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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 3  |  Page : 328-332

Association of RB1 rs9568036 and CDKN1A rs1801270 polymorphisms with retinoblastoma susceptibility

1 Eye Research Center, The Five Senses Institute, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran
2 Eye Research Center, The Five Senses Institute, Rassoul Akram Hospital; Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
3 Department of Genetic and Molecular Biology, Iran University of Medical Sciences, Tehran, Iran

Date of Submission16-Apr-2022
Date of Decision25-Jun-2022
Date of Acceptance26-Jul-2022
Date of Web Publication30-Nov-2022

Correspondence Address:
Golnaz Khakpoor
Rassoul Akram Hospital, Niyayesh Ave., Sattarkhan St. Tehran 14455-364
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/joco.joco_125_22

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Purpose: To investigate the association of polymorphisms (rs9568036 and rs1801270) in the RB1 and P21 genes with susceptibility to retinoblastoma (RB).
Methods: This case–control study was designed with 50 patients with RB and 50 controls. Polymerase chain reaction was performed to amplify the intron 17 of RB1 rs9568036 and exon 2 of P21 rs1801270. Then, all the amplified fragments were subjected to directional sequencing, and finally, the association between genotypes and the development of RB risk and invasion was studied.
Results: A statistically significant difference in genotypic or allele frequencies of single-nucleotide polymorphisms (SNPs) (rs1801270 and rs9568036) was found between Iranian RB patients and the controls (P > 0.05). However, the frequency of genotype RB1 rs9568036 observed a statically significant difference in the RB patients compared to the control group, and the nonwild-type allele A increased the chance of susceptibility to developing RB by 2.92 times.
Conclusion: The rs9568036 SNP in the RB1 gene may increase susceptibility to the development of RB in the affected patients. In spite of that, this polymorphism does not influence RB patient's invasion. Further investigation with a large enough sample size is recommended to validate this hypothesis.

Keywords: CDKN1A rs1801270, Genetics, Polymorphism, RB1 rs9568036, Retinoblastoma

How to cite this article:
Azimi F, Naseripour M, Sedaghat A, Kachoei ZA, Khakpoor G. Association of RB1 rs9568036 and CDKN1A rs1801270 polymorphisms with retinoblastoma susceptibility. J Curr Ophthalmol 2022;34:328-32

How to cite this URL:
Azimi F, Naseripour M, Sedaghat A, Kachoei ZA, Khakpoor G. Association of RB1 rs9568036 and CDKN1A rs1801270 polymorphisms with retinoblastoma susceptibility. J Curr Ophthalmol [serial online] 2022 [cited 2023 Jun 9];34:328-32. Available from: http://www.jcurrophthalmol.org/text.asp?2022/34/3/328/362450

  Introduction Top

Retinoblastoma (RB) (OMIM#180200) is the most frequent intraocular tumor in children, with 5000–8000 new pediatric cases every day worldwide. In 95% of cases, including both hereditary (40%) and sporadically (60%) types, it affects children under 5 years[1],[2],[3] by inactivating two alleles of the RB1 gene in the 13q14 locus via translocations, point mutations, insertions, and deletions (INDELs).[4],[5] In the hereditary type, these mutations are present in the germline, but they occur only in the tumor tissue in the sporadic type.[6],[7]

Deletion of members of the RB family results in a compensatory increase in the 21 kD protein (p21) (cyclin-dependent kinase inhibitor),[8] which the CDKN1A gene encodes at 6p21.2. After activation of p53 in response to cellular DNA damage, the p21 protein is upregulated. p21 is an important protein that plays a vital role in cell cycle check by inhibiting the activity of cyclin complexes (E/cdk2 and A/cdk2).[9],[10]

Polymorphisms are present in all individuals, and because they are kind of changes in the genomic sequence, they are always passed on to the next generation. In fact, the reason they are called polymorphisms is that although they are changes in everyone's genomes, they often do not cause a specific disease or phenotype. However, some of them can be associated with some traits or diseases such as RB. The most common type of polymorphism is single-nucleotide polymorphisms (SNPs). These changes can either protect people or make them susceptible to certain diseases. In RB, in addition to mutations in the RB1 gene, polymorphisms have been reported in several genes that are associated with disease onset and invasiveness.[11],[12]

p21 SNP converts serine to arginine in codon 31 (rs1801270 C > A), which is part of the zinc finger region with highly conserved,[13] it can lead to a lack of p21 accumulation during cellular DNA damage.[14] Thus, this p21 SNP may affect the activity and expression of p21. As a result, it disrupts the activity of the p53 pathway and plays a role in susceptibility to different types of cancers, including RB.[15],[16]

In the following, RB1 rs9568036 and P21 rs1801270 were selected for this experiment from the HapMap SNP database. For the first time, we studied these SNPs in the RB1 and P21 genes to investigate their association with RB cancer risk and invasiveness in an Iranian population.

