoflavonoid. The estrogenic potency of each isoflavonoid was presented at EC50 levels with a 17beta-estradiol equivalent concentration (EEQ) based on the dose response of each chemical. The EC50s and EEQs of genistein, biochanin A and daidzein were 4.15, 0.89 and 0.18 microM, and 15.0, 5.12 and 1.83 microM/M, respectively. Our data clearly demonstrated that the pERE-luciferase reporter gene assay was suited for the sensitive and quantitative measurement, and large scale screening, of the estrogenicity of chemicals in vitro.

Assessment of interactions of diverse ternary mixtures in an estrogen receptor-alpha reporter assay.

This study used an MCF-7 cell based ER-alpha reporter gene assay to assess chemical interactions within the following ternary mixtures: (1) three synthetic pesticides, methoxychlor (MXC), o,p-DDT, and dieldrin; (2) three polyaromatic hydrocarbons, benzo[a]pyrene (BAP), 1,2-benzanthracene (BENZ), and chrysene (CHRY); and (3) an endogenous estrogen, [17beta-estradiol, (E(2))]; a phytoestrogen, genistein (GEN); and a synthetic estrogen, o,p-DDT. A full factorial design in which four concentrations of each chemical were assessed in all possible combinations (64 treatment groups) was utilized. In addition, mixtures were tested in both a low range (concentrations near the individual chemical response thresholds) and a high range ( approximately 2-10x higher) experiment. A response surface was estimated using a nonlinear mixed model, and the cumulative response in each mixture was evaluated for departure from additivity. The mixture of E(2), GEN, and DDT exhibited antagonistic interactions (p < 0.001) in both concentration ranges. However, specific interactions between E(2)/GEN and E(2)/DDT differed between the low and high range concentrations. The BAP/BENZ/CHRY mixture did not depart significantly from additivity (p = 0.66) in either concentration range, although response levels were generally low. The MXC/DDT/dieldrin mixture did not depart significantly from additivity in either the high (p = 0.065), or low dose range (p = 0.506), with generally minimal responses dominated by MXC and DDT. This methodology has allowed for a rigorous statistical evaluation of potential departures from additive interactions in endocrine active mixtures. In no case was a significantly greater-than-additive (synergistic) interaction observed.

Assessment of pMTL construct for detection in vivo of luciferase expression and fate of the transgene in the zebrafish, Brachydanio rerio.

Early embryos of the zebrafish Brachydanio rerio were cytoplasmically microinjected with pMTL plasmid containing firefly luciferase gene, in both linearized- and supercoiled-plasmid forms, to evaluate in vivo expression, pattern of integration and germ-line transmission of the transgene in the host fish. It was possible to detect luciferase expression in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong luciferase activity was detected 15-20 hours after injection, coinciding with early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F1 progeny. In vivo screening for expression of the transgene in the developing embryo using luciferase assay as a method for detecting the presence of the transgenic fish compares favourably, with PCR and Southern blot analysis (SBA). No integration of the introduced DNA into the genome of treated fish and their progeny, was detected, instead it remained in extrachromosomal form. Most of the first generation founders were mosaic. Germline transmission was observed in one individual only. A probable reason for the absence of integration in this study when compared to the varying frequencies reported earlier in the same fish is discussed.