Blood Res.  2019 Jun;54(2):144-148. 10.5045/br.2019.54.2.144.

An investigation of methylation pattern changes in the IKZF1 promoter in patients with childhood B-cell acute lymphoblastic leukemia

• Rahmani M
• Fardi M
• Farshdousti Hagh M
• Hosseinpour Feizi AA
• Talebi M
• Solali S

• KMID: 2451016
• DOI: http://doi.org/10.5045/br.2019.54.2.144

Abstract

BACKGROUND
Ikaros family zinc finger 1 (IKZF1) is a transcription factor with an important role in controlling hematopoietic proliferation and function, particularly lymphoid cell differentiation. It was previously shown that various mechanisms and expression patterns of Ikaros are linked to a variety of cancers. We hypothesized that aberrant methylation (hypomethylation) of the IKZF1 promoter region might be one of the causes of B-cell acute lymphoblastic leukemia (B-ALL). In B-ALL patients, an increased expression of this gene is a potential cause of B-cell differentiation arrest and proliferation induction. Therefore, as more than 90% of patients with ALL are <15 years old, we investigated the methylation pattern of the IKZF1 promoter in childhood B-ALL.
METHODS
Twenty-five newly diagnosed B-ALL cases were included (all younger than 15 yr). In addition, we selected 25 healthy age- and sex-matched children as the control group. We collected the blood samples in EDTA-containing tubes and isolated lymphocytes from whole blood using Ficoll 1.077 Lymphosep. Next, we extracted genomic DNA with the phenol/chloroform method. Two microgram of DNA per sample was treated with sodium bisulfite using the EpiTect Bisulfite Kit, followed by an assessment of DNA methylation by polymerase chain reaction (PCR) analysis of the bisulfite-modified genomic DNA.
RESULTS
Our data highlighted a hypomethylated status of the IKZF1 promoter in the ALL cases (96% of the cases were unmethylated). In contrast, the control group samples were partially methylated (68%).
CONCLUSION
This study demonstrated a hypomethylated pattern of the IKZF1 promoter region in childhood B-ALL, which might underlie the aberrant Ikaros expression patterns that were previously linked to this malignancy.

Keyword

Leukemia; Ikaros; IKZF1; DNA methylation; Hematological malignancy

MeSH Terms

B-Lymphocytes*
Child
DNA
DNA Methylation
Ficoll
Hematologic Neoplasms
Humans
Leukemia
Lymphocytes
Methods
Methylation*
Polymerase Chain Reaction
Precursor Cell Lymphoblastic Leukemia-Lymphoma*
Promoter Regions, Genetic
Sodium
Transcription Factors
Zinc Fingers
DNA
Ficoll
Sodium
Transcription Factors

Figure

• 

  Fig. 1 Methylation patterns differed significantly between the patient and control groups. As shown in this figure, 24 out of 25 patients with B-ALL had an unmethylated pattern in their IKZF1 promoter genomic region, while this was 6 out of 25 normal cases. It shows a significant (P<0.05) unmethylation pattern in our patients with childhood B-ALL. On the other hand, it shows that most normal cases have methylated or partially methylated statues in their IKZF1 gene promoter site.

• 

  Fig. 2 PCR products were subjected to electrophoresis. IKZF1-associated bands are visible at the 200 bp marker. (A) MSP results with IKZF1 unmethylated primers for the control group. As can be seen, in most of the control samples, the unmethylated pattern was observed. (B) MSP results with the methylated primers of the IKZF1 gene for the control group. As shown in this figure, the majority of these samples exhibited a relative methylation state. (C) MSP results with IKZF1-specific methylated primer for the patient group. As can be seen, samples from patients didn't have any band with methylated primer. (D) MSP results with IKZF1 unmethylated primers for the patient group. All samples of the patients group had recognizable bands with the unmethylated primers of IKZF1 promoter.

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