Polymorphisms of the collagen genes of the fibrous ring rs1800012, rs2276454, rs1793953 , and the VDR gene rs2228570 with intervertebral disc degeneration

The intervertebral disc (IVD) functions through the integration of the annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates. Key biomechanical properties of the IVD, such as resistance to rupture, stretching, shear, and static axial loads, are largely determined by collagens. The AF, primarily composed of collagen types I (COLI) and II (COLII), forms the outer structure of the IVD. COLI is located in the outer AF, providing tensile strength and resistance to deformation, while COLII in the inner AF contributes to elasticity and mobility. The ratio of type I to type II collagen changes gradually, with type II decreasing and type I increasing as one moves farther from the NP [5].


Introduction
Spinal disorders are increasing in many developed countries, with about 8 % of individuals becoming disabled each year due to back pain.Degenerative changes are believed to result from the gradual accumulation of micro-injuries during normal physiological loading, and genetic factors can accelerate these processes [1].Identifying candidate genes associated with early intervertebral disc degeneration is a promising method for early diagnosis and prognosis [2].Although matching genetic modifications to functional disorders is challenging, many structural changes that determine clinical diagnosis are already known [3,4].
The intervertebral disc (IVD) functions through the integration of the annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplates.Key biomechanical properties of the IVD, such as resistance to rupture, stretching, shear, and static axial loads, are largely determined by collagens.The AF, primarily composed of collagen types I (COLI) and II (COLII), forms the outer structure of the IVD.COLI is located in the outer AF, providing tensile strength and resistance to deformation, while COLII in the inner AF contributes to elasticity and mobility.The ratio of type I to type II collagen changes gradually, with type II decreasing and type I increasing as one moves farther from the NP [5].

