| Abstract |
Acute lymphoblastic leukemia is the most common type of leukemia in
children. It accounts for one‐third of all cancers diagnosed in children under
15 years old. Despite the high survival rates (overall survival over 90%), a
noteworthy number of children relapse, and for them, the outcome remains
poor. Epidemiological studies that examined possible risk factors for acute
leukemias showed that inherited genetic variants can play an important role
in leukemogenesis. Recent gene association studies for cancer risk have
focused on the effects of single nucleotide polymorphisms (SNPs) of genes
that are responsible for the regulation of inflammation and tumor
suppression, such as chemokines, TP53 and P450 cytochrome. CXCL12 is a
chemokine expressed in various tumors and its polymorphism rs1801157 may
influence the ALL risk in children. The tumor protein p53 (TP53) is a tumor
suppressor protein whose polymorphism rs1042522 has been studied among
risk factors for malignant diseases. Additionally, previous studies have shown
a possible association between SNPs of Cytochrome P450 Family 1 Subfamily
A Member 1 (CYP1A1) gene and risk of leukemia. More specifically, rs1048943
of CYP1A1*2C has been reported to increase the risk of ALL in Caucasians. The
FAS gene, also known as TNFSF6 / CD95 / APO‐1, belongs to the family of
tumor necrosis factor receptors (TNF receptors) and is one of the essential
molecules of the apoptosis pathway. FAS rs2234767 polymorphism is a
replacement of G from A at position ‐1377, which has been shown to reduce
promoter activity and FAS expression levels. Decreased expression of the FAS
gene leads to a decrease in FAS‐mediated apoptosis and is associated with an
increased risk of cancer. This polymorphism has been shown to reduce the risk
of carcinogenesis.
In this study, we attempt to investigate the association between genetic
polymorphisms in FAS, CYP1A1, CXCL12 and TP53 genes in children and adolescent patients with ALL in the island of Crete, Greece. The case group
consisted of 86 patients, children and adolescents, (51 males and 35 females)
which were diagnosed with ALL in the department of Pediatric Hematology‐
Oncology in the University Hospital of Heraklion in Crete from 1990 to January
2018. The control group consisted of 105 adult blood donors and 20 children,
from the same region of patients. Whole blood was collected in EDTA–
containing tubes. Genomic DNA was isolated from peripheral blood
leukocytes, was quantified by spectrophotometry and stored at ‐20οC until
analyzed. Amplification and genotyping for the FAS rs2234767, CXCL12
rs1801157, TP53 (codon 72) rs1042522 and CYP1A1*2C rs1048943 SNPs was
performed by restriction fragments lengths polymorphism (RFLPs). Concerning
TP53, the Pro allele yielded a 178‐ bp product, while the Arg allele yielded a
136‐bp PCR product. Regarding CXCL12, the G allele yielded a 193‐bp and a
100‐bp product, while the A allele yielded a 293‐bp product and concerning
CYP1A1, the A allele yielded 149‐bp and 55‐bp products, while the G allele
yielded a 204‐bp product. Finally, concerning FAS, the A allele yielded a
product of 122‐bp and the G allele yielded products of 103‐bp and 19‐bp.
Data manipulations and statistical analyses were conducted using (a) the IBM
SPSS Statistics Version 25 statistical package, and (b) a set of MS Excel
applications developed in the laboratory of Bio‑Medical Data Analyses, Digital
Applications and Interdisciplinary Approaches in Medical School of Crete.
Chi‑square and Fischer's exact tests were also performed for data analysis. P
values <0.05 were considered as statistically significant.
The rs1048943 of CYP1A1 gene was genotyped in 84 patients (49 males, 35
females) and 125 controls, rs1801157 of CXCL12 was genotyped in 83 patients
(49 males, 34 females) and 120 controls and rs1042522 of TP53 gene was
genotyped in 86 patients (51 males, 35 females) and 121 controls. The
rs2234767 of FAS was genotyped in 103 patients and 118 controls. CYP1A1: We observed a frequency of 51/84 (60.7%) and 105/125 (84%) for
AA genotype, 33/84 (39.3%) and 19/125 (15.2%) for AG genotype and 0/84
(0%) and 1/125 (0.8%) for GG genotype among patients and controls,
respectively. A significant correlation was found between the AG genotype
and ALL patients (OR=3,5759; 95% CI: 1.8553‐6.8918). A person with AG
genotype has 3,57 times chances of being ALL patient compared with AA
genotype, which is statistically significant.
