Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers.

Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.

BRAF 1799T>A Mutation Frequency in Mexican Mestizo Patients with Papillary Thyroid Cancer.

Thyroid cancer is the most frequent endocrine malignancy, and its incidence and prevalence are increasing worldwide. Despite its generally good prognosis, the observed mortality rates are higher in the less-developed regions. This indicates that timely diagnosis and appropriate initial management of this disease are important to achieve a positive outcome. We performed an observational study in order to describe the frequency of the BRAF 1799T>A mutation in Mexican mestizo patients with thyroid nodules, a scarcely studied ethnic group with large populations. Competitive allele-specific Taqman PCR was performed in 147 samples of thyroid tissue DNA obtained from patients histologically diagnosed with papillary thyroid cancer (PTC), colloid goiters, and follicular adenomas. The BRAF 1799T>A mutation frequency was 61.1% in PTC samples (p = 4.99 × 10-11). Potential diagnostic values were as follows: sensitivity, 61.1%; specificity, 96%; PPV, 94.2%; NPV, 69.5%; accuracy, 77.9%. Taking into account the fact that this mutation is not frequently found in cytologically indeterminate nodules, we suggest that the BRAF mutational analysis should be implemented in the clinical setting along with other diagnostic criteria such as USG, in order to contribute to diagnosis and to surgical decision-making during the initial management of thyroid nodules in Mexican public hospitals.

The molecular fingerprint of human papillomavirus infection and its effect on the Langerhans cell population in squamous cell carcinomas of the genital skin.

BACKGROUND: Information is scarce about the presence of molecular alterations related to human papillomavirus (HPV) infection in squamous cell carcinomas of the genital skin and about the effect of this infection in the number of Langerhans cells present in these tumors. AIMS: To determine the presence of HPV in genital skin squamous cell carcinomas and to see the relationship between HPV infection and changes in the expression of Ki-67 antigen (Ki-67), p53 protein (p53), retinoblastoma protein (pRb) and E-cadherin and to alterations in Langerhans cell density, if any. METHODS: A descriptive, comparative, retrospective and cross-sectional study was performed with all the cases diagnosed as squamous cell carcinomas of the genital skin at the Dermatopathology Service from 2001 to 2011. The diagnosis was verified by histopathological examination. The presence of HPV was examined using chromogenic in situ hybridization, and protein expression was studied via immunohistochemical analysis. RESULTS: The 34 cases studied were verified as squamous cell carcinomas and 44.1% were HPV positive. The degree of expression of pRb was 17.50% ±14.11% (mean ± SD) in HPV-positive cases and 29.74% ±20.38% in HPV-negative cases (P = 0.0236). The degree of expression of Ki-67 was 47.67% ±30.64% in HPV-positive cases and 29.87% ±15.95% in HPV-negative cases (P = 0.0273). CONCLUSION: HPV infection was related to lower pRb expression and higher Ki-67 expression in comparison with HPV negative samples. We could not find a relationship between HPV infection and the degree of expression of p53 and E-cadherin or with Langerhans cell density.

Reporting detection of Chlamydia trachomatis DNA in tissues of neonatal death cases.

OBJECTIVE: to determine whether C. trachomatis was present in neonates with infection, but without an isolated pathogen, who died during the first week of life. METHODS: early neonatal death cases whose causes of death had been previously adjudicated by the institutional mortality committee were randomly selected. End-point and real-time polymerase chain reaction of the C. trachomatis omp1 gene was used to blindly identify the presence of chlamydial DNA in the paraffinized samples of five organs (from authorized autopsies) of each of the dead neonates. Additionally, differential diagnoses were conducted by amplifying a fragment of the 16S rRNA of Mycoplasma spp. RESULTS: in five cases (35.7%), C. trachomatis DNA was found in one or more organs. Severe neonatal infection was present in three cases; one of them corresponded to genotype D of C. trachomatis. Interestingly, another case fulfilled the same criteria but had a positive polymerase chain reaction for Mycoplasma hominis, a pathogen known to produce sepsis in newborns. CONCLUSION: the use of molecular biology techniques in these cases of early infant mortality demonstrated that C. trachomatis could play a role in the development of severe infection and in early neonatal death, similarly to that observed with Mycoplasma hominis. Further study is required to determine the pathogenesis of this perinatal infection.