Оригінальні дослідження / Original Researches
Collagen type 1 is a fibrillar heterotrimer composed of two α1 chains and one α2 chain.The genes for the α1 (COL1A1) and α2 (COL1A2) chains are located at 17q21.31-q22 and 7q22.1, respectively.The primary amino acid sequence of α chains in COL1 is crucial for the strength of the extracellular matrix (ECM) [6].The COL1A1 polymorphism (rs1800012) in the Sp1 binding site of the first intron (G/T substitution at position +1245) increases mRNA expression and COL1A1 protein levels.This disrupts the COL1A1 and COL1A2 ratio (2 : 1), forming rigid homotrimers composed of three COL1A1 chains, which have a rope-like appearance and resistance to protease action [7,8].These homotrimers and heterotrimers create heterofibrils, potentially leading to accelerated ECM degeneration and early intervertebral disc degeneration (IVDD) [9,10].
Population studies confirmed the significance of the COL1A1rs1800012 polymorphism in IVDD.Among Greek military persons with early lumbar disc degeneration, 33.3 % were TT genotype carriers, while the control group had no TT genotype carriers [11].Dutch research showed that TT genotype carriers aged 65 and older had a 3.6-fold higher incidence of IVDD compared to GT or GG carriers [12].Patients with the TT genotype also exhibited a higher Pfirman stage of degeneration and more severe IVDD compared to the control group, indicating that the COL1A1 Sp1 polymorphism is associated with increased risk and severity of IVDD [13].
Collagen type 2 is a fibrillar homotrimer encoded by the COL2A1 gene located at 12q13.11-q13.2.COL2A1 is crucial for IVD health, particularly in the interlamellar space of the AF, where it helps restore disc parameters after loading and is associated with lower levels of degeneration.Changes in COL2 quantity and quality are linked to various IVD disorders, such as herniations [14][15][16].Polymorphisms COL2A1rs2276454 and COL2A1rs1793953 are associated with IVD degeneration, with significant differences in allele frequencies between degenerated and control groups.COL2A1rs2276454 is an independent risk factor for IVDD [17,18].
Vitamin D receptor (VDR) belongs to the nuclear receptor superfamily of transcription factors.The VDR gene is located on the long (q) arm of chromosome 12 at position 13.11.Binding VDR and the active form of vitamin D to its nuclear receptor leads to the formation of an active transcriptional complex, which increases the expression of several proteins [19,20].The Fok1 polymorphism (missense initiator codon variant) is one of the functional and important polymorphisms of VDR gene that may correlate with IVDD.Fok1 polymorphism results in different translation initiation sites due to thymine to cytosine (T/C) substitution in the first translation initiation codon ATG.In the VDR ff variant, initiation of translation occurs at the first ATG site, giving rise to a full-length VDR protein comprised of 427 amino acids.In the VDR FF variant, translation begins at the second ATG site due to the substitution at the first site, resulting in a truncated protein with 424 amino acids.It has also been reported that FF, the shorter form of VDR protein is the more active form than its full-length protein, ff [21,22].The Fok1 polymorphism thus may affect function of VDR, resulting in altered efficiency of binding to vitamin D and transcription of the corresponding genes.Such sites have been identified in the promoter's collagen genes of type I, II, III, and V [22][23][24].
Some studies have found a connection between the VDR gene polymorphism rs2228570 (Fok1) and IVDD [25,26].However, there existed some diversities in Northern Europe, suggesting that carrying the VDR Fok1 F allele may be a protective factor against IVDD [27].
At the current stage, a concept is being formed about the multifactorial nature of IVDD.According to this concept, individual genetic variants may not manifest in the phenotype (manifestations of the disease), and the accidental combination of several genetic variants can determine a clearly expressed phenotype.The result of the combination of genes can manifest in the form of disruption of the interaction of gene products with polymorphisms, which can lead to pathological changes in tissues, organs, and their functioning, but without clear Mendelian inheritance.A person who has inherited such a combination of genes is also more sensitive to the influence of adverse external factors.To detect the association of combinations of genetic variant pairs with disease traits, research on linkage disequilibrium (LD) is conducted.LD manifests as a tendency for the frequency of allele combinations to deviate from the frequencies that would be expected under the condition of maintaining Hardy-Weinberg equilibrium [28].
The purpose was to study the association of polymorphisms COL1A1rs1800012, COL2A1rs2276454, COL2A1rs1793953 (collagen genes) as the structural components of the fibrous ring, and VDRrs2228570 with dege neration of L 4 -L 5 , L 5 -S 1 , C 5 -C 7 IVD among ethnic Ukrainians.
Most patients had a prolonged (on average, a year-long) pain syndrome that did not disappear with conservative treatment, which led to their referral to a medical institution and surgical intervention.The case group consisted mostly of patients with the third and fourth stages of osteochondrosis.The acute phase of the disease was most commonly observed in men and women aged 30-40.There was no statistically significant difference in the number of patients with smoking, diabetes, hypothyroidism, excessive physical exertion, spinal trauma, excessive weight, and motor homogeneity between the case and control groups.The erythrocyte sedimentation rate of all examined patients was within the physiological norm.Spinal injury preceded degenerative changes in approximately 10 % of patients, and the number of cases did not differ statistically between the groups.All study participants underwent magnetic resonance imaging (MRI).The control group included the persons without registered MRI pathologies.All examined individuals resided in the territory of Ukraine and belonged to the ethnic Ukrainians.

Оригінальні дослідження / Original Researches
The permission for the study was obtained from the bioethics committee of the Romodanov Neurosurgery Institute of the National Academy of Medical Sciences of Ukraine.All patients gave informed written consent to participate in the study.

Methods
In this study, the TaqMan Universal PCR Master Mix (Applied Biosystems, USA) and TaqMan SNP Genotyping Assays (Applied Biosystems, USA) were used for the determination of polymorphisms.Real-time PCR was conducted using the CFX96 instrument (BioRad, USA).Context sequences for the determination of collagen gene polymorphisms were as follows: - Genotype determination of polymorphic loci was carried out using allele discrimination method with the CFX96 Real-Time PCR Detection System software.

