The gene polymorphisms of COL1A1 and VDR in children with scoliosis

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Introduction

Scientists and clinicians have been involved in the search for etiologic factors of spinal deformities in children for several centuries. The emergence of molecular, genetic, and biochemical technologies enabled evaluation of scoliosis (which is far from complete at the present stage of medical development) from more fundamental positions [1, 2].

Spinal deformities that are found in the postnatal period are congenital. However, curvature of the spine also can be due to vertebral anomalies that may manifest during growth and development of a child with anatomically normal structure, in which case the scoliosis is idiopathic (IS). Regardless of the nature of the deformity, scoliosis is a multifactorial disease, the formation of which is due to genetic determinants and environmental factors [3].

In connection with the development of IS and congenital scoliosis, the candidate genes were studied; that is, the epidermal growth factor gene [4], DLL3 [5], WNT3A [6], TBX6 [7], and so forth. The genes whose products are involved in the processes of bone metabolism and osteogenesis are considered as “candidate” genes that contribute to the formation of spinal deformities. Among many genes in this group, the vitamin D receptor gene (VDR) and the type I collagen gene (COL1A1) are considered to be particularly significant [8].

It is known that vitamin D and its active metabolites have a key role in phosphoric calcium homeostasis and bone metabolism, and regulate the growth and differentiation of cells in various target organs [9-10]. Vitamin D is a ligand for a nuclear receptor encoded by the VDR gene and it is a regulator of the activity of many target genes through its interaction with specific DNA sequences in the promoter regions of these genes [11]. Under the influence of calcitriol or vitamin D3, the expression of the type I collagen gene decreases [12, 13].

The organic matrix of bones is represented by 95% type I collagen, and its amino acid structure is encoded by the COL1A1 gene [14].

The VDR gene is located on chromosome 12q12-14. It consists of 60,000 nucleotide pairs and includes 10 exons and 8 introns [14]. Several polymorphisms of the length of the restriction fragments (RFLPs) in the VDR gene have been identified (BsmI, TaqI, ApaI, FokI, polyadenyl mononucleotide [poly (A) repeat]). Functional mutations in this gene affect mineral metabolism and mineral density of bone tissue [14]. The site for the TaqI-restrictase is located inside the 9th exon. This polymorphism is characterized by the replacement of thymine (T) with cytosine (C; rs731236). In a number of studies, the connection of this polymorphism of the VDR gene with bone mineral density [8] has been noted, as well as with diseases, such as osteoporosis [14], chronic periodontitis [15], and type 1 diabetes mellitus [16].

The Cdx2 polymorphism (rs11568820) is located in the promoter region of the VDR gene and is associated with decreased bone mineral density [17], an increased risk of fractures, in particular, the spine [18], and colorectal cancer [19]. Suh et al. [20] analyzed the distribution of alleles and genotypes according to the Cdx2 polymorphism of the VDR gene in 198 girls with IS of II to IV degrees and in healthy girls. There were no significant differences in their frequencies, or in bone mineral density values in children with IS carriers of different alleles and genotypes of Cdx2 polymorphism of the VDR gene [20]. At the same time, a number of studies have revealed a decrease in bone mineral density values in children with IS [21, 22], and osteopenia has been considered as a risk factor for the progressive course of IS [23].

In studies on the polymorphism of Sp1 (rs1800012) located in the first intron of the COL1A1 gene, an association of allele s with reduced bone mineral density, osteoporosis, and fracture risk was revealed [24]. Some studies have proved that in carriers of the allele s in the homozygous and heterozygous states there is an impairment of the collagen function and a predisposition to osteoporosis [25]. In addition, the decrease in bone mineral density was observed in carriers of certain combinations of genotypes, −1997G/T (PCOL2) and + 1245G/T (Sp1) of the COL1A1 gene [26, 27].

Analysis of the data of previous studies led to the conclusion that structural changes in the genes of the VDR and type I collagen can be associated with the development of congenital and acquired spinal deformities. The aim of this study was to evaluate the molecular and genetic testing of the −3131A/G (Cdx2) and +61968T/C (TaqI) VDR genes and −1997G/T (PCOL2) and +1245G/T (Sp1) of the COL1A1 gene in patients with scoliotic deformity of various etiologies and healthy children. We subsequently studied the distribution of frequencies of alleles and genotypes according to the polymorphisms studied, and analysis of the relationship between the studied molecular and genetic markers with the development of scoliosis.

