Contributions of IKZF1,DDC,CDKN2A,CEBPE,and LMO1 Gene Polymorphisms to Acute Lymphoblastic Leukemia in a Yemeni Population

Introduction

Acute lymphoblastic leukemia (ALL) is a neoplasm of early stage lymphoid cells with a peak occurrence at 2-5 years of age. It represents ~25% of malignancies in children younger than 15 years of age (Eden, 2010; Kaatsch, 2010). ALL is the main cause of tumor-related mortality among children (Stiller and Parkin, 1996). In Asia, it has been estimated that the incidence of childhood ALL is 1.25/100,000 individuals each year (Yeoh et al., 2013). Epidemiological and biological information on childhood ALL is limited in Yemen, partly due to the lack of hospital- and/or national-based registries and inadequate diagnostic methodologies (Vandenberg et al., 2009). The only data available, according to the World Health Organization (WHO) report, are that 10% of all tumors in the Yemeni population are attributed to leukemia, with a mortality rate of 12.3% (World Health Organization, 2014).

The initiation of ALL has been hypothesized to occur in utero or during early infancy (Kaatsch, 2010). However, the exact etiology and development of childhood ALL remain poorly understood. Nevertheless, it is widely recognized that genetic variations, infections, and environmental factors play important roles in the development of ALL (Belson et al., 2007; Guo et al., 2014; Ma et al., 2014).

Genetic variations in both Ikaros family zinc-finger (IKZF1) and dopa decarboxylase aromatic l-amino acid (DDC) genes, located on chromosome 7p12, have been identified as risk loci for ALL susceptibility (Papaemmanuil et al., 2009; Treviño et al., 2009; Prasad et al., 2010). The intronic single-nucleotide polymorphism (SNP) rs3731217 in CDKN2A (cyclin-dependent kinase inhibitor) was found to be strongly associated with ALL risk in the European population (Sherborne et al., 2010). Genetic variations in the CEBPE gene, encoding the CCAAT/enhancer-binding protein transcription factor, have been found to be associated with ALL risk in the European and Caucasian populations (Papaemmanuil et al., 2009; Treviño et al., 2009; Pastorczak et al., 2011). CCAAT/enhancer-binding protein transcription factor plays an important role in the proliferation and differentiation of cells, particularly myeloid cells (Nerlov, 2004). LMO1, located on chromosome 11, encodes a cysteine-rich two LIM domain transcriptional regulator (Boehm et al., 1991). Genetic variations in LMO1 have been shown to be associated with ALL in Caucasian children (Beuten et al., 2011). Differences in the incidence of ALL across different regions and populations are recognized. For instance, the incidence of ALL is higher among Europeans than Asians (Parkin et al., 1988). Also, the incidence rate of ALL in Hispanic children is more than that reported in non-Hispanic whites (Matasar et al., 2006).

To better understand the differences in susceptibility to ALL and how genetic and environmental factors interact together to increase or decrease ALL risk, it is crucial to study the impact of genetic polymorphisms among different ethnicities.

Currently, there are limited genetic studies examining ALL susceptibility in the Middle East population. Replicating studies conducted in other populations is imperative to confirm a possible universal impact of these polymorphisms with regard to ALL, in addition to discovering possible genetic differences among various populations to bettering treatment and ALL diagnosis. At present, no study examining the effects of IKZF1, DDC, CDKN2A, CEBPE, and LMO1 on ALL exist in Yemen. As such, the primary aim of this case-control study is to evaluate the influence of polymorphisms in these five genes on childhood ALL susceptibility in the Yemeni population of Arab-Asian ancestry.

Materials and Methods

Results

The current study is comprised of 136 children diagnosed with ALL and 153 healthy children. The distribution of demographic and hematological variables among ALL cases and controls are listed in Table 1.

