The role of cholinesterases in chagas disease.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze acetylcholine (ACh) in neural synaptic clefts and are primarily found in erythrocytes and blood plasma, respectively. Besides inactivating ACh, cholinesterases may play a non-classical role in inflammation and in immune response. In a previous study, we reported that BChE levels were decreased in chronic Chagas disease patients presenting the mega syndromes. In this series, we reported that: i) the activity of AChE did not differ between patients and controls, irrespective of the presence or not of the 1057C > A ACHE polymorphism, and ii) the increased BChE levels modestly influenced the AChE activity in Chagas disease.

Correlation of plasma butyrylcholinesterase concentration with Acethylcholinesterase H353N polymorphism in the inflammatory response of Chagas disease patients.

Trypanosoma cruzi infection triggers an intense production of pro-inflammatory cytokines mediated by T helper 1 lymphocytes, inducing the anti-inflammatory reflex of acetylcholine (ACh). The ACh concentration modulation is associated to the two major esterases, the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). AChE H353N protein polymorphism is related to low Chagas chronic disease prognostic. In order to evaluate the correlation of plasmatic BuChE concentration and the presence of AChE H353N polymorphism in Chagas disease patients and healthy individuals, we studied two groups of individuals, one of 61 Chagas disease patients and another of 74 healthy individuals. Plasma concentration of BuChE was measured by the chemiluminescent method and AChE H353N polymorphism was investigated by PCR-RFLP and sequencing of the respective encoding AChE gene fragment. The BuChE concentration was statistically higher in Chagas disease patients, with no AChE genotype significant influence. AChE genotypes YT*A/YT*A, YT*A/YT*B and YT*B/YT*B, respectively, were expressed in 53 (86.88%), 7 (11.46%) and one (1.64%) chagasic patients, and in 68 (91.89%), 6 (8.10%) and none healthy individuals. BuChE activity may represent an important marker for chronic Chagas disease inflammatory process and prognostic. Lower BuChE concentration correlated with AChE YT*B allele, although without statistical power.

Molecular studies of DO alleles reveal that JO is more prevalent than HY in Brazil, whereas HY is more prevalent in New York.

Because of the scarcity of anti-Hy and anti-Jo(a), hemagglutination typing for the Dombrock blood group system antigens, Hy and Jo(a), is not feasible. The molecular bases associated with these antigens have been determined, making it possible to distinguish HY and JO from wild-type DO. This provides a tool to predict the probable phenotype of patients and to screen for antigen-negative donors. PCR-RFLP assays and a microchip assay were used to determine the frequency of HY and JO alleles in donors from Brazil and New York. DNA from random Brazilian donors, 288 by PCR-RFLP and 599 by the bead array method (BeadChip, BioArray Solutions, Warren, NJ), was tested to determine 323G/T (HY+/HY-) and 350C>T (JO+/JO-) single-nucleotide polymorphisms. In New York, 27,226 donors who self-identified as being African American were tested by hemagglutination with anti-Gy(a). Nonreactive and weakly reactive samples were tested by PCR-RFLP for the same alleles as listed above. In Brazil, 30 (3.4%) of the samples were JO/DO and 13 (1.4%) were HY/DO. In New York, of the samples that had HY or JO alleles, 14 were homozygous HY/HY 132 were heterozygous HY/DO, 13 were heterozygous HY/JO, 14 were heterozygous JO/DO, and 3 were homozygous JO/JO. These results show that in donors from Brazil, JO (30 alleles) is more than twice as prevalent as HY (13 alleles), whereas in donors from New York, HY (173 alleles) was more than five times more common than JO (33 alleles).

Dombrock gene analysis in Brazilian people reveals novel alleles.

BACKGROUND AND OBJECTIVES: The Doa and Dob polymorphisms are associated with three single nucleotide polymorphisms (SNPs) in exon 2 of the DO gene: 378C/T, 624T/C and 793A/G for the DOA and DOB alleles, respectively. The SNPs 350C/T (JO allele) and 323G/T (HY allele) are associated with the Jo(a-) and Hy-negative phenotypes. Recently, two new DO alleles [DOB-SH (378C, 624C, 793G) and DOA-HA (378T, 624T, 793A)] were identified using microarray technology. Although the molecular background of Dombrock alleles is well defined, no studies have been conducted in the Brazilian population. MATERIALS AND METHODS: We employed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based assays and a microarray assay to determine the frequency of the DO alleles (DOA, DOB, HY1, HY2 and JO) in Brazilians. We tested DNA of 288 Brazilians from three different ethnic groups by PCR-RFLP to determine the 793A/G (DOA/DOB), 323G/T (HY), 350C/T (JO) and 898C/G (HY1/HY2) SNPs. We also tested DNA from 162 blood donors by using the HEA Beadchip assay to determine the 378C/T, 624T/C, 793A/G (DOA/DOB), 350C/T (JO allele) and 323G/T (HY) SNPs. RESULTS: Two novel allele combinations were found in our samples: the DOB allele (793G and 323G) associated with 898G (DOB-WL); and an allele carrying the nucleotides 378C, 624C, 793A and 323G (DOA-SH). We also found the DOB-SH and DOA-HA.alleles recently reported. CONCLUSIONS: Our data demonstrate high heterogeneity of DO alleles in the Brazilian population. Our study also highlights the importance of testing a cohort of different populations to determine DO haplotypes and of establishing reliable genotyping tests for predicting Doa/Dob status.

A novel DI*A allele without the Band 3-Memphis mutation in Amazonian Indians.

BACKGROUND AND OBJECTIVES: The blood-group antigens Dia and Dib are carried on erythrocyte band 3 and are defined by a single amino acid substitution at position 854 (Leu for Dia and Pro for Dib). The Band 3-Memphis variant has a point mutation (166A>G) in the SLC4A1 gene, which encodes the amino acid substitution Lys56Glu. Two types of Band 3-Memphis, variants I and II, are distinguished by their susceptibility to covalent labelling with 4,4'-diisothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS). Memphis II is more readily labelled than Memphis I or normal band 3. It is reported that Memphis II is associated with Dia. In a study designed to determine the frequency of the DI*A/DI*B and 166A>G polymorphisms in different populations in Brazil, we found a new DI*A allele. MATERIALS AND METHODS: We studied DNA samples from 70 Amazonian Indians, 71 individuals of Japanese descent, 93 random Brazilian blood donors and 84 blacks with sickle cell disease. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses were performed on all samples, using MspI for DI*A/DI*B (exon 19) and MnlI for 166A>G (exon 4). Exon 4 and exon 19 from four outliers were sequenced. RESULTS: Among Amazonian Indians, DI*A and 166G mutations both had a high frequency (0.57 and 0.54, respectively). In individuals of Japanese descent, these alleles were moderately frequent (0.07 and 0.19, respectively). We identified a new allele with DI*A and 166A (56Lys) in four Amazonian Indians. CONCLUSIONS: Our results revealed that DI*A does not have a strict association with 166G. They also show the relevance of testing a cohort of different populations.