Materials and methods

From 2008 to 2011, we examined 195 children 6 months to 17 years old with congenital scoliosis. Children with scoliotic spinal deformity in the structure of various genetic syndromes were excluded from the study (41 patients). Comparison group 1 consisted of 145 patients (12–18 years old) with IS of II to IV degrees of severity, who were treated on an inpatient basis in the clinic of spine pathology and neurosurgery of the Turner Scientific and Research Institute for Children’s orthopedics. Comparison group 2 included 278 children (aged 1–18 years) who did not show signs of spinal deformity at the time of examination (the unstable impaired posture was allowed). These patients underwent outpatient examination for diseases not related to skeletal pathology. All patients voluntarily signed the informed consent to participate in the study.

Table 1 shows the distribution of the study groups of children by sex and age.

 

Table 1. Distribution of children in the study groups by sex and age

Sex group	Congenital scoliosis n, %	Average age (years)	IS n, %	Average age (years)	Control n, %	Average age (years)
Boys	63 (40.9)	7.2 (+0.4)	15 (10.3)	15.4 (+0.1)	124 (44.7)	11.2 (+0.2)
Girls	91 (59.1)		130 (89.7)		154 (55.3)
Total	154		145 (100)		278 (100)

 

DNA for the study was isolated from peripheral blood leukocytes via a standard phenol-chloroform extraction method. The TaqI and Cdx2 polymorphisms of the vitamin D receptor gene (VDR) were determined via the RFLP method with the use of the restrictases TaqI and Cdx2. The Sp1 polymorphism of the αI gene of the collagen type I chain (COL1A1) was determined via the RFLP method with the use of the Bse1I restrictase. The 1997G/T (rs1107946) polymorphism of the αI gene of the collagen type I chain (COL1A1) was determined by the RFLP method using the BstMAI restrictase.

Statistical data processing was performed using Statistica 5.5 software (Stat-Soft, Inc.). The Hardy–Weinberg equilibrium was used to assess the correspondence of the distribution of genotypes on the polymorphic markers studied. The Pearson χ² criterion was used for comparative analysis of the frequency distribution of alleles and genotypes based on the studied polymorphisms in the patients with congenital and idiopathic scoliosis, as well as in children without scoliotic deformity. The differences at P < 0.05 were considered statistically significant.

The presence of the relationship of certain genotypes with the development of scoliotic deformity was determined by the odds ratio (OR) value, which is an indicator reflecting how many times the probability of being in the “case” group (patients) differs from the probability of being in the “control” group (healthy) for the carrier of a certain genotype (allele) according to the studied polymorphisms of the VDR and COL1A1 genes: OR = (A/B)/(C/D), where A and B are the number of patients having and not having a mutant allele, respectively; and C and D are the number of healthy patients having and not having a mutant genotype or allele, respectively. According to OR, this genotype is supposed to be associated with the risk of development of the disease: OR = 1 indicates the lack of relation of this genotype with the risk of development of the disease, OR > 1 indicates the increased risk of development of the disease, while OR < 1 indicates a negative association of the DNA marker with development of the pathology. For calculations, the Calculator software for calculating statistics in case-control studies was used (available in the public domain at http://test.tatotili.ru/calculator.php). For OR, a confidence interval (CI) at a 95% significance level was calculated.

Results and discussion

Distribution of frequencies of alleles and genotypes of VDR and COL1A1 genes on all the studied polymorphisms in the analyzed samples of the patients examined corresponded to the Hardy–Weinberg equilibrium. The results of the allele and genotype distribution on polymorphisms of the VDR gene as +61968T>C,–3731А>G, and those of the COL1A1 gene as +1245 G>T,–1997 G>T in patients with congenital and acquired scoliosis, and children without spinal deformities are shown in Table 2.

 

Table 2. The distribution of frequencies of alleles and genotypes of the genes VDR (+61968T>C, −3731A>G) and COLA1 (+1245 G>T, −1997 G>T) in groups of patients T with congenital and IS, and children without spinal deformity