IKZF1 SNPs (rs4132601, rs6964969, rs11978267, rs7789635, and rs10235796), DDC SNPs (rs3779084, rs880028, and rs7809758), CDKN2A SNPs (rs3731246 and rs3731217), CEBPE SNPs (rs2239633 and rs12434881), and LMO1 SNPs (rs442264, rs3794012, and rs4237770) conformed to HWE in the control group. However, IKZF1 SNPs rs6964823 and rs6944602 showed departure from HWE (p-value = 0.04, 0.007, respectively), hence these two SNPs were excluded from further analysis. The logistic regression model adjusted for age and gender revealed that IKZF1 rs10235796 C allele was a risk factor for ALL (OR = 2.50; p = 0.002) in comparison to the T allele. The results also showed that IKZF1 rs6964969 A>G variant was significantly associated with ALL (OR = 2.14; p = 0.048, GG vs. AA) (Table 2).

Our results revealed borderline association between ALL risk and IKZF1 SNP rs4132601 T>G (OR = 2.07; p = 0.050, GG vs. TT). On the other hand, we found no associations between IKZF1 rs11978267 and rs7789635 genotypes with ALL (Table 2). Similarly, there were no associations between DDC SNPs (rs3779084, rs880028, and rs7809758) and ALL risk (Table 2). The findings indicated that CDKN2A rs3731246 G>C polymorphism was associated with ALL risk (OR = 2.01; p = 0.047, GC+CC vs. GG). Specifically, the rs3731246 C allele demonstrated increased risk for ALL (OR = 1.55; p = 0.007) compared to the G allele. However, CDKN2A SNP rs3731217 was not associated with ALL. In addition, we found no association between CEBPE SNPs (rs2239633 and rs12434881) and LMO1 SNPs (rs442264, rs3794012, and rs4237770) with childhood ALL (Table 2).

Upon performing haplotype analysis, two-SNP haplotypes and diplotype blocks were determined to exhibit significant LD. The constructed block consisted of IKZF1 SNPs rs11978267 and rs6964969 (Fig. 1). Two haplotypes and three diplotypes with frequency >4% in the total sample were analyzed for their association with increased risk of ALL (Table 3). The most common haplotype (AA) was composed of the protective alleles of rs11978267 and rs6964969. This haplotype revealed protection from ALL (OR = 0.43, p = 0.015), whereas the risk allele-haplotype (GG) showed borderline association with ALL (OR = 1.97, p = 0.05). Likewise, the diplotype (GG-GG) had borderline association with ALL risk (OR = 1.97, p = 0.05) (Table 3).

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FIG. 1.

Pairwise linkage disequilibrium among IKZF1 SNPs in Yemeni children. SNPs, single-nucleotide polymorphisms.

Discussion

ALL is a heterogeneous disease influenced by both genetic and non-genetic factors. In the present study, we assessed whether polymorphisms within IKZF1, DDC, CDKN2A, CEBPE, and LMO1 genes have any influence on susceptibility to ALL in Yemeni children of Arab-Asian ancestry. We found that IKZF1 SNPs rs10235796 and rs6964969 were associated with increased risk of ALL, whereas SNP rs4132601 merely has a marginal association with ALL. The association between rs6964969 in IKZF1 and ALL has been reported in the Hungarian population (Lautner-Csorba et al., 2012). Of interest, we found a strong association between CDKN2A SNP rs3731246 with increased risk of ALL, a novel result that has never been reported prior. Our results also revealed that IKZF1 SNPs rs7789635 and rs11978267, DDC SNPs, CDKN2A SNP rs3731217, CEBPE SNPs, and LMO1 SNPs have no effect on the susceptibility to childhood ALL. Our findings were inconsistent with previous studies that showed association between IKZF1 rs11978267 and ALL (Treviño et al., 2009; Lautner-Csorba et al., 2012; Orsi et al., 2012; Linabery et al., 2013; Xu et al., 2013). Noteworthy, Bahari et al. (2016) found no association between rs11978267 and childhood ALL in the Iranian population.

