Association of KDR (rs2071559, rs1870377), CFH (rs1061170, rs1410996) genes variants and serum levels with pituitary adenoma

Abstract Introduction Pituitary adenomas (PA) are slow‐growing, benign tumors that usually do not metastasize to other body organs. Although they are referred to as benign, tumor growth can eventually put pressure on nearby structures, spread to surrounding tissues, and cause symptoms. The exact cause of PA is unknown, and the pathogenesis is multifactorial. Methods Our study included PA patients and healthy volunteers. Genomic DNA was extracted using the DNA salting‐out method. All participants were genotyped for the KDR rs2071559, rs1870377, CFH rs1061170, and rs1410996 polymorphisms. Serum levels of KDR and CFH were examined using the ELISA method. Results The results of the present study showed that KDR rs2071559 A allele was associated with the occurrence of PA, hormonally active PA, invasive PA, and PA without recurrence development. KDR rs1870377 increased the probability of invasive PA and PA recurrence. CFH rs1061170 C allele was associated with hormonally active PA and the T allele was associated with non‐invasive PA development. Conclusion KDR rs2071559, rs1870377, and CFH rs1061170 could be potential biomarkers associated with PA.

headaches, changes in visual function, or endocrine disorders such as infertility, decreased libido, and galactorrhea (Lake et al., 2013).PA accounts for about 10%-25% of all intracranial tumors, and the occurrence of clinically diagnosed PA is 1 per 1000 cases in the general population (Daly & Beckers, 2020;Yang & Li, 2019).Adenomas are classified according to size, location, cell type, neoplastic behavior, and hormonal activity (Ho et al., 2021;Holmes et al., 2007;Ken et al., 2021).Clinically active PAs (prolactinomas, somatotroph, corticotroph, thyrotrophin, and gonadotroph adenomas) make excess hormones, whereas non-functioning tumors do not show signs and symptoms of hypersecretion (Mehta & Lonser, 2017;Trouillas et al., 2020;Varlamov et al., 2019).The exact cause of PA is unknown, and its pathogenesis is multifactorial.Genetic predisposition implies the existence of germline DNA mutations that have a variety of impacts on pituitary cellular biology (Jaffrain-Rea et al., 2011).
Abnormal gene expression is linked to genetic changes such as gene amplification or allele loss and epigenetic changes such as promoter methylation (Vandeva et al., 2010).While thousands of genetic variants, including single nucleotide polymorphisms (SNPs), are related to different types of cancer, the molecular mechanisms of the diseases are still not fully understood (Bakhtiari et al., 2020).SNP analysis is a valuable predictor of cancer risk in particular populations where this polymorphism is frequently detected (Deng et al., 2017).More data on PA factors are needed to make it easier to predict tumor occurrence, formation, and invasiveness.New molecular markers can optimize treatment and increase patients' survival (Alwi, 2005).
Vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) control angiogenesis -the later creation of blood vessels from pre-existing vessels, and vasculogenesis -the early embryonic development of blood vessels from precursor cells.VEGF-A and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) play major roles in physiological as well as pathological tumor angiogenesis (Shibuya, 2011).VEGFR2/KDR is more widely distributed and expressed in all blood vessels at endothelial contact sites and, therefore, could be found in tumor vasculature.Kinase insert domain receptor (KDR) is widely regarded as having a crucial part in mediating VEGF-induced responses in angiogenesis (Rydén et al., 2003;Fu et al., 2014).KDR gene mutations, insertions, and deletions are associated with cancer occurrence, and alterations are observed in 3.02% of all cancers including lung adenocarcinoma, melanoma, colon adenocarcinoma, and conventional glioblastoma (The AACR Project GENIE Consortium, 2017).
The complement system is a component of innate immunity that plays diverse roles in the inflammatory response, hemostasis, and embryogenesis and is implicated in various stages of tumorigenesis and cancer progression (Afshar Kharghan, 2017).Complement factor H (CFH) is one of the key regulators in the alternative complement pathway, which has been known to inhibit the complement pathway by binding to C3b and destroying the C3 convertase (Ezzeldin et al., 2015).Changes in the CFH gene are associated with lung cancer (Yoon et al., 2019).Therefore, this research aims to identify SNPs of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 as well as KDR and CFH serum levels in PA patients and healthy subjects and relate the obtained results to PA hormonal activity, invasiveness, and recurrence.

