Wilson Baleotti

DARC (Duffy) and BCAM (Lutheran) reduced expression in thyroid cancer

Duffy or DARC (Duffy Antigen Receptor for Chemokines) is a glycosylated membrane protein that selectively binds angiogenic chemokines. Previous in vivo and in vitro studies of DARC function in cancer have associated DARC over expression with better prognosis, decreased metastatic potential, and inhibition of tumor-associated neovascularization. Another carcinogenesis-associated antigen is Lutheran or BCAM (basal cell adhesion molecule), a surface glycoprotein that acts as a receptor for the extracellular matrix protein, laminin. BCAM is a protein related to tumor progression; and, its over expression is associated with skin, ovarian and pancreatic cancers. We explored DARC and BCAM functions and investigated whether or not their expressions were altered in thyroid cancer. The expression of DARC and BCAM were evaluated by quantitative real-time PCR (qPCR) in a set of 18 normal thyroid tissues (NT), 15 follicular adenomas (FTA), 17 follicular carcinomas (FTC), and 122 papillary thyroid carcinomas (PTC), including 78 classical (CVPTC) and 44 follicular variant (FVPTC). RNA was isolated, reverse transcribed to cDNA, and used in qPCR reactions containing SYBR Green. The relative expression value was calculated using ribosomal protein S8 as an internal control. When we compared benign (NT and FTA) versus malignant samples (FTC, CVPTC and FVPTC) we observed a significant decrease of DARC and BCAM relative expression in malignant cases. Additionally, we correlated clinic-pathological features (tumor size, presence of metastasis, presence of lymphocyte infiltrate) with DARC and BCAM expression. We found a diminished expression of DARC in PTC samples, which was correlated with tumor size and presence of a lymphocyte infiltrate. We, also, found a correlation between decreased BCAM expression and tumor size or presence of metastasis. DARC and BCAM expression was associated with pathogenesis of thyroid carcinoma and correlated with clinical-pathological features.

HNA-3 gene frequencies in Brazilians and a new polymerase chain reaction-restriction fragment length polymorphism method for HNA-3a/3b genotyping

HNA‐3 antigens are the result of a rs2288904 single‐nucleotide polymorphism (SNP) in the CTL2, and the HNA‐3a and HNA‐3b variants are encoded by a guanine and adenine at Nucleotide Position 461. Anti‐HNA‐3 are involved in severe transfusion‐related acute lung injury reactions and in neonatal alloimmune neutropenia. Since the distribution of the HNA‐3 system was unknown in South Americans, in this study we determined the frequency of the HNA‐3 alleles in Brazilians.

HLA-DRB1*07:01 allele is primarily associated with the Diego a alloimmunization in a Brazilian population

The Diego blood group presents a major polymorphic site at Residue 854, causing a proline (Dib antigen) to leucine (Dia antigen) substitution. Dia alloimmunization has been observed among Asian and Native South American populations. Considering that Brazilians represent a genetically diverse population, and considering that we have observed a high incidence of Dia alloimmunization, we typed HLA‐DRB1 alleles in these patients and performed in silico studies to investigate the possible associated mechanisms.

A PCR-Based Strategy for Dombrock Screening in Brazilian Blood Donors Reveals a Novel Allele: The DO{*}A-WL

Background: Determination of the molecular basis underlying the antigens in the Dombrock blood group system has shown various rearrangements between the alleles associated with DO*A and DO*B. Based on this, we employed a PCR‐based strategy to screen DO alleles (DO*A, DO*B, HY*1, HY*2 and JO) in Brazilians. Methods: We tested DNA of 278 Brazilian blood donors by PCR‐RFLP on plates of 96 wells to determine the 793A/G (DO*A/DO*B), 323G/T (HY), 350C/T (JO) and 898C/G (HY*1/HY*2) single nucletide polymorphisms. In order to confirm the results sequence analysis was also performed. Results: When samples of these donors were analyzed, a novel allele combination, the DO*A allele (793A and 323G) associated with 898G was identified and designated as DO*A‐WL allele. This new allele encoding 300Val is the same as HY*1 at nucleotide 898 on the molecular background of DO*A. Among the 556 alleles analyzed by PCR‐RFLP, 3 were DO*A‐WL and 78 were DO*B‐WL. This represents an overall frequency of 0.5% for DO*A‐WL and 14% for DO*B‐WL across the population studied. Conclusion: Molecular screening of Brazilians revealed one novel allele, the DO*A‐WL. Our data highlight the importance of testing a cohort of different populations to determine DO haplotypes and to establish reliable genotyping tests for predicting Doa/Dob status. J. Clin. Lab. Anal. 25:79–82, 2011.