Statistical analysis
The statistical analysis was conducted using the publicly available online program SNPStats [29,30].Genotype distribution frequencies were checked for deviations from Hardy-Weinberg equilibrium using the Pearson chi-squared (χ 2 ) test.The program used various models for genotype frequency analysis: codominant, dominant, recessive, overdominant, and additive.The likelihood of a clinical feature occurring depending on the presence of polymorphic variants in binary analysis (case-control) was determined using logistic regression analysis.The results were summarized in terms of genotype frequencies, odds ratios (OR) with a 95% confidence interval (CI).The difference in comparisons was considered statistically significant at a p-value < 0.05.When more than one SNP is included in the analysis, SNPStats offers the possibility of performing linkage disequilibrium (LD).For LD, matrices with selected statistics (D', r and associated p-values) are shown.D' values between 0 and 1 indicate the degree of LD between the two loci.A value close to 1 suggests strong LD, while a value close to 0 indicates weak or no LD.The sign of r ("+" or "-") indicates the direction of the association.Positive r indicates that knowing the presence of one allele increases the probability of finding the other allele, while negative r indicates that knowing the presence of one allele decreases the probability of finding the other alleles.

Results
A study of the distribution of patients with L 4 -L 5 , L 5 -S 1 , C 5 -C 7 IVDD by age and gender showed no significant differences compared with the control group.The frequencies of reference and alternative alleles and genotypes in the control group are consistent with genotyping data obtained by other authors (Table 1).The frequency of the minor allele T (COL1A1rs1800012) in our study was 0.15; the minor A allele (COL2A1rs2276454) was 0.36; the allele A (COL2A1rs1793953) was 0.61; alternative allele VDRrs2228570 -0.53.
Results of the association study of COL2A1rs2276454 with IVDD are presented in Table 2.In men there was a negative association of L 5 -S 1 IVDD with the G/A genotype (COL2A1rs2276454): OR (95% CI) 3.50 (1.26-9.72).The fact that the G/A genotype was more frequently observed among individuals in the control group suggests its potential protective value.This observation was also confirmed by the fact that the G/G (COL2A1rs2276454) genotype among Оригінальні дослідження / Original Researches male patients with L 5 -S 1 IVDD, is 4 times less often observed in the control group compared to the female group, OR (95% CI) 0.25 (0.09-0.71); р = 0.008 (Table 2).Analysis of the association of L 5 -S 1 IVDD with VDRrs2228570 showed statistical significance in a general group of patients with different genetic models.Recessive model: Т/Т+C/T vs. СС showed that genotype СС is associated with L 5 -S 1 IVDD: OR (95% CI) 2.255 (1.089-4.670).Overdominant model: individuals had 2.39 times higher odds of having the C/T genotype compared to C/C+T/T in the control group than those with L 5 -S 1 IVDD, OR (95% CI) 0.418 (0.217-0.802), which might have indicated a protective value for heterozygotes (Table 3).
Comparison of association of L 5 -S 1 IVDD with VDRrs2228570 in groups of men and women showed a statistically significant excess of the OR for C/C vs. (T/T+T/C) in recessive model among men: OR (95% CI) 3.441 (1.039-11.399)and in overdominant genetic model among men a statistically significant increase by 2.7 times of the OR for T/C vs. (T/T+C/C): 0.367 (0.144-0.936) (Table 4).
No statistically significant associations of L 5 -S 1 IVDD with polymorphisms COL1A1rs1800012 and COL2A1rs1793953 were found.A study of the presence of polymorphic alleles rs1800012, rs2276454, rs1793953, rs2228570 in individuals with L 5 -S 1 IVDD showed the absence of statistically significant associations.
A study of the presence of polymorphic alleles rs1800012, rs2276454, rs1793953 in individuals with C 5 -C 7 IVDD showed the absence of statistically significant associations with IVDD.