| Patients and ethical requirements
Permission (No. BE-2-47; approved date: 25 December 2016) to perform the research was approved by the Ethics Committee for Biomedical Research at the Lithuanian University of Health Sciences (LUHS).All subjects have signed an agreement according to the Declaration of Helsinki.The study was conducted at the Laboratory of Ophthalmology, Neuroscience Institute, LUHS.Study subjects were divided into two groups: • Group I: patients with PA (N = 100) comprised 39 men and 61 women.Inclusion criteria for the study were: adult (age ≥18 years); PA diagnosed and confirmed by magnetic resonance imaging (MRI); suitable patients' general health and absence of other tumors.Moreover, PA patients were distributed into subgroups by invasiveness, hormonal activity, and recurrence.Invasiveness, hormonal activeness, and recurrence evaluation were described previously in our studies (Glebauskiene et al., 2017;Sidaraite et al., 2020;Yoon et al., 2019).• Group II: healthy control group comprised 112 women and 88 men, with good general health on the examination date.

| SNP selection
The KDR gene is located on the long arm of chromosome 4 (4q12), and the CFH gene is located on the long arm of chromosome 1 (1q31  et al., 2021;Neto et al., 2021;Suchankova et al., 2009).Increased VEGF/KDR signaling promotes angiogenesis and plays a key role in chronic inflammation (Paradowska-Gorycka et al., 2019).The gene CFH is involved in the removal of waste and damaged cells from the body.It is suggested that deficiency of CFH and accumulation of waste and damaged cells in the body may lead to oxidative stress and inflammation (He & Karin, 2011;Armento et al., 2021).Since both KDR and CFH genes are associated with inflammation, they may also be linked to cancer development and progression (Singh et al., 2019).Because cancer cells have a relatively high metabolic demand for oxygen and nutrients to continue growing, angiogenesis is important for tumor development.The tumor vasculature has been found to express KDR at higher levels than the normal vasculature, and it has been hypothesized that inhibition of angiogenesis may lead to tumor growth arrest (Holmes et al., 2007).CFH is likely involved in the resolution of inflammation in lipid-rich deposits that have accumulated in the arteries, brain, eyes, and kidneys (Meri & Haapasalo, 2020).This accumulation can activate the complement system, which triggers the development of an inflammatory phenotype in microglia, and phagocytic cells in the brain (Shen et al., 2003).Therefore, poor complement regulation may be involved in carcinogenesis, and complement activation may also interfere with tumor clearance by the immune system (Laskowski et al., 2020;Reis et al., 2018).
The pathogenesis of diseases associated with variations in the KDR and CFH genes involves multiple complex pathways and mechanisms, and further research is needed to fully understand the underlying processes of PA.

| DNA extraction and genotyping
Blood for DNA extraction was collected in EDTA tubes.Genomic DNA was extracted using the DNA saltingout method from 200 μL peripheral blood (white blood cells).The concentrations and purity indexes of DNA in each blood sample were evaluated by UV spectrophotometry (Agilent Technologies, Cary 60 UV-Vis) as the ratio absorbance 260/280 nm.All samples presented a purity index between 1.8 and 2.0.All participants were genotyped for KDR gene rs2071559, rs1870377, CFH gene rs1061170, and rs1410996 polymorphisms at the Laboratory of Ophthalmology, LUHS.SNPs were determined using TaqMan® genotyping assays (Applied Biosystems; Thermo Fisher Scientific), C__15869271_10 (rs2071559), C__11895315_20 (rs1870377), C___8355565_10 (rs1061170), C___2530294_10 (rs1410996).The genotyping was conducted using the real-time polymerase chain reaction (RT-PCR) method according to the manufacturer's recommendations using a Step One Plus RT-PCR system (Applied Biosystems).

| ELISA
For serum preparation, peripheral venous blood was collected.The blood samples were incubated for 30 min at room temperature and centrifuged.The serum was removed from the pellet, transferred into 2 mL tubes, frozen, and stored at −80°C until analysis.Following the manufacturer's instructions, serum KDR levels were evaluated in PA patients and control subjects.The assay was performed using an Aviva Systems Biology KDR ELISA Kit (Human) based on standard sandwich ELISA technology, with a sensitivity of <0.236 ng/mL.Also, serum CFH levels were determined following the manufacturer's instructions in PA patients and control subjects, using a sandwich-type Invitrogen Complement Factor H ELISA Kit (Human), with a sensitivity of 2 ng/mL.