CXCL12: We observed a frequency of 9/83 (10.8%) and 16/120 (13.3%) for AA
genotype, 50/83 (60.2%) and 55/120 (45.8%) for AG genotype and 24/83
(28.9%) and 49/120 (40.8%) for GG genotype among patients and controls,
respectively.
TP53: We observed a frequency of 35/86 (40.7%) and 64/121 (52.9%) of
homozygous prevalent (Arg/Arg genotype), 37/86 (43%) and 47/121 (38.8%)
of heterozygotes (Arg/Pro genotype) and 14/86 (16.3%) and 10/121 (8.3%) of
rare homozygous (Pro/Pro genotype) among patients and controls,
respectively. We noticed that a person with TP53 Pro/Pro has 2,56 times
chance of being ALL patient compared with TP53 Arg/Arg (OR= 2.5600, 95%
CI= 1.0303‐6.3607), which is statistically significant.
FAS: We observed a frequency of 2/103 (1.9%) and 58/118 (49.2%) for AA
genotype, 19/103 (18.4%) and 9/118 (7.6%) for AG genotype and 82/103
(79.6%) and 51/118 (43.2%) for GG genotype among patients and controls,
respectively. A significant correlation was found between AG genotype and
ALL patients (OR = 61.2222, 95% CI = 12.1470 ‐ 308.5663) and GG genotype
and risk of ALL (OR = 46.6275 95% CI = 10.9122 ‐ 199.2382). An individual with
AG genotype has 20,35 times the risk of being a patient than those who
carries AA genotype and an individual with GG genotype has 18,49 times the
risk of being a patient than those who carries AA genotype, and these results
are statistically significant. Moreover, we tested the possible combinations of CYP1A1, CXCL12 and TP53
in pairs and in all three together. FAS rs2234767 polymorphism was not
included in the genotype combinations as it was analyzed at a later time. We
showed that an individual with CYP1A1 AG and TP53 Arg/Pro genotype has
3,14 times the risk of being a patient with ALL compared to those who carries
the wild/major type for both genes OR= 4.6184, 95% CI= 1.6584‐12.8620.
Moreover, an individual with CYP1A1 AG and CXCL12 GG has 8,29 times the
risk of being a patient than those who carries AA genotype for both genes
(OR= 11.3684, 95% CI= 2.2156‐58.3335), whereas an individual with CYP1A1
AG and CXCL12 AG genotype has 1,9 times the risk of being a patient
compared to the wild type (OR= 5.4000, 95% CI= 1.6899‐17.2557).
Finally, an individual with CYP1A1 AG, CXCL12 AG and TP53 Pro/Pro genotype
has 11,2 times chances of being ALL patient compared to those who have AA
genotype for CYP1A1 and CXCL12 and Arg/Arg genotype for TP53 (OR=
11.2000, 95% CI= 1.0421‐120.3681), which is statistically significant.
Our study is the first report that associates CYP1A1*2C heterozygotes with an
increased risk for ALL in Caucasian children. To our knowledge, it is also the
first study that investigated the different combinations of the polymorphisms
of CYP1A1*2C (rs1048943), CXCL12 (rs1801157) and TP53 (rs1042522) genes,
leading to some interesting results. The risk of developing pediatric ALL is
significantly increased when an individual has the following combinations of
genotypes i) CYP1A1 AG and TP53 Arg/Pro, ii) CYP1A1 AG and CXCL12 GG, iii)
CYP1A1 AG and CXCL12 AG, iv) CYP1A1 AG, CXCL12 AG and TP53 Pro/Pro. In
addition, our study correlated heterozygotes (AG) and homozygotes (GG) for
FAS rs2234767 polymorphism with an increased risk of ALL, a result that is
consistent with literature data on this polymorphism.
The main limitation of our study is the small study population, which does not
allow the generalization of this results' interpretation. Thus, studies with a
larger number of both patients and controls are needed to support further the data presented. We consider that the investigation of the above genotypes’
combinations that increased the ALL risk in our study population may be a
promising future screening tool for increased ALL risk in children.
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Μελέτη του ρόλου των πολυμορφισμών των γονιδίων FAS, CXCL12, TP53, kai CYP1A1,στην οξεία λεμφοβλαστική λευχαιμία παιδιών και εφήβων