Discussion
A study examining the distribution of patients with L 4 -L 5 , L 5 -S 1 , and C 5 -C 7 IVDD by age and gender found no significant differences when compared to the control group.The frequencies of reference and alternative alleles and genotypes in the control group align with genoty ping data reported by other researchers.The average frequency of the COL1A1rs1800012 minor T allele was 0.16-0.091[31].In our study, among individuals in the control group, it was 0.15.The frequency of the minor A allele In our study, the frequency of the A allele was 0.36.The frequency of the A allele COL2A1rs1793953 was 0.16-0.61[33].In our study, the frequency of the A allele was 0.61.The average frequency of the alternative VDRrs2228570 allele was 0.51-0.74[34], in our study, the frequency of the alternative allele was 0.53.
A study of the association of the polymorphism of COL2A1rs2276454 with IVDD L 5 -S 1 indicated its possible protective effect on the development of degenerative changes in AF.One explanation may be that substituting G for A at position chr12:g.47982508results in the replacement of the glycine codon GGC, which in the mRNA of the COLII alpha-1 chain has a frequency of occurrence of 29 % with the preferred glycine codon GGU (47 %).It is known that the rate of incorporation of amino acids into more frequent codons is higher.The benefits of the translation process may be important in the repair of degenerative processes [35][36][37].
It is known that complexes involving VDR regulate the expression of more than 900 genes in various tissues, including the collagen genes COL1A1 and COL2A1 [38,39].

Оригінальні дослідження / Original Researches
The results of studies of the association of Fok1 VDR polymorphism and IVDD do not provide a clear answer.Study of association of L 5 -S 1 IVDD with VDRrs2228570 conducted by us showed statistical significance in different genetic models: with overdominant model (overdominantthe heterozygote produces a phenotype better adapted than that of the homozygote) and with recessive model (traits are expressed only if both alleles are recessive).In men, there is a negative association of L 5 -S 1 IVDD with the T/C genotype (VDRrs2228570).The fact that the T/C genotype (VDRrs2228570) is observed 2.7 times more often among the control group indicates its potential protective value.
The potential protective value of the heterozygous T/C genotype (VDRrs2228570) is also correlated with a higher incidence of heterozygotes among males with C 5 -C 7 IVDD than in females.In contrast, the C/C genotype in males in the recessive model is associated with L 5 -S 1 IVDD.
A study of the association of pairs of polymorphic alleles with IVDD showed that COL1A1rs1800012/VDRrs2228570 (p = 0.01); COL2A1rs1793953/VDRrs2228570 (p = 0.0002); COL2A1rs2276454/COL2A1rs1793953 (p = 0.0048) are associated with C 5 -C 7 IVDD and COL2A1rs2276454/ COL2A1rs1793953 are associated with L 4 -L 5 IVDD (p = 0.001).It should be noted that the COL2A1rs2276454 polymorphisms in the group of men with L 5 -S 1 IVDD can potentially have a protective value, but two polymorphisms in the COLII gene rs2276454 and rs1793953 are associated with C 5 -C 7 and L 4 -L 5 IVDD in the general group of patients.The data obtained give reason to assume that random combinations of polymorphic alleles of different collagen genes are important in the formation of degenerative processes in the intervertebral disc.
Prospects for further research.The importance of genetic research for the implementation of personalized medicine and prognosis dictates the need to establish associations of genetic variants with socially significant degenerative processes of intervertebral discs.To further improve the results, it is necessary to increase the studied groups, as well as to introduce a more advanced polygenic analysis.
Study limitations.It is necessary to conduct research with the involvement of a larger contingent of individuals, especially with degenerative processes in the cervical spine.
ConclusionsC/C genotype of VDRrs2228570 in males is associated with L 5 -S 1 IVDD.COL2A1rs2276454 variant may have a protective effect on the development of degenerative changes L 5 -S 1 IVDD in males.T/C genotype of VDRrs2228570 indicates its potential protective value in men with L 5 -S 1 IVDD.The pairs of alleles COL1A1rs1800012/ VDRrs2228570, COL2A1rs1793953/VDRrs2228570, COL2A1rs2276454/COL2A1rs1793953 are associated with C 5 -C 7 IVDD.The pair of alleles COL2A1rs2276454/ COL2A1rs1793953 is associated with L 4 -L 5 IVDD.