Computational protocol: Association of germline genetic variants in RFC, IL15 and VDR genes with minimal residual disease in pediatric B-cell precursor ALL

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Protocol publication

[…] Selection of 23 polymorphic markers for the association study was based on the combination of two approaches. In a candidate gene approach, the genes were selected based on their involvement in metabolic pathways of drugs used in ALL therapy, as evidenced by Pharmacogenomics Knowledgebase ( and literature data. The polymorphisms included: GSTM1 and GSTT1 gene deletions, GSTP1 (rs1695), MTHFR (rs1801133), TYMS 28-bp tandem repeats (rs3474033: allele TSER*2, allele TSER*3, and a G > C SNP within the TSER*3 allele; TSER*3G > C), RFC (rs1051266), NR3C1 (rs6198, rs41423247), MDR1 (rs3789243, rs2235046, rs1045642), and VDR (rs2228570, rs1544410). The second approach allowed inclusion of polymorphisms in the genes earlier reported to be associated with the MRD-positive status: CCR5 (rs333 also known as 585del32), TPMT (rs1800460, rs1142345); other six loci from the genome-wide association study (GWAS): IL15 (rs10519613), NALCN (rs7992226), CCDC85C (rs11160533), and three intergenic (rs3862227, rs4888024, rs9871556). The six loci selected from the GWAS study were chosen with regard to their P-values for association with MRD, odds ratio (OR) for the MRD-positive status associated with carrying the risk allele, P-values for association with other phenotypes (e.g. methotrexate clearance), and minor allele frequency (MAF) in Caucasians.Genotyping of polymorphic loci was performed in all 174 patients. DNA was isolated with the use of QIAamp DNA Blood Mini Kit (Qiagen, Germany) from blood/bone marrow samples obtained at the time of hematological remission (in most cases, week 12). High Resolution Melting (HRM) analysis was used for genotyping of 11 loci. Six loci were studied with the use of TaqMan Genotyping Assays (Life Technologies), namely: NALCN, CCDC85C, three intergenic variants selected from GWAS and one polymorphism in NR3C1 (rs41423247). 7900HT Fast Real-Time PCR System was used for both HRM and TaqMan Genotyping assays. Conventional PCR and agarose gel detection was used for genotyping of CCR5 (585del32), GSTM1 and GSTT1 gene deletions, TYMS 28-bp tandem repeats (alleles TSER*2, TSER*3, TSER*3G > C) and RFC, the latter two followed by restriction fragments length polymorphism analysis. For each polymorphic locus, 10% of results were verified by repeating the genotyping analysis or by direct sequencing.All polymorphic loci were screened for linkage disequilibrium (LD) using SNAP software (SNP Annotation and Proxy Search, Broad Institute) based on Haploview 4.0 and genotype data from International HapMap Project and the 1000 Genomes Project. Compliance of genotypes distribution with the Hardy-Weinberg equilibrium (not applicable to GSTM1 and GSTT1 gene deletions) was tested using the Court Lab Calculator ( PolyPhen-2 ( and SIFT ( were used for prediction of functional effects of selected polymorphic variants. [...] MRD results were analyzed as a categorical variable (MRD-positive vs. MRD-negative, with a cut off level 10−4) and as a continuous variable (MRD levels).Pearson χ2 test or, if expected frequencies were <5, 2-sided Fisher’s exact probability test (2 × 2 tables) and Freeman-Halton extension of 2-sided Fisher’s exact probability test (2 × 3 tables), with 2-sided Z-test for difference in proportions as a post hoc test were used to estimate the significance of: 1/differences in genotypes distribution between patients with MRD-positive and MRD-negative status; 2/association of MRD status with the allele frequency and allele positivity (genotypes pooled into binary groups and analyzed in dominant and recessive mode, e.g. GG + AG < > AA and AA + AG < > GG); odds ratio (OR) and confidence intervals (CI) for MRD-positivity calculated for the allele frequency and allele positivity; 3/association of the clinical features with MRD status, defined as ‘MRD-associated clinical covariates’; 4/additive effects between polymorphic variants (differences between MRD-positive and MRD-negative patient groups in the distribution of patients carrying risk allele in 0, 1 or 2 loci identified to be associated with the MRD status at day 33).Univariate logistic regression was used to confirm the association of polymorphic variants with MRD status. Multivariate logistic regression was used to adjust for MRD-associated clinical covariates and for identification of the best regression models predicting the MRD status.Non-parametric test (Kruskal-Wallis ANOVA on ranks) was used for analysis of association of polymorphic variants (genotypes and binary groups representing allele positivity) with MRD levels (MRD as a continuous variable).Two-sided analysis of statistical power was performed using Quanto software v.1.2.4.P-values < 0.05 defined statistical significance in all tests applied.Benjamini-Hochberg procedure was applied to control for false discovery rate (FDR) in case of multiple comparison tests (association of the MRD status with the distribution of genotypes, allele frequency and allele positivity). This adjustment was not applied to tests involving clinical covariates and additive effects of carrying risk alleles, since they did not comply with the Benjamini-Hochberg assumption of the independence of multiple tests.Statistica v. 10 (StatSoft Inc.) and VassarStats: Website for Statistical Computation ( were used for statistical analyses. […]

Pipeline specifications

Software tools SNAP, Haploview, PolyPhen, Statistica, VassarStats
Databases International HapMap Project PharmGKB
Applications Miscellaneous, GWAS
Organisms Homo sapiens
Diseases Neoplasms, Precursor Cell Lymphoblastic Leukemia-Lymphoma