  Methods Top

Our study included 50 RB patient samples (36 bilateral and 14 unilateral) who were treated between April 2018 and August 2020. The 50 control samples were selected from normal individuals (25 females and 25 males) with similar socioeconomic status and geographical origins that had been referred to the hospital for examination of other problems (without eye involvement). Clinical data of the RB patients (gender, age-onset of disease, laterality, status of global, focality, and international classification of retinoblastoma (ICRB) classification was collected from their medical records.

Our study with the ethics code 1398.723 was approved by the Ethical Committee of the Iran University of Medical Sciences, and written informed consent was received from the caregivers of the patient and control groups.

Three ml of blood samples was collected in EDTA anticoagulant tubes from all the participants.

Genomic DNA was extracted from peripheral blood samples using extraction kit (BIORAN AccuPrepk, Germany). Then, the extracted DNA was preserved at −20°C for the next step of the study. A spectrophotometer (Thermo Scientific™ NanoDrop™ 2000/2000c, USA) was used to measure the amount, purity, and concentration of the extracted DNA.

The exon 2 of the CDKN1A gene and intron 17 of the RB1 gene fragments were amplified using gene-specific oligonucleotide primers. The primer sequences and polymerase chain reaction (PCR) conditions are shown in [Table 1].
Table 1: Primer sequences of polymorphisms and polymerase chain reaction conditions for target amplification

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PCR was conducted in a 30 μl mixture containing 10 pM of each primer, 200 ng of genomic DNA, 25 μl of 2× Master Mix containing Taq DNA polymerase, MgCl2, dNTPs, and reaction buffers. PCR products were conducted in 1% agarose gel electrophoresis with constant V90 voltage and alternating current for 60 min with GelRed and then observed under ultraviolet light.

Then, 25 μl of each PCR product with forward primer for direct sequencing of P21 SNP and RB1 SNP (exon 2 and intron 17, respectively) was sent to South Korea. All samples were sent for Sanger sequencing on an ABI 3730 sequencer (Bioneer, South Korea), and the results were analyzed using Chromas and Bio Edit software.

Electropherograms were used to find the polymorphisms in sense directions.

Statistical analysis

Molecular data were analyzed using logistic regression (P < 0.05 was assumed to be statistically significant). Odds ratios (ORs) were checked with a 95% confidence interval (CI) using SPSS version 22 software (IBM Corp. Armonk, NY, USA). The Hardy–Weinberg equilibrium (HWE) was measured by the Guo and Thompson method.[17]

  Results Top

Fifty RB patients (mean of 22.5 ± 17 months) and the control group of 50 individuals without RB (mean of 20.5 ± 17 months), with age onset of disease of 0–60 months, were included in the study. Both the groups had an almost equal ratio of both sexes. According to the ICRB, 15 eyes from 50 patients in the RB group were enucleated, including 9 eyes (60%) in Group D and 6 eyes (40%) in Group E.

Genotype distribution and the frequencies of allelic variants of RB1 and CDKN1A genes in patients and controls are presented in [Table 2]. [Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e, [Figure 1]f demonstrates the sequencing results using Chromas software.
Table 2: Genotypes distribution and allele frequencies for p21 rs1801270 and RB1 rs9568036 polymorphisms

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Figure 1: Sequence chromatogram of some individuals showing single nucleotide polymorphism (SNP) sites. The SNP sites are indicated by symbol*. (a) Homozygous wild-type for RB1 rs9568036 (G > A); (b) Heterozygous nonwild-type for RB1 rs9568036 (G > A); (c) homozygous nonwild-type for RB1 rs9568036 (G > A); (d) Homozygous wild-type for p21 rs1801270 (C > A); (e) Heterozygous nonwild-type or p21 rs1801270 (C > A); (f) Homozygous nonwild-type for p21 rs1801270 (C > A)

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The polymorphisms studied in this experiment were observed in the HWE in the control groups. There was a significant difference between the minor allele frequency (MAF) of the two selected SNPs between RB patients and the control group (all P > 0.05).

In RB patients, the frequency of nonwild-type allele A for rs9568036 was significantly higher than in the control group (OR = 2.92, 95% Confidence interval [CI]: [0.35–4.3], P = 0.014). Conversely, there was no significant difference in RB patients compared to the control groups for the frequency of nonwild allele A in rs1801270 (OR = 1.21, 95% CI: [0.37–3.8], P = 0.766).