| Statistical analysis
Statistical data analysis was performed using SPSS/W 27.0 software (Statistical Package for the Social Sciences for Windows, Inc.) statistical data analysis program.The genotype distribution of SNPs was analyzed using the Chi-square test.To estimate the association between KDR rs2071559, rs1870377, CFH rs1061170, and rs1410996 and PA development, odds ratios (ORs) and 95% confidence intervals (CIs) were computed using binary logistic regression analysis.A two-sided p < 0.05 was considered to be statistically significant.The Akaike information criterion (AIC) selected the best genetic model.Statistically significant differences were considered when p < 0.05.Genotypes were assessed using indicator variables with the common homozygote as a reference.The serum KDR and CFH levels were examined in the groups of PA patients and healthy individuals using the Mann-Whitney U test.Also, we performed haplotype association analysis in PA and control groups.It was performed using the online SNPStats website (https:// www.snpst ats.net/ snpst ats/ ).Linkage disequilibrium (LD) was measured using D′ and r 2 .The associations between the haplotypes and PA were calculated using logistic regression.The associations were introduced as ORs and 95% CI and measures in PA and healthy subjects.

| RESULTS
A case-control study was conducted involving 300 individuals who were divided into two groups: the control group (N = 200) and a group of subjects with pituitary adenoma (N = 100).After forming the groups of subjects' an analysis of single nucleotide polymorphisms of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 was performed.The PA group consisted of 100 persons: 39 men (39.0%) and 61 women (61.0%).The average age of patients was 52.4 years.The patients' group was later divided into subgroups by PA's hormonal activity, invasiveness, and recurrence.The control group consisted of 200 individuals: 88 men (44.0%) and 112 women (56%).The average age of the control group was 69.1 years.Further analysis was adjusted by age after considering the statistically significant age difference in the comparison control and PA groups.The demographic data of the subjects are presented in Table 1.
3.1 | KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 associations with PA occurrence After analyzing the genotypes and alleles of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996, it was found that only the KDR rs2071559 AA genotype and A allele were statistically significantly more frequent in PA patients compared to the control group individuals (39.0 vs. 26.5, p = 0.027, 60.5 vs. 51.0,p = 0.028) (Table 2).There were no statistically significant differences between KDR rs1870377, CFH rs1061170, and rs1410996 distribution of genotypes and alleles for patients with PA and the control group.
Binominal logistic regression analysis of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 showed no statistically significant differences between PA and control group (Table S1).
3.2 | KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 associations with PA hormonal activity After evaluating the distribution of genotypes and alleles of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in hormonal active PA and the control groups, it was found that both KDR rs2071559 AA genotype and A allele were statistically significantly more frequent in hormonal active PA group compared to a control group (41.8 vs. 26.5, p = 0.028, 62.7 vs. 51.0,p = 0.029, respectively).Also, CFH rs1061170 CC genotype and C allele were statistically significantly less frequent in the hormonal active PA group than in the control subjects (3.6 vs. 15.0,p = 0.024, 28.2 vs. 39.5, p = 0.030, respectively).Analysis of KDR rs1870377 and CFH rs1410996 polymorphisms revealed no statistically significant differences between hormonal active PA and the control groups (Table 3).
We analyzed KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 associations with PA without hormonal activity.There were no statistically significant differences between genotype distribution in patients with PA without hormonal activity and the  control group (Table S2).Also, binary logistic regression of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in PA with and without hormonal activity showed no statistically significant results (Tables S3 and  S4, respectively).4).
After evaluating the distribution of genotypes and alleles of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in patients with invasive PA and the control group, it was found that KDR rs2071559 both AA genotype and A allele were statistically significantly more frequent in patients with invasive PA group compared to the control group (42.2 vs. 26.5., p = 0.018; 63.3 vs. 51.0,p = 0.015, respectively).However, KDR rs1870377, and CFH rs1061170, rs1410996 showed no statistically significant results (Table 5).
After analyzing the influence of PA invasiveness on the disease manifestation, we found that the KDR rs1870377 was associated with 4.4-and 4.1-fold increased odds of invasive PA development in codominant and recessive models (OR = 4.407; 95% CI: 1.157-16.785;p = 0.030; OR = 4.141; 95% CI: 1.159-14.802;p = 0.029, respectively) (Table 6).Moreover, binary logistic regression analysis of these SNPs in non-invasive PA patients and the control group revealed no statistically significant results (Table S5).

| KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 associations with PAs recurrence
Analysis of the distribution of genotypes and alleles of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 showed that KDR rs2071559 AA genotype and A allele were statistically significantly less frequent in the group of PA patients without recurrence compared to the control group (14.9 vs. 26.5, p = 0.043, 38.5 vs. 51.0,p = 0.009, respectively).While the GG genotype was statistically significantly more frequent in PA patients with no recurrence compared to control group individuals (37.8 vs. 24.5, p = 0.029) (Table 7).Binary logistic regression analysis revealed no statistically significant results of SNPs in PA without recurrence patients and control group (Table S6).

rs1061170, rs1410996 haplotypes analysis
We performed a haplotype association analysis of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in patients with PA.Pairwise linkage disequilibrium between SNPs in PA patients is shown in Table 9.We analyzed haplotype frequencies, and statistical analysis of PA has shown that individuals carrying rs2071559, and rs1870377 haplotype A-A were associated with 1.7-fold increased odds of PA occurrence (OR = 1.67; 95% CI: 1.02-2.72;p = 0.041) (Table 10).Haplotype analysis of PA and CFH rs1061170 and rs1410996 showed no statistically significant results (Table S8).

| KDR and CFH serum levels
During the study, the concentration of KDR in the blood serum was examined in the groups of PA patients, and  1a).Also, we evaluated CFH serum level, but no statistically significant differences were found (median (IQR): 18.23 (63.98) vs. 13.66 (24.43), p = 0.596) (Figure 1b).

