PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil.

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs

First identification of the benzimidazole resistance-associated F200Y SNP in the beta-tubulin gene in Ascaris lumbricoides.

The main control strategy for Ascaris lumbricoides is mass drug administration (especially with benzimidazoles), which can select strains of parasites resistant to treatment. Mutations in the beta-tubulin isotype-1 gene at codons 167, 198 and 200 have been linked to benzimidazole resistance in several nematodes. The mutation in codon 200 is the most frequent in different species of parasites, as previously observed in Necator americanus and Trichuris trichiura; however, this mutation has never been found in populations of A. lumbricoides. This study aimed to screen for single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codon 200 in A. lumbricoides. We developed a technique based on an amplification refractory mutation system (ARMS-PCR) for the analysis of 854 single A. lumbricoides eggs collected from 68 human stool samples from seven Brazilian states. We detected the mutation in codon 200 at a frequency of 0.5% (4/854). This is the first report of this mutation in A. lumbricoides. Although the observed frequency is low, its presence indicates that these parasite populations have the potential to develop high levels of resistance in the future. The methodology proposed here provides a powerful tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations

Structural variation and missense mutation in SBDS associated with Shwachman-Diamond Syndrome

Submitted by Nuzia Santos ([email protected]) on 2015-02-19T14:31:17Z No. of bitstreams: 1 2014_082.pdf: 2566126 bytes, checksum: 4b3fcff3506178c29fa9f993d17b1b72 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-19T14:31:29Z (GMT) No. of bitstreams: 1 2014_082.pdf: 2566126 bytes, checksum: 4b3fcff3506178c29fa9f993d17b1b72 (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-19T14:39:14Z (GMT) No. of bitstreams: 1 2014_082.pdf: 2566126 bytes, checksum: 4b3fcff3506178c29fa9f993d17b1b72 (MD5)Made available in DSpace on 2015-02-19T14:39:14Z (GMT). No. of bitstreams: 1 2014_082.pdf: 2566126 bytes, checksum: 4b3fcff3506178c29fa9f993d17b1b72 (MD5) Previous issue date: 2014Baylor College of Medicine. Department of Molecular and Human Genetics. Houston, TX, USA/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brazil.Baylor College of Medicine. Department of Molecular and Human Genetics. Houston, TX, USA.Baylor College of Medicine. Department of Pediatrics. Houston, TX, USA.Baylor College of Medicine. Department of Molecular and Human Genetics. Houston, TX, USA.Baylor College of Medicine. Human Genome Sequencing Center. Houston, TX, USABaylor College of Medicine. Human Genome Sequencing Center. Houston, TX, USABaylor College of Medicine. Human Genome Sequencing Center. Houston, TX, USABaylor College of Medicine. Human Genome Sequencing Center. Houston, TX, USABaylor College of Medicine. Human Genome Sequencing Center. Houston, TX, USAHospital for Sick Children. Research Institute. Program in Physiology and Experimental Medicine. Toronto, Ontario, Canada.Texas Children’s Hospital. Houston, TX, USA/ Baylor College of Medicine. Department of Radiology. Houston, TX, USA.Baylor College of Medicine. Department of Molecular and Human Genetics. Houston, TX, USA/ Baylor College of Medicine. Department of Pediatrics. Houston, TX, USA/Texas Children’s Hospital. Houston, TX, USABaylor College of Medicine. Department of Molecular and Human Genetics. Houston, TX, USA/ Baylor College of Medicine. Department of Pediatrics. Houston, TX, USA/Texas Children’s Hospital. Houston, TX, USABACKGROUND: Shwachman-Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused mainly by compound heterozygous mutations in SBDS. Structural variation (SV) involving the SBDS locus has been rarely reported in association with the disease. We aimed to determine whether an SV contributed to the pathogenesis of a case lacking biallelic SBDS point mutations. CASE PRESENTATION: Whole exome sequencing was performed in a patient with SDS lacking biallelic SBDS point mutations. Array comparative genomic hybridization and Southern blotting were used to seek SVs across the SBDS locus. Locus-specific polymerase chain reaction (PCR) encompassing flanking intronic sequence was also performed to investigate mutation within the locus. RNA expression and Western blotting were performed to analyze allele and protein expression. We found the child harbored a single missense mutation in SBDS (c.98A > C; p.K33T), inherited from the mother, and an SV in the SBDS locus, inherited from the father. The missense allele and SV segregated in accordance with Mendelian expectations for autosomal recessive SDS. Complementary DNA and western blotting analysis and locus specific PCR support the contention that the SV perturbed SBDS protein expression in the father and child. CONCLUSION: Our findings implicate genomic rearrangements in the pathogenesis of some cases of SDS and support patients lacking biallelic SBDS point mutations be tested for SV within the SBDS locus