Mutations in Genes Producing Nitric Oxide and Hydrogen Sulfide and Their Connection With Apoptotic Genes in Chronic Myeloid Leukemia

Abstract

Background Despite advances in chronic myeloid leukemia (CML) genetics, the role of nitric oxide (NO) and hydrogen sulfide (H₂S) gene mutations and their relationship to apoptotic genes is unclear. Therefore, this study investigated NO- and H₂S-producing genes' mutations and their interactions with apoptotic genes using Sanger sequencing and next-generation sequencing (NGS). Methodology A complete blood count (CBC) was carried out to measure the total number of white blood cells, while IL-6 levels were assessed in both control and CML patients using an ELISA technique. Sanger sequencing was used to analyze mutations in the CTH and NOS3 genes, whereas NGS was applied to examine mutations on all chromosomes. Results White blood cell (WBC) and granulocyte counts were significantly higher in CML patients compared to controls (p<0.0001), and monocyte counts were similarly higher (p<0.05). Interleukin-6 (IL-6) levels were significantly elevated in CML patients than controls (p<0.0001), indicating a possible link to CML etiology or progression. Multiple mutations have been identified in both genes, notably in CTH exon 12 and the NOS3 genes VNTR, T786C, and G894T. This study also measured IL-6 concentrations using IL-6 assays, identifying its potential as a CML prognostic diagnostic. WBC counts, granulocyte counts, and mid-range absolute counts, or MID counts, were significantly higher in CML patients than in normal control individuals. NGS identified 1643 somatic and sex chromosomal abnormalities and 439 actively expressed genes in CML patients. The findings imply a genomic landscape beyond the BCR-ABL1 mutation in CML development compared to other databases. Conclusion In conclusion, this study advances the understanding of the genetic characteristics of CML by identifying mutations in the NO- and H₂S-producing genes and their complex connections with genes involved in apoptosis. The comprehensive genetic profile obtained by Sanger sequencing and NGS provides possibilities for identifying novel targets for therapy and personalized treatments for CML, therefore contributing to developments in hematological diseases.

Keywords: chronic myeloid leukaemia; cth gene; next generation sequencing (ngs); next-generation sequencing (ngs); nos3 gene; sanger sequencing.

Figure 1. A comparison of hematological parameters and IL-6 levels between controls and chronic myeloid leukemia (CML) patients

Patients with CML had significantly greater (p<0.001) total WBC (A) and granulocyte counts (B). (C) Patients with CML had a significantly higher monocyte count (p<0.05). (D) In CML patients, IL-6 levels were significantly higher (p<0.001). *p<0.05; ****p<0.0001 vs. healthy individuals

Figure 2. Sanger sequence analysis through the cBioPortal database

(A) A lollipop mutational map showing the CTH gene mutation. (B) A lollipop mutational map showing the NOS3 gene (VNTR, T786C, and G894T). PTM, post-translational modification

Figure 3. Electropherograms showing the mutational sample with reference

(A) A Sanger sequence chromatogram for the CTH gene showing the missense mutation (dbSNP:1021737), and amino acid change (serine>isoleucine) in position (28400G>GT); (B) a Sanger sequence chromatogram for the NOS3 gene showing the mutation in the splice region on T786C that changes the nucleotide (8535G>GC); (C) a Sanger sequence chromatogram for the NOS3 gene showing the mutation in the splice region on T786C that changes the nucleotide (8371 C>T); (D) a Sanger sequence chromatogram for the NOS3 gene showing the substitution mutation (dbSNP:2070744) that changes the nucleotide (2436C>T) located on G894T; (E) a Sanger sequence chromatogram for the NOS3 gene showing the duplication mutation 6725_6751het_dupAGTCTAGACCTGCTGCG GGGGTGAGGA) located in VNTR; (F) a Sanger sequence chromatogram for the NOS3 gene showing the duplication mutation 6751_6752het_INSAGTCTAGGACCTGCTGCGGGGGTGAGGA) located in VNTR.

Figure 4. Next-generation sequencing (NGS) analysed through the SRplot database

(A) The chromosome distribution map illustrates the highest concentration of chromosomes; (B) a two-dimensional Circos plot displaying four columns, with the first indicating chromosomes, the second showing starting coordinates, the third indicating end coordinates, and the fourth representing fold change reflecting gene upregulation and downregulation during cancer progression and GC content variability; (C) an RCircos diagram (version 1.2.2, an R package for Circos 2D track plots) depicting gene names to showcase expressed genes and copy number variation, and includes information on chromosome location in the first three columns, along with gene name locations, while log2fc is displayed in another column.