Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project

Abstract The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.

Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project.

The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.

Haplotypes from the SLC45A2 gene are associated with the presence of freckles and eye, hair and skin pigmentation in Brazil.

The Solute Carrier Family 45, Member 2 (SLC45A2) gene encodes the Membrane-Associated Transporter Protein (MATP), which mediates melanin synthesis by tyrosinase trafficking and proton transportation to melanosomes. At least two SLC45A2 coding SNPs [E272K (rs26722) and L374F (rs16891982)] were reported influencing normal variation of human pigmentation. Here we aimed at evaluating the influence of haplotypes of 12 SNPs within SLC45A2 in the determination of eye, hair and skin pigmentation in a highly admixed population sample and comparing their frequencies with the ones found in data retrieved from the 1000 Genomes Project. To achieve this goal, 12 SLC45A2 SNPs were evaluated in 288 unrelated individuals from the Ribeirão Preto city area, Southeastern Brazil. SNPs were genotyped by PCR-RFLP or Allele-specific PCR, followed by polyacrylamide gel electrophoresis. Haplotypes of each individual were inferred by two independent computational methods, PHASE and Partition-Ligation-Expectation-Maximization (PL-EM) algorithms, and 34 different haplotypes were identified. The hp9 haplotype was the most frequent (58.3%) and was associated with the presence of blond/red hair, pale skin, blue eyes and freckles. All haplotypes significantly associated with dark or light pigmentation features harbor the 374L and 374F alleles, respectively. These results emphasize the role played by haplotypes at SLC45A2 in the determination of pigmentation aspects of human populations and reinforce the relevance of SNP L374F in human pigmentation.

IL-18, TNF, and IFN-γ alleles and genotypes are associated with susceptibility to chronic hepatitis B infection and severity of liver injury.

This study evaluated the association of polymorphisms in the IL-18 (-607C/A and -137C/G), IFNγ (+874 A/T), and TNF (-238 A/G and -308 A/G) genes with susceptibility to HBV infection and severity of liver injury. A total of 259 chronic HBV-infected patients followed at the University Hospital, Faculty of Medicine of Ribeirão Preto, São Paulo, Brazil, and 202 healthy individuals were studied. Four Single Nucleotide Polymorphisms (SNPs) were amplified by Polymerase Chain Reaction (PCR). Liver biopsy was performed in 212 HBV-infected patients and classified according to severity of liver fibrosis (scores 0-4) and necroinflammatory activity (HAI scores 0-18). TNF-308*A allele (P < 0.001; OR = 2.16) and TNF -308 AA genotype (P = 0.026; OR = 5.43) were associated with susceptibility to HBV infection. An association was found between severe liver fibrosis when compared to mild fibrosis and the following polymorphisms: Alleles IL-18 -137*G (P = 0.004; OR = 3.45), TNF -308*A (P < 0.001; OR = 3.39), and IFNγ +874*T (P = 0.029; OR = 1.85) and IL-18 -137 GG genotype (P = 0.009; OR = 3.70). No significant association was found between IL-18 (-607 A/C) polymorphism and severity of liver fibrosis. Alleles IL-18 -137*G (P = 0.028; OR = 2.64) and TNF-308*A (P = 0.002; OR = 3.06) and IL-18 -137 GG genotype (P = 0.011; OR = 4.20) were associated with severe necroinflammatory activity (HAI>12) when compared to mild necroinflammatory activity (HAI 1-8). The results suggest that IL-18 -137C/G, TNF-308 G/A and IFNγ +874 A/T SNPs were associated to more severe liver injury in chronic HBV infection. TNF -308*A allele and TNF -308 AA genotype could play a role in the susceptibility to HBV infection.