An easy and efficient strategy for KEL genotyping in a multiethnic population

Background The Kell blood group system expresses high and low frequency antigens with the most important in relation to transfusion including the antithetic KEL1 and KEL2; KEL3 and KEL4; KEL6 and KEL7 antigens. Kell is a clinically relevant system, as it is highly immunogenic and anti-KEL antibodies are associated with hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Although required in some situations, Kell antigen phenotyping is restricted due to technical limitations. In these cases, molecular approaches maybe a solution. This study proposes three polymerase chain reaction genotyping protocols to analyze the single nucleotide polymorphisms responsible for six Kell antithetic antigens expressed in a Brazilian population. Methods DNA was extracted from 800 blood donor samples and three polymerase chain reaction-restriction fragment length polymorphism protocols were used to genotype the KEL*1/KEL*2, KEL*3/KEL*4 and KEL*6/KEL*7 alleles. KEL*3/KEL*4 and KEL*6/KEL*7 genotyping was standardized using the NlaIII and MnlI restriction enzymes and validated using sequencing. KEL*1/KEL*2 genotyping was performed using a previously reported assay. Results KEL genotyping was successfully implemented in the service; the following distribution of KEL alleles was obtained for a population from southeastern Brazil: KEL*1 (2.2%), KEL*2 (97.8%), KEL*3 (0.69%), KEL*4 (99.31%), KEL*6 (2.69%) and KEL*7 (97.31%). Additionally, two individuals with rare genotypes, KEL*1/KEL*1 and KEL*3/KEL*3, were identified. Conclusion KEL allele genotyping using these methods proved to be reliable and applicable to predict Kell antigen expressions in a Brazilian cohort. This easy and efficient strategy can be employed to provide safer transfusions and to help in rare donor screening.

576 Hla and Chronic Urticaria with Positive Autologous Serum Skin Test among Brazilians

Background Many autoimmune diseases are associated with certain alleles of the human leukocyte antigen (HLA) system, and recent studies have shown that, in many cases, chronic urticaria has autoimmune etiology. An association between class I and II alleles of the major histocompatibility complex (MHC) and idiopathic chronic urticaria (ICU) has previously been observed in different populations, but there are still no studies on Brazilian populations in this respect. The involvement of MHC classes I and II (loci A, B and DR) in Brazilian patients with ICU and a positive autologous serum skin test (ASST) was investigated and compared with a healthy population group. Methods DNA was extracted from the blood of 42 patients with ICU (28 women; mean age ± SD: 44 ± 12 years; range: 19 to 88 years) and MHC classes I and II alleles were determined using the polymerase chain reaction (PCR) and a laboratory test for oligonucleotide hybridization using a single-filament probe. The frequencies of these alleles in patients with chronic urticaria were compared with the frequencies in 1000 genetically unrelated voluntary blood donors from the same region of Brazil. The diagnosis of idiopathic chronic urticaria was based on the patients’ clinical histories and routine laboratory tests. Only the patients with positive ASSTs were selected. The allele distribution results from the patient and control groups were analyzed using odds ratios and 95% confidence intervals. Results No statistically significant differences were found between the ASST-positive patients with chronic urticaria and the control group, in relation to the MHC classes I and II alleles studied. Conclusions We found that in this population group, there was no specific association between the HLA alleles studied (HLA-A, HLA-B and DRB1) and ASST-positive chronic urticaria. We believe that further population studies are needed in order to investigate the possible existence of this association.

Molecular characterization of the Dombrock blood group system in a Brazilian population

Abstract The molecular basis of the Do a and Do b polymorphisms areassociated with three single-nucleotide polymorphisms (SNPs) onexon 2 of the DO gene: 378C>T, 624T>C and 793A>G, the DOA and DOB alleles. The SNPs 350C>T ( JO allele) and 323G>T (HYallele) are associated with: Jo(a-) and Hy-negative phenotypes.Recently, two new DO alleles were identified using microarraytechnology, DOB-SH (378C, 624C, 793G) and DOA-HA (378T,624T, 793A). Although the molecular background of the Dombrocksystem is well defined, no studies have been carried out in theBrazilian population.We used PCR-RFLP based assays and a microarray assay todetermine the frequency of the DO alleles ( DOA, DOB, HY1, HY2 and JO ) in Brazilians. We tested the DNA of 288 individuals byPCR-RFLP to determine the 793A>G ( DOA/DOB ), 323G>T (HY),350C>T ( JO ) and 898C>G ( HY1/HY2 ) SNPs. We also tested DNAfrom 162 blood donors using HEA Beadchip TM (BioArray Solutions,USA) to determine the 378C>T, 624T>C, 793A>G (