Similarly, there was no statistically significant difference between the frequency of the combination of polymorphisms RB1 rs9568036 and p21 rs1801270 in the RB patients and the control group (OR = 1.22, 95% CI: [0.35–4.3], P = 0.75).

After establishing the above information, we examined the association of significant polymorphism (RB1 rs9568036) and p21 rs1801270 with RB invasion in Iranian patients using logistic regression analysis. The results are shown in [Table 3] and [Supplementary Table 1].
Table 3: The association between the RB1 rs9568036 with sex, laterality, diagnosis age, status of global and focality

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There was no statistically significant association between RB1 rs9568036 polymorphism and gender (P = 0.730), laterality of the disease (unilateral or bilateral) (P = 0.638), age onset of disease (<24 and >24) (P = 0.164), enucleating or preserved globe (P = 0.125), or in terms of focality (unifocal or multifocal) involvement (P = 0.979). Similarly, we found no association between p21 rs1801270 and the risk of RB invasion [Supplementary Table 1].

Finally, we analyzed the frequencies of the RB1 rs9568036 in the nonwild-type allele A based on ICRB classification. The results were as follows: 3 A (6%), 12 B (24%), 9 C (18%), 6 D (12%), and 8 E (16%). Furthermore, in the wild-type allele G, the frequencies of this SNP were 3 A (6%), 2 B (4%), 2 C (4%), 3 D (6%), and 2 E (4%). Similarly, the nonwild-type allele A in p21 rs1801270 was 2 A (4%), 1 B (2%), and 3 C (6%), and the wild-type allele C was 18 A (36%), 13 B (26%), 9 C (18%), 2 D (4%), and 2 E (4%).

  Discussion Top

RB is a malignant eye cancer that affects children under 5 years old. 1 In addition to translocations, point mutations, and INDLs, genetic polymorphisms can play a role in the development of this type of cancer as well.[4],[5]

To the best of our knowledge, this is the first study that has evaluated the SNPs rs9568036 and rs1801270 in p53 and RB signaling pathways in the Iranian. Our results for RB rs9568036 revealed that the frequency of the nonwild-type A allele in patients was higher than in the controls. Compatible to our study, Anaya-Pava et al. also showed that the frequency of the nonwild-type A allele in patients with RB was higher than control.[18] We found that allele A in the nonwild-type increases the chance of developing RB by 2.92 fold. However, our results did not display any association between RB rs9568036 and RB invasion. Published studies on polymorphism RB1 rs9568036 have demonstrated that it was not only associated with better tumor response to chemotherapy[18] but also can result in increased survival rates of nonsmall lung cancer cells patients.[19]

Although for p21 rs1801270, our finding did not reveal any statistically significant difference in frequency of the nonwild-type A allele in patients compared to the control group, Carvalho et al. reported that the frequency of the nonwild-type A allele in patients was greater than in controls.[16] The frequency of the A allele (4.5) was relatively high in RB patients in Chen et al.'s report, compared to our study (0.07).[20]

The frequency assessment indicates the MAFs of rs1801270 among ethnic groups displays a wide range of difference from 0.04 in Swedish Caucasians to 0.5 in Chinese. Relatively higher prevalence was observed in African and Indians than in Swedish Caucasians.[21] Similar to our study, a meta-analysis of case–control studies by Cao et al. indicated no statistically significant association between p21 rs1801270 polymorphism and susceptibility to RB.[22]

In contrast to several studies reporting that p21 rs1801270 SNP is associated with the increased risk of various types of cancer,[23],[24],[25],[26] in our study, the difference was not statistically significant for the chance of developing RB in the presence of the nonwild-type allele A of p21 rs1801270. In addition, Carvalho et al. found an increased RB risk in the CA genotype of Brazilian carriers.[16]

Despite a 38% decrease in p21 expression due to SNP rs1801270,[26] in our study, no statistically significant association was found between p21 rs1801270 and RB invasion.

We also investigated the association between the double heterozygous polymorphisms (rs9568036 [G > A] and rs1801270 [C > A]) and susceptibility to RB and invasion. No association was found using logistic regression analysis on double heterozygotes between a group of patients and control children.

The first major limitation of the study is the selection of controls, which may be an important source of bias, and the second limitation is the small sample size that reduces the available statistical power, which makes it difficult to study low-frequency genotypes as well as any small effect size SNPs.

In summary, based on these results, we demonstrate that the minor alleles of the polymorphism RB1 rs9568036 may play as risk factors for the development of RB in our samples. However, it does not seem to affect the invasion. Further functional studies on larger samples are needed for different ethnic groups to confirm that this polymorphism does indeed affect the development of RB.

Financial support and sponsorship

This research was financially supported by the Iran University of Medical Sciences, Tehran, Iran.

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1]

  [Table 1], [Table 2], [Table 3]


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