The IKZF1 gene, located at 7p12, encodes the Ikaros zinc-finger transcription factors, which are important in normal lymphoid maturation and tumor suppression (Sellars et al., 2011; Kastner et al., 2013; Yoshida and Georgopoulos, 2014). Mutations of Ikaros are common in B cell ALL (Nakase et al., 2000) and poor prognosis is associated with IKZF1 alterations (Feng and Tang, 2013). The activity of hematopoietic stem cells were reduced in Ikaros-null mice (Ng et al., 2009) and deletion of IKZF1 leads to the development of a high-risk form of childhood ALL (Mullighan et al., 2008, 2009; Den Boer et al., 2009). It has been suggested that mutations in IKZF1 affects expression of this gene and disruption of normal Ikaros function has been implicated in the leukemogenesis of ALL in infants (Papaemmanuil et al., 2009).

IKZF1 rs4132601 SNP has been shown to be significantly associated with susceptibility to childhood ALL in both the Caucasian and Hispanic populations (Papaemmanuil et al., 2009; Prasad et al., 2010; Pastorczak et al., 2011; Lautner-Csorba et al., 2012; Orsi et al., 2012; Hsu et al., 2015). However, we found borderline association between rs4132601 and ALL risk in Yemeni children. Contentious findings have been reported by study groups in Taiwan, China, and Korea that show no association between rs4132601 and ALL risk in the Asian population (Han et al., 2010; Wang et al., 2013; Lin et al., 2014). On the other hand, Vijayakrishnan and Houlston (2010) have found that rs4132601 SNP was associated with increased risk of ALL in the Thai population (Vijayakrishnan and Houlston, 2010). Also, the findings in the Tunisian and Iranian population supported the association between this SNP and ALL risk (Bahari et al., 2016; Gharbi et al., 2016). Two recent meta-analysis reports have suggested an association between IKZF1 rs4132601 and increased ALL risk among Caucasians, but not among Asians (Dai et al., 2014; Li et al., 2015). These inconsistencies might be attributed to differences in genetic and environmental factors, as well as variations in LD.

IKZF1 SNP rs4132601 is located in exon 8 and may affect messenger RNA (mRNA) stability (Kastner et al., 2013). Papaemmanuil et al. (2009) found that IKZF1 rs4132601 increases the risk of B cell ALL (Papaemmanuil et al., 2009). They found that the risk genotype of this SNP was associated with reduced expression of IKZF1 in lymphocytes. Upon evaluating the impact of the IKZF1 haplotypes on ALL susceptibility, haplotype GG, containing risk alleles of both IKZF1 SNPs rs11978267 and rs6964969, was found to be marginally associated with ALL risk. Conversely, haplotype AA, containing nonrisk alleles, showed a protection against ALL risk.

DDC is a prime candidate gene to study given its contiguous involvement with leukocytogenesis. DDC gene expression in human peripheral white blood cells leads to l-DOPA decarboxylase synthesis. l-DOPA is subsequently decarboxylated to dopamine, an important regulator for leukocyte proliferation and maturation (Kokkinou et al., 2009). DDC polymorphisms have been previously reported to be associated with ALL susceptibility in the Caucasian population (Papaemmanuil et al., 2009). In our study, we found no association between DDC SNPs and ALL risk. Our findings are in line with that of Healy et al. (2010) who could not replicate the association between DDC SNPs and ALL risk in a cohort of French Canadians of European descent (Healy et al., 2010). Of interest, Trevino et al. (2009) revealed that two DDC SNPs, rs2167364 and rs2242041, were associated with ALL. In addition, they found that these two SNPs in combination with IKFZ1 rs11978267 were able to distinguish B cell from T cell ALL in the European population (Treviño et al., 2009).

