Investigation of genetic variations regulating cell signaling pathways in leukemia

Abstract

Leukemia is a heterogeneous group of hematological cancer responsible for a multitude of morbidities and mortalities worldwide. It is one of the most prevalent cancers among pediatric malignancies in Pakistan and causes a huge economic burden. Epidemiological integrated transdisciplinary research is needed for the estimation of the actual leukemia burden and contributing risk factors in Pakistan. Leukemia incidence, subtypes distribution, and survival variance point towards the strong involvement of population genetics. There are numerous yet diverse numbers of genomic variations, single nucleotide polymorphisms (SNPs), mutations (point, insertions, deletions), and chromosomal aberrations actively elucidated for a role in triggering and increasing susceptibility of leukemia. Screening of the genetic factors contributing to leukemia in diverse and distinct populations is of particular importance. Genetic factors affect personalized drug decisions, treatment outcomes, and patient survival. Revealing the genomic landscape of individual patients, with modern genetic techniques, can help design personalized treatment plans leading to highly effective treatment protocols. In silico interaction studies, involving docking of compounds having inhibitory potential can be helpful in the development of effective, and specific inhibitors. The present study was designed to investigate the epidemiological, environmental, and lifestyle related risk factors, mutational status of selected JAK-STAT pathway genes, and screening of mutational hot spots in key genes of crucial signaling pathways in leukemia patients in the Pakistani population. The present study also included a virtual exploration of the molecular interaction of potential inhibitors with their mutated products. A total of 1500 subjects, including 616 patients and 884 controls were recruited through a consented, retrospective cross-sectional sampling design from tertiary care hospitals in Pakistan. DNA was extracted from blood samples obtained from the study participants. For the risk factors identification, the data of 594 patients and 884 controls were processed. Descriptive summaries were generated, and risk factors were analyzed through logistic regression. For allele-specific PCR, the DNA samples of 276 patients, were processed. The genetic status of JAK1V623A, JAK2 S473, and STAT5BN642H was screened through allele-specific PCR. Targeted next-generation sequencing (NGS) was performed for selected patient’s samples by using Thermofisher Ion S5TM Systems, Ion Torrent, using Ion AmpliSeq™ Library Kit 2.0, with Ion Ampliseq Cancer hotspot panel 2. The panel comprised 50 crucial human genes involved in cell signaling pathways. The sequencing results were analyzed through Thermofisher Scientific, based Ion Reporter™ Software and interpreted. Microarray was performed on a patient’s DNA sample through Cyto Scan Cytogenetics Suite by Thermofisher as per manual instructions and the data was analyzed through Thermofisher Chromosome Analysis Suite (ChAS) 4.3 software. In-silico analysis was performed through molecular docking of wildtype and mutant protein retrieved from the protein databank. Selected protein inhibitor structures were retrieved from the PubChem database. The ligands and protein were prepared, and docking was performed through Auto Dock Vina 123 and visualized through Discovery Studio Visualizer 2021. The present study observed that acute lymphoblastic leukemia comprised almost 70% of the total patients, with male preponderance. We identified Pathan ethnicity (OR=2.85; 95%CI=2.29-3.54), no formal education (OR=3.36; 95%CI=2.62-4.32), poor diet (OR=2.34; 95%CI=1.79-3.06), lower BMI (OR=1.95; 95%CI=1.50-2.60), parental consanguinity (OR=2.13; 95%CI=1.67-2.71), positive family history (OR=4.24; 95%CI=2.18-8.26), rural residential setup (OR=2.93; 95%CI=2.10 4.10), drinking of groundwater (OR=2.25; 95%CI=1.6479-3.0964), wooden fuel (OR= 3.97; xx 95%CI=3.14-5.01), carbonated drinks (OR=1.25, 95%CI=1.00-1.57) and tobacco usage (OR=1.57, 95%CI=1.24-1.98) as significant risk factors for leukemia. However, odds ratios were significantly lower for patients using a microwave oven (OR=0.25; 95%CI=0.18-0.35), and perfumes (OR=0.42; 95%CI=0.33-0.53). Males exhibit an increased risk for lymphoid leukemia as compared to myeloid leukemia (OR=1.97; 95%CI=1.38-2.80). Paraclinical parameters indicated that 71% of the cases had >50% of blast cells. Leukocytosis (OR= 9.06; 95% CI=6.46-12.71), anemia (OR= 15.84; 95% CI=11.84-21.21), low hemoglobin (OR=8.11; 95% CI=6.35-10.37), thrombocytopenia (OR=32.40; 95% CI=21.57-48.68), lymphocytosis (OR= 3.41; 95% CI=2.55-4.57), and neutropenia (OR=7.32; 95% CI=5.59-9.60) had significantly higher odd ratio for leukemia patients. The allele-specific amplification showed that all the patients were homozygous normal for JAK1V623A, JAK2 S473 major allele. However, 6 patients (5 male, 1 female) in the age range of 10-11 years, with ALL were STAT5BN642H+. The NGS data identified the presence of 26 genetic variations on different genomic loci, including 14 exonic mutations; comprised of 3 missense variations (TP53; c.215C>G/ p.Pro72Arg, ALK; c.3551G>A/ p.Gly1184Glu, SMAD4; c.767A>T/ p.Gln256Leu), and 11 synonymous variations (FGFR3; c.1953G>A/Thr651Thr, PDGFRA; c.2472C>T/p.Val824Val, & c.1701A>G/ Pro567Pro, RET; c.2307G>T/ Leu769Leu, APC; c.4479G>A/p.Thr1493Thr, PIK3CA/p.Thr1025Thr; c.3075C>T, EGFR; c.2361G>A/p. Gln787Gln, EGFR-AS1; c.2361G>A/p.Gln787Gln, MET; c.534C>T/ p.Ser178Ser, ABL1; c.1149C>T/ p.Gly383Gly, HRAS; c.81T>C/ p.His27His). The remaining variation included 9 intronic variations (rs3738868, rs5030613, rs7692791, rs10006115, rs3729674, rs2491231, rs2075606, rs839541, INDEL rs1412472143/rs67894136) and a downstream UTR_3 multiple nucleotide variation (rs38669350). The microarray analysis identified the presence of 97 loss of heterozygosity (LOH) loci, 2 copy number loss at chromosome 8 (p11.22, q24.23), and a copy number gain on chromosome 14 (q32.33). The molecular docking of the ligands to wildtype and STAT5BN642H+revealed that AC-4-130, Pimozide, Indirubin, and Stafib-2 have higher, but differential docking affinities for both normal and mutated STAT5B. However, AC-4-130 has a higher affinity for wildtype and Stafib-2 has stable molecular interaction with STAT5BN642H+. Risk factors associated with leukemia are mainly related to exposure due to rural residence, poor lifestyle, and family history of the disease. The disease incidence can be minimized by designing and implementing risk mitigation strategies. The aggressive form of pediatric leukemia, carrying STAT5BN642H+ mutation has been identified in the studied population. The molecular docking study predicted that AC-14-30 and stafib-2 are potential ligands for inhibition of constitutively active STAT5B if modified and optimized for use in combination therapy. NGS and microarray are efficient platforms, to perform sensitive, reliable, and simultaneous detection of genomic/genetic aberrations in one setting. technologies are valuable for identifying individual genetic profiles and clustering the patients for targeted therapies.