Genes Profiling of a Patient with Chronic Myeloid Leukemia on Illumina MiSeq Platform: A Cases Report

Ibraheem Ashankyty^1,2* & Edem Nuglozeh³

¹Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, University of Ha’il, Ha’il, Saudi Arabia
²Department of Medical Laboratory Technology, College of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
³Department of Biochemistry, College of Medicine, University of Ha’il, Ha’il, Saudi Arabia

*Correspondence to: Dr. Ibraheem Ashankyty, Department of Medical Laboratory Technology, College of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.

Copyright © 2018 Ibraheem Ashankyty, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Case Report
A 55 years old tribal ma n came to King Khalid Hospital, Ha’il, KSA, suffering from left abdominal pain, weight loss and extreme fatigue. CBC showed increased WBC and differential count revealed the presence of elevated neutrophils with the presence of precursors. He was then suspected of suffering from chronic myelocytic leukemia (CML). The patient was transferred to King Faisal Specialist Hospital (KFSH) in Riyadh, where cytogenetic study reveals the presence of the Philadelphia chromosome and underwent subsequent treatment. Whole blood sample was sent to our lab for genetic profiling using the whole genome sequencing (WGS) technology.

Exome Library Preparation and DNA Sequencing
High quality gDNA was purified from whole blood using a Genomic DNA Purification Kit (QIA amp DNA Blood Mini Kit from Qiagen, Hilden, Germany as reported initially [1]. Brief, the library construction was conducted using the Illumina Nextera Kit (V3 Chemistry).50 ng of gDNA was utilised using transposase based chemistry to create exome libraries. 2x 75 paired end cycles were performed (+1 cycle to each forward and reverse read to allow for phasing/pre-phasing).

Bioinformatics Analysis
fastq files were concatenated from multiple runs

Adapter trimming and base quality scores (less than Q30) were removed using Cutadapt

FastQC was used to check primary and post trimmed sequences

Alignments to the reference human genome (hg19) were conducted using BWA (version 0.7.15)

The Genome Analysis Tool Kit (version 3.0.0) was used for base quality score recalibration, variant calling following by hard filtering to identify high quality variants for downstream analyses

SnpEffv4.1 was exploited to determine in silico impacts upon protein function of candidate genes

Discussion
The pathophysiology and the mechanisms leading to CML development still remained completely ununderstood. CML is a malignant disease of the pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome (Ph) [2] (25) and a rearrangement between the BCR gene (break-point cluster region) and the ABL gene [3] (26). Clinically, CML is characterized by a massive expansion of immature progenitors and precursors that leave the marrow microenvironment prematurely [4] (27). Although normal progenitors coexist with the malignant clone in CML, their growth seems to be inhibited, possibly as consequence of some anomalies in CML microenvironment itself [5] (28). The mechanisms that underlie the massive expansion of Ph + hematopoietic progenitors, precursors, and mature cells are also not understood and why CML progenitors circulate prematurely in the blood is not completely understood neither [6].

We undertook WES to access the genetic profile of this patient suffering from leukemia. The variants profile in table 1 corresponding to exonic reduction shows multiple genes of interest like: BCL6, CARD8, CASP7, FBXW7, IL1A, IL4R and RAG1. Indeed, CASP7 is strongly involved in cancer development. Park et al [7] have demonstrated an association between CASP7 and CASP14 in the genetic polymorphisms with the risk of childhood leukemia development by running the minimum P-value (minP) and the false discovery rate (FDR) test. This approach confirms our findings characterized by exonic frameshift deletion of two TT in CASP 7 located on chromosome 10.

The development and growth of any types of cancer is under the control of cancer-initiating cells (CICs), a cell population with the same attribute like stem cells [8]. In addition to their self-renewal and multipotency properties, stem cells are endowed with their own cell cycle features. Uncovering key mechanisms underlying their cell cycle control will shed the light in the regulation of the self-renewal and differentiation processes [9]. The (Fbxw7) is a key regulator gene of the cell cycle involved in the maintenance of normal stem cells and CICs Takeishi et al. [10,11] The Fbxw7 gene products are characterized by three isoforms (Fbxw7α, β, and -γ) that differ only at their amino termini, with each isoform possessing the dimerization domain. Fbxw7 plays pivotal roles in cell division, growth, and differentiation by targeting several proteins - including: c-Myc, Notch1, Notch4, c-Jun, and cyclin E - for degradation [12,13]. Fbxw7 binds each of these substrates through a conserved phosphorylated domain known as the Cdc4 phosphodegron. Given that most of these proteins targeted by Fbxw7 for degradation are proto-oncoproteins, Fbxw7 has been thought to function as a tumor suppressor. Indeed, in heterozygous mutations Fbxw7 have been detected in several types of human cancer, including T-cell acute lymphoblastic leukaemia (T-ALL), T-cell lymphoma, and cholangiocarcinoma [13,14]. Nearly three-quarters of these mutations are point mutations that result in amino-acid substitutions at key positions in the WD40 repeats and consequent disruption of substrate binding. These clinical observations thus indicate that Fbxw7 is crucial for preventing carcinogenesis as a result of its role in cell cycle regulation. Our reductional variants list after filtration of intronic variants indicates frameshift deletion of G.

Conclusion
In summary, we have run the whole exome sequencing on Saudi male adult blood genomic DNA using Illumina platform. Analysis and subsequent filtration identified 244 unique genes have been found to be associated with Leukemia. Exonic filtration reveals 14 gene of interest that are associated to the development and progression of CML. Of importance is the identification of two variants: JAK1 and MLP which are associated with Myeloproliferative Disorders Philadelphia Chromosome. More studies remained to be conducted via SNPs genotyping specifically associate these variants to CML and ultimately conduct clinical studies.

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