Based on its biology, CDKN2A is a strong candidate ALL-susceptibility gene. Abnormalities in CDKN2A/CDKN2B tumor suppressor genes is present in up to 40% of B-precursor ALL and plays a role in the deregulation of cell cycle in leukemia (Mullighan et al., 2007). CDKN2A-CDKN2B genes encode three tumor suppressor proteins (p16^INK4A, p14^ARF, and p15^INK4B). p16^INK4A and p15^INK4B are inhibitors of cyclin-dependent kinases, which are important regulators of cell cycle through the retinoblastoma protein (Rb)-E2F pathway (Krug et al., 2002). We investigated the association of CDKN2A SNPs with ALL and found that rs3731246 is the only SNP associated with ALL risk. To the best of our knowledge, this is the first study to demonstrate the contribution of rs3731246 to increase risk of ALL in Yemeni children of Arab-Asian descent. Our data also revealed no association between rs3731217 and ALL, consistent with results from earlier studies that have failed to identify an association between this SNP and ALL (Vijayakrishnan et al., 2010; Pastorczak et al., 2011). A study examining a Tunisian population of Arab-African ancestry similarly found no association between SNP rs3731217 and ALL risk (Gharbi et al., 2016). A former GWAS in the European population revealed no significant association between CDKN2A variations and childhood ALL (Papaemmanuil et al., 2009). However, another GWAS using larger sample size in the European population showed significant association (Sherborne et al., 2010).

CEBPE is a tumor suppressor gene that inhibits myeloid leukemogenesis and may contribute to B cell ALL development (Papaemmanuil et al., 2009). We did not identify any significant associations between CEBPE SNPs rs2239633 and rs12434881 with ALL. Our findings are consistent with the results from the Asian (Vijayakrishnan et al., 2010; Wang et al., 2013) and Polish studies (Pastorczak et al., 2011), which exhibited an absence of association between rs2239633 and childhood ALL. Healy et al. (2010) showed that rs2239633 was not associated with childhood ALL in French Canadians of European descent (Healy et al., 2010). Contradictorily, a Korean study showed moderate association of this SNP with the risk of ALL (Han et al., 2010). Also, two CEBPE SNPs that were not included in this study, rs4982731 and rs10143875, have been found to be associated with increased risk for ALL in the Hispanic population (Hsu et al., 2015). Former studies in Caucasians have shown association between CEBPE variations and childhood ALL risk (Papaemmanuil et al., 2009; Prasad et al., 2010; Orsi et al., 2012). Xu et al. (2013) showed strong association between CEBPE SNPs and ALL risk in a European-American cohort, with dissimilar results in the non-European population (Xu et al., 2013). Wang et al. (2013) found differences in locus of rs2239633 in LD blocks between Chinese and European populations (Wang et al., 2013). Interestingly, SNP rs2239632 located in the promoter region of CEBPE has been hypothesized to increase susceptibility to leukemia as a result of its risk allele increasing the expression of the final gene product (Ryoo et al., 2013).

CEBPE and CDKN2A variants were found to be more restricted to European population (Xu et al., 2012, 2013; Perez-Andreu et al., 2013). The disparities might be explained by the presence of population-specific disease variants and different LD between tag SNPs and causal SNPs. No significant associations were detected between LMO1 SNPs and childhood ALL in this study. Our results contradict those obtained from a previous study in a Caucasian population which showed that LMO1 SNP rs442264 was strongly associated with ALL risk (Beuten et al., 2011). In the same study, LMO1 SNPs rs11041815, rs11041830, and rs11041831 were also found to be associated with ALL; a 14-SNP haplotype block within LMO1 gene was shown to increase susceptibility to ALL (Beuten et al., 2011). High expression of this gene is seen in several tissues, such as bone marrow (Liu et al., 2008), and plays a role in protein-protein interaction (Rabbitts, 1994). Abnormal LIM protein expression contributes to T cell leukemogenesis (Boehm et al., 1991; Izraeli, 2004) and about 45% of T-ALL has aberrant LMO1 expression (Aifantis et al., 2008). The role of LMO1 variants in ALL pathogenesis has yet to be thoroughly investigated.

Conclusion

We have performed the first study investigating the relationship between IKZF1, DDC, CDKN2A, CEBPE, LMO1 gene polymorphisms, and childhood ALL risk in the Yemeni population. We have found that IKZF1 SNPs rs10235796 and rs6964969 and CDKN2A SNP rs3731246 were significantly associated with increased risk of childhood ALL. Replication studies in the Arab population with larger sample sizes are necessary to validate the extent of our findings.