Genes. alleles. genotypes	Frequencies of alleles and genotypes
	Congenital scoliosis	Control		IS
VDR
TaqI (+61968T/C)	n = 154	n = 271		n = 145
T/T	0.396	0.498		0.448
T/t (T/C)	0.455	0.421		0.428
t/t (C/C)	0.149	0.081		0.124
Statistics	χ² = 6.79; d. f. = 2; p = 0.03		χ² = 2.31; d. f. = 2; p = 0.31
T*	0.623	0.708		0.662
t**	0.377	0.292		0.338
Statistics	χ² = 6.51; d. f. = 1; p = 0.01		χ² = 1.91; d. f. = 1; р = 0.17
Cdx2(–3731А/G)	n = 154	n = 269		n = 145
A/A	0.052	0.026		0.028
A/G	0.266	0.279		0.310
G/G	0.682	0.695		0.662
Statistics	χ² = 1.94; d. f. = 2; p = 0.38		χ² = 0.48; d. f. = 2; p = 0.79
A	0.185	0.165		0.183
G	0.815	0.835		0.817
Statistics	χ² = 0.53; d. f. = 1; р = 0.47		χ² = 0.40; d. f. = 1; р = 0.53
COL1A1
Sp1 (+1245 G/T)	n = 154	n = 276		n = 145
S/S (G/G)	0.675	0.714		0.724
S/s (G/T)	0.292	0.272		0.248
s/s (T/T)	0.032	0.014		0.028
Statistics	χ² = 1.88; d. f. = 2; p = 0.39		χ² = 1.07; d. f. = 2; р = 0.59
S*	0.821	0.850		0.848
s**	0.179	0.150		0.152
Statistics	χ² = 1.17; d. f. = 1; p = 0.28		χ² = 0.00; d. f. = 1; p = 0.96
PCOL2
(–1997 G/T)	n = 154	n = 268		n = 145
G/G	0.701	0.690		0.697
G/T	0.253	0.280		0.262
T/T	0.045	0.030		0.041
Statistics	χ² = 0.94; d. f. = 2; р = 0.62		χ² = 0.48; d. f. = 2; p = 0.79
G	0.828	0.830		0.828
T	0.172	0.170		0.172
Statistics	χ² = 0.01; d. f. = 1; р = 0.93		χ² = 0.01; d. f. = 1; р = 0.92

 

Note: d.f. is the number of degrees of freedom; T* and S* are alleles having no site for the restrictase; t** and s** are alleles having a site for the restrictase.

Comparative analysis of frequencies of alleles and genotypes on polymorphism +61968T>C of the VDR gene in the children with and without scoliotic deformity of the spine revealed differences in their distribution. Among patients with congenital scoliosis, children who were carriers of allele t(C) (0.377 vs 0.292; χ² = 6.51; d.f. = 1; P = 0.01) and the genotype tt(CC) (0.149 vs 0.081; χ² = 6.79; d.f. = 2; P = 0.03) were more common compared to children without scoliotic deformity. The OR for carriers of the TT genotype was 0.66 (95% CI, 0.44–0.99), and for the genotype tt(CC) it was 1.99 (95% CI, 1.07–3.70). It is possible that carriership of the VDR gene allele t can be regarded as a marker of increased risk of the development of congenital scoliosis. Comparative analysis of the distribution of alleles and genotypes on polymorphism −3731A>G of the gene VDR and the polymorphisms (+1245 G>T, −1997 G>T) of the COLA1 gene revealed no significant differences in the children with congenital and IS. There also were no significant differences in the distribution of alleles and genotypes on the studied polymorphic markers when comparing their frequencies among patients with different types of scoliosis and the control group.

Analysis of the distribution of alleles and genotypes combinations of the VDR gene (+61968T>C,−3731А>G) was conducted in patients with congenital and IS and in the children without spinal deformity (Table 3). Carriership of the genotype tt/GG of the VDR gene (+61968T>C,− 3731А>G) is found in the children with congenital scoliosis more than two times more often than in the children without scoliosis (11% and 5.2% of cases, respectively; χ² = 4.17; d.f. = 1; P = 0.04). This genotype was detected in 8.7% of children with IS. There were no statistical differences in the carriership of this genotype in the patients with IS and in children without scoliotic deformity of the spine.

Analysis of the distribution of combinations of alleles and genotypes of the COLA1 gene (+1245 G>T,−1997 G>T) was performed in patients with congenital and IS and children without spinal deformity (Table 4).

