Identification of Compound Heterozygous EVC2 Gene Variants in Two Mexican Families with Ellis-van Creveld Syndrome.

BACKGROUND: Ellis-van Creveld syndrome (EvCS) is an autosomal recessive ciliopathy with a disproportionate short stature, polydactyly, dystrophic nails, oral defects, and cardiac anomalies. It is caused by pathogenic variants in the EVC or EVC2 genes. To obtain further insight into the genetics of EvCS, we identified the genetic defect for the EVC2 gene in two Mexican patients. METHODS: Two Mexican families were enrolled in this study. Exome sequencing was applied in the probands to screen potential genetic variant(s), and then Sanger sequencing was used to identify the variant in the parents. Finally, a prediction of the three-dimensional structure of the mutant proteins was made. RESULTS: One patient has a compound heterozygous EVC2 mutation: a novel heterozygous variant c.519_519 + 1delinsT inherited from her mother, and a heterozygous variant c.2161delC (p.L721fs) inherited from her father. The second patient has a previously reported compound heterozygous EVC2 mutation: nonsense mutation c.645G > A (p.W215*) in exon 5 inherited from her mother, and c.273dup (p.K92fs) in exon 2 inherited from her father. In both cases, the diagnostic was Ellis-van Creveld syndrome. Three-dimensional modeling of the EVC2 protein showed that truncated proteins are produced in both patients due to the generation of premature stop codons. CONCLUSION: The identified novel heterozygous EVC2 variants, c.2161delC and c.519_519 + 1delinsT, were responsible for the Ellis-van Creveld syndrome in one of the Mexican patients. In the second Mexican patient, we identified a compound heterozygous variant, c.645G > A and c.273dup, responsible for EvCS. The findings in this study extend the EVC2 mutation spectrum and may provide new insights into the EVC2 causation and diagnosis with implications for genetic counseling and clinical management.

Human papillomavirus infection and seroprevalence among female university students in Mexico.

Human papillomavirus (HPV) is one of the most common causes of sexually transmitted diseases, and the main etiology of cervical cancer. This study was aimed to assess type-specific cervical HPV prevalence and their association with HPV-specific antibodies in a cohort of female university students. HPV genotyping was performed by amplifying and sequencing a fragment of the L1 protein. A BLAST search was performed to identify HPV types. HPV-specific IgG antibodies were measured by ELISA in serum samples. A total of 129 women participated, with an average age of 21.75 years. The prevalence of vaginal HPV infection was 74.42%. The most predominant high-risk HPV types were 18 (13.95%), 31 (10.85%), and 16 (9.3%). We found that early age at coitarche and a higher number of sexual partners were significantly associated with a high prevalence of HPV infection. In addition to sexual behavior, we observed that the presence of serum-specific IgG antibodies against HPV can impact the prevalence of the virus. Seropositivity to HPV-16 and HPV-18 was associated with a lower prevalence of HPV-16, but not for other HPV types. Of note, there was a lower proportion of HPV-specific seropositivity in women who had the presence of the same HPV type in a cervical specimen, suggesting an immunoregulatory mechanism associated with the viral infection. In conclusion, the prevalence of HPV in university women was higher than expected and it was associated with early age of sexual debut, an increasing number of sexual partners, and a low proportion of HPV seropositivity.

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

The combination astemizole-gefitinib as a potential therapy for human lung cancer.

Lung cancer is a major cause of cancer mortality. Thus, novel therapies are urgently needed. Repositioning of old drugs is gaining great interest in cancer treatment. Astemizole is an antihistamine proposed to be repositioned for cancer therapy. This drug targets several molecules involved in cancer including histamine receptors, ABC transporters and the potassium channels Eag1 and HERG. Astemizole inhibits the proliferation of different cancer cells including those from cervix, breast, leukemia and liver. Gefitinib is widely used to treat lung cancer; however, no response or drug resistance occurs in many cases. Here, we studied the combined effect of astemizole and gefitinib on the proliferation, survival, apoptosis and gene and protein expression of Eag1 channels in the human lung cancer cell lines A549 and NCI-H1975. Cell proliferation and survival were studied by the MTT method and the colony formation assay, respectively; apoptosis was investigated by flow cytometry. Gene expression was assessed by real-time polymerase chain reaction (RT-PCR), and protein expression was studied by Western blot analysis and immunocytochemistry. We obtained the inhibitory concentrations 20 and 50 (IC20 and IC50, respectively) values for each drug from the cell proliferation experiments. Drug combination at their IC20 had a superior effect by reducing cell proliferation and survival in up to 80% and 100%, respectively. The drugs alone did not affect apoptosis of H1975 cells, but the drug combination at their IC20 increased apoptosis roughly four times in comparison to the effect of the drugs alone. Eag1 mRNA levels and protein expression were decreased by the drug combination in A549 cells, and astemizole induced subcellular localization changes of the channel protein in these cells. Our in vitro studies strongly suggest that the combination astemizole-gefitinib may be a novel and promising therapy for lung cancer patients.

HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.

BACKGROUND: Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. METHODS: HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). RESULTS: We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrindim), transitory amplifying cells (α6-integrinbri/CD71bri) and progenitor cells (α6-integrinbri/CD71dim). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrinbri/CD71dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. CONCLUSIONS: Our data demonstrated the presence of a small subpopulation with epithelial "progenitor cells" characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.