| DISCUSSION
PA tumorigenesis is not a fully understood process involving oncogene activation, tumor suppressor gene inactivation, and abnormal pituitary cell growth (Farrell & Clayton, 2000).PAs are sporadic in more than 95% of cases.Although whole genome sequencing studies have made significant progress in identifying their pathogenesis, the genetics of a significant fraction of pituitary tumors is still unclear (Melmed et al., 2022).The impact of KDR and CFH polymorphisms on the development of various tumors has been analyzed in many studies.To our knowledge, this is the first study targeting the association between KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 polymorphisms and PA occurrence.
As KDR regulates tumor angiogenesis, migration, and vascular permeability, it is associated with different types of cancers, including coronary heart disease, rheumatoid arthritis (RA) severity, age-related macular degeneration (AMD), and recurrent miscarriage (Gao et al., 2016;Jinnin et al., 2008;Kaira et al., 2022;Lazzeri et al., 2012;Pădureanu et al., 2017;Paradowska-Gorycka et al., 2019;Su et al., 2011;Vasconcelos et al., 2019;Wang et al., 2007).Pădureanu et al. have analyzed KDR rs2071559 polymorphism association with pancreatic cancer.The study showed that the GG genotype and G allele were more frequent among pancreatic cancer patients than controls (p = 0.036; p = 0.032, respectively), suggesting that the rs2071559 G allele may confer an increased risk of the disease (Pădureanu et al., 2017).According to a study done by Vasconcelos et al., inherited abnormalities in blood vessel formation related to KDR SNPs influence the risk and aggressiveness of high-grade glioma (HGG) in an ethnically diverse population from Southeastern Brazil (Vasconcelos et al., 2019).Fraga with co-authors investigated that carriers of the KDR rs2071559 A allele were more prone to higher VEGFR2/KDR expression in prostate epithelial cells (p = 0.006) (Fraga et al., 2017).Another study showed that higher KDR expression levels afford a very poor prognosis of colorectal cancer (Zhang et al., 2018).Also, the KDR rs2071559 G allele correlates with an increased risk of astrocytomas, while individuals with the A allele and genotype TA +AA of rs1870377 showed a protective effect against astrocytomas (Gao et al., 2016).Moreover, Qian et al. showed that the KDR rs2071559 AA genotype is associated with a fast baseline peritoneal solute transfer rate (PSTR), which is related to local membrane inflammation (p = 0.039) (Qian et al., 2022).As studies reveal controversial results, we observed that the KDR rs2071559 Abbreviations: PA, pituitary adenoma; p-value, significance level (differences considered significant when p < 0.05).
Note: Significant results are indicated in bold.a AA vs. AG+GG, p = 0.018.
AA genotype and A allele are statistically significantly more frequent in PA patients than in the control group (p = 0.027; p = 0.028, respectively).
Complement factor H (CFH) is a primary regulator of the alternative complement pathway that controls C3 activation and modulates innate immune responses (Kopp et al., 2012).C3a stimulates anterior pituitary hormone release and activates the hypothalamic-pituitary-adrenal axis, a key regulator of inflammation.The complement molecules modulate systemic inflammatory responses through communication with the pituitary gland (Francis et al., 2003).However, CFH is associated with neurodegenerative diseases, decreased risk of recurrent pregnancy loss, periodontitis, myocardial infarction (MI), and a smoking-related risk factor for lung cancer (Cho et al., 2019;Ezzeldin et al., 2015;Kardys et al., 2007;Maugeri et al., 2019;Salminen et al., 2022;Zhang et al., 2016).A study done by Zhang et al showed that CFH rs1061170 has strong associations with Alzheimer's disease (AD) in the European population and Han Chinese and European populations together (p < 0.001, p meta = 0.0005, respectively) (Zhang et al., 2016) Abbreviations: AIC, akaike information criterion; OR, odds ratio; PA, pituitary adenoma; p-value, significance level (differences considered significant when p < 0.05).
Note: Significant results are indicated in bold.
T A B L E 6 Binary logistic regression analysis of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in patients with invasive PA and control groups.
rs3753394 CT/TT genotype and T allele in P. aeruginosa-induced septic patients was significantly higher than in the control group (p = 0.033'; p = 0.014, respectively) (Li et al., 2020).The CFH rs1061170 independently predicted mortality at discharge and 6 months and survival duration after spontaneous intracerebral hemorrhage (p = 0.019; p = 0.041, respectively) (Appelboom et al., 2011).It is conceivable that the CFH rs1061170 could be used to predict poor disease prognosis.We found a correlation between KDR gene rs2071559 and PA hormonal activity, invasiveness, and recurrence.As far as we know, no studies analyzed the association between the KDR and CFH gene polymorphisms and PA invasiveness, hormonal activity, and recurrence.We revealed that the KDR polymorphism rs1870377 genotype had a 4.4-and 4.1-fold increase in the odds of invasive PA development in codominant and recessive models (p = 0.030; p = 0.029, respectively).Also, The KDR rs1870377 polymorphism increases the odds of PA recurrence by 7.2-and 9.3-fold in codominant and recessive models (p = 0.020; p = 0.007, respectively).
After analyzing the haplotype associations with pituitary adenoma, we revealed that individuals carrying KDR rs2071559, and rs1870377 haplotype A-A had increased odds of PA occurrence.A study done by Gao et al. showed that haplotype C-C-T and C-T-T were linked to an increased risk of astrocytomas in the haplotype-specific analysis.Variants of KDR rs2071559-C and rs2305948-T may increase the risk of astrocytomas, whereas mutants of KDR rs1870377-A may reduce the risk of astrocytomas (Gao et al., 2016).These results support the hypothesis that KDR and CFH genes are associated with inflammation in different organs, which can lead to an oncogenic process (Greten & Grivennikov, 2019).Further studies with larger sample sizes are warranted to evaluate KDR's and CFH's predictive and prognostic value.
Demographic characteristics of study subjects.
T A B L E 2 a AA vs. AG+GG p = 0.027.
Distribution of genotypes and alleles of KDR rs2071559, rs1870377 and CFH rs1061170, rs1410996 polymorphisms in patients with an invasive PA and control group.
T A B L E 5 . Li with co-authors investigates the genetic relationship between CFH SNPs and susceptibility to sepsis caused by bacterial infections in Chinese Han populations.The frequency of the Binary logistic regression analysis of KDR rs2071559, rs1870377, and CFH rs1061170, rs1410996 in PA with recurrence and control groups.Linkage disequilibrium between studied polymorphisms in patients with PA.Abbreviations: D′, the deviation between the expected haplotype frequency and the observed frequency; r 2 , the square of the haplotype frequency correlation coefficient; SNP, single nucleotide polymorphism; p-value, significance level when p < 0.05.
Abbreviations: AIC, Akaike information criterion; OR, odds ratio; PA, pituitary adenoma; p-value, significance level (differences considered significant when p < 0.05).Note: Significant results are indicated in bold.T A B L E 8 T A B L E 9