 

Table 3. The distribution of genotype combinations of alleles and genotypes of VDR (+61968T>C,–3731А>G) in patients with congenital and IS and in children without spinal deformity

Gene, polymorphisms	Genotype combinations	Congenital scoliosis n (%)	IS n (%)	Control n (%)
VDR +61968T>С–3731А>G	TT/GG	42 (27.2)	45 (31.0)	93 (34.5)
	TT/AA	3 (1.9)	0	3 (1.2)
	TT/AG	16 (10.4)	20 (13.7)	40 (14.8)
	tt/GG	17 (11.0)	12 (8.7)	14 (5.2)
	tt/AA	1 (0.7)	1 (0.4)	1 (0.4)
	tt/AG	5 (3.2)	5 (3.5)	6 (2.3)
	Tt/GG	44 (28.6)	39 (26.9)	79 (29.3)
	Tt/AA	4 (2.7)	3 (2.0)	5 (1.9)
	Tt/AG	22 (14.3)	20 (13.8)	28 (10.4)

 

Table 4. The distribution of combinations of alleles and genotypes of COLA1 (+1245 G>T,−1997 G>T) in patients with congenital and IS and in children without spinal deformity

Gene, polymorphisms	Genotype combinations	Congenital scoliosis (n, %)	IS (n, %)	Control (n,%)
COL1A1 +1245 G > T / –1997 G > T	ss/GG	5 (3.2)	4 (2.7)	8 (3.0)
	ss/TT	0	0	0
	ss/TG	0	0	0
	SS/GG	67 (43.5)	68 (46.9)	120 (44.9)
	SS/TT	6 (3.9)	6 (4.1)	7 (2.6)
	SS/TG	31 (20.1)	32 (22.2)	63 (23.5)
	Ss/GG	36 (23.5)	29 (20.0)	57 (21.2)
	Ss/TT	1 (0.6)	0	0
	Ss/TG	8 (5.2)	6 (4.1)	13 (4.8)
	N	154	145	268

 

It should be noted that ss/TT and ss/TG genotypes were not found in children either with or without scoliosis. There was no difference in the distribution of combinations of alleles and genotypes of COLA1 (+1245 G>T,−1997 G>T) in patients with congenital scoliosis and children without spinal deformity (χ² = 3.08; d.f. = 6; P > 0.05), as well as in patients with IS and children without scoliosis (χ² = 1.07; d.f. = 6; P > 0.05). There was no difference in the distribution of combinations of alleles and genotypes of COLA1 (+1245 G>T,−1997 G>T) in patients with congenital and IS (χ² = 1.92; d.f. = 6; P > 0.05).

Conclusions

Thus, our results indicated that there were significantly more children who were carriers of allele t(C) and genotype tt(CC) among patients with congenital scoliosis compared to children without scoliotic spinal deformity. The genotype tt/GG of the gene VDR was found in children with congenital scoliosis more than 2 times more often than in children without scoliotic spinal deformity.

For a more reliable assessment of the contribution of gene polymorphism, the products of which are involved in bone metabolism and in the development of congenital and acquired spine deformities, further studies involving more extensive patient samples are required.

Information on funding and conflict of interest

The work was performed on the basis of the Children’s Regional Clinical Hospital in Tver and with the support of the Turner Scientific and Research Institute for Children’s orthopedics of the Ministry of Health of Russia. The authors declare the absence of obvious and potential conflicts of interest related to the publication of this article.

About the authors

Sergey V. Vissarionov

The Turner Scientific and Research Institute for Children’s Orthopedics

Author for correspondence.
Email: turner01@mail.ru

MD, PhD, professor, Deputy
Director for Research and Academic Affairs, head of the
department of spinal pathology and neurosurgery

Russian Federation

Valentina I. Larionova

The Turner Scientific and Research Institute for Children’s Orthopedics

Email: turner01@mail.ru

MD, PhD, professor, senior
research associate of the genetic laboratory

Russian Federation

Irina V. Kazarian

The Turner Scientific and Research Institute for Children’s Orthopedics

Email: turner01@mail.ru

MD, research associate of the genetic
laboratory of the Center for rare and hereditary diseases in
children

Russian Federation

Alexandra N. Filippova

The Turner Scientific and Research Institute for Children’s Orthopedics

Email: alexandrjonok@mail.ru

MD

Russian Federation

Mikhail M. Kostik

Saint Petersburg State Pediatric Medical University

Email: turner01@mail.ru

MD, PhD, pediatrician, cardiorheumatologist,
associate professor

Russian Federation

Anna N. Voitovich

Saint Petersburg State Pediatric Medical University

Email: turner01@mail.ru

consultant molecular technologies Academy of molecular medicine

Russian Federation

Elena V. Rotchev

Children’s Regional Clinical Hospital

Email: turner01@mail.ru

MD, orthopedic and trauma surgeon

Russian Federation

References

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