The COL27A1 Gene Polymorphism in Children with Tic Disorder in Southern China and Its Clinical Association

Background: The three single nucleotide polymorphisms(SNPs) (rs4979356, rs4979357 and rs7868992) of COL27A1 has been reported to correlate with Tourette Syndrome (TS) patients in European countries as well as northern Chinese population, though there are lack of relevant studies in southern Chinese population. In this study we aimed to evaluate the distribution of COL27A1(rs4979356, rs4979357 and rs7868992) gene polymorphism and its clinical relationship in children with Tic disorder (TD) in southern China. Methods: The children with TD between November 2018 to August 2019 from Department of Neurology, Guangzhou Women and Children’s Medical Center were recruited and followed up. The control children from normal primary school were also recruited. The SNP of COL27A1 (rs4979356, rs4979357 and rs7868992) was detected in child from each groups by polymerase chain reaction(PCR) and Sanger sequencing. Results: A total of 114 children with TD and 100 healthy control children were included. 111 out of 114 TD’s children were followed up more than one year, including 32 with transient tic disorder (TTD) (Age:7.58±2.402;), 45 with chronic tic disorder (CTD) (Age:: 8.72±2.312;) and 34 with Tourette Syndrome (TS)(Age:: 8.85±2.720) respectively. The genotype frequency and gene frequency of rs4979356, rs4979357 and rs7868992 from COL27A1 showed no statistically signicant difference (P > 0.05) between the both groups (cid:0) The distribution of genotype CG of COL27A1 rs4979356 in TS group was signicantly higher than that in TTD+CTD group( P=0.002) and in control group (P=0.001) (cid:0) there was no signicant difference in allele frequency among each groups (P (cid:0) 0.05) (cid:0) Genotype TC of COL27A1

Methods: The children with TD between November 2018 to August 2019 from Department of Neurology, Guangzhou Women and Children's Medical Center were recruited and followed up. The control children from normal primary school were also recruited. The SNP of COL27A1 (rs4979356, rs4979357 and rs7868992) was detected in child from each groups by polymerase chain reaction(PCR) and Sanger sequencing.
Results: A total of 114 children with TD and 100 healthy control children were included. 111 out of 114 TD's children were followed up more than one year, including 32 with transient tic disorder (TTD) (Age:7.58±2.402;), 45 with chronic tic disorder (CTD) (Age:: 8.72±2.312;) and 34 with Tourette Syndrome (TS)(Age:: 8.85±2.720) respectively. The genotype frequency and gene frequency of rs4979356, rs4979357 and rs7868992 from COL27A1 showed no statistically signi cant difference (P > 0.05) between the both groups The distribution of genotype CG of COL27A1 rs4979356 in TS group was signi cantly higher than that in TTD+CTD group( P=0.002) and in control group (P=0.001) there was no signi cant difference in allele frequency among each groups (P 0.05) Genotype TC of COL27A1 rs4979357 was signi cantly more distributed in TS group than in TTD+CTD group (P=0.004) and control group (P=0.001) There was no signi cant difference in allele frequency among three groups (P 0.05);Genotype GA of COL27A1 rs7868992 was signi cantly more distributed in TS group than in TTD+CTD group (P=0.018) and control group (P=0.035). The distribution of G allele frequency in CTD group was signi cantly higher than that in TS group ( P=0.025), while the difference of other allele frequency between the groups was not statistically signi cant(P 0.05).
Conclusion: The genotype CG of rs4979356, the genotype TC of rs4979357 and the genotype GA of rs7868992 might serve as the high risk factors in children with TS in southern China.

Background
Tic disorder (TD) is a common neuropsychiatric disease in childhood, and the prevalence of TD in China is 6.1% [1]. According to the symptoms and duration, TD can be divided into transient TD (TTD), chronic TD (CTD) and Tourette syndrome (TS), with TS incidence rate of 0.3-1.0% and refractory cases increasing, which has caused distress to children and their families.
At present, the pathogenesis of TD is not clear, and the research on genetic factors has attracted much attention. In recent years, genome-wide association studies (GWAS) have discovered the genetic association recognition of many common complex traits. In 2013, Scharf and colleagues conducted the rst GWAS of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. They found the strongest correlation with the single nucleotide polymorphism (SNP) rs7868992 in collagen XXVII type α 1 gene (COL27A1) in European samples [2]. COL27A1 is located on chromosome 9q32-33, approximately 156 KB in length, and has 61 exons, encoding a long trihelical domain, a carboxy-terminal propeptide and a large spherical amino-terminal propeptide. COL27A1 is strongly expressed in developing cartilage and is currently mainly related to iron and steel syndrome [3,4]. It is weakly expressed in many other tissue types such as skin, stomach, gonad and brain [5]. Some studies have found that microRNA-455-3P encoded by COL27A1 gene is considered as a potential biological and/or therapeutic target for Alzheimer's disease (AD) [6] . However, little is known about the role of COL27A1 in neural development, particularly in neural circuits. The diagnostic criteria of TD based on DSM-5 as follows: TTD: One or more motor twitch and/or vocal twitch; The course of disease is shorter than 1 year; Onset before the age of 18; exclude certain drugs or medical diseases caused by; Do not meet the diagnostic criteria for chronic TD or TS; CTD: One or more motor twitch or vocal twitch, only one twitch form appears in the course of the disease; Since the rst twitch, the frequency of twitch can be increased or decreased, the course of disease is more than 1 year; Onset before the age of 18; exclude certain drugs or medical diseases caused by; Does not meet the diagnostic criteria of TS; TS: multiple motor twitches and one or more vocal twitches, but not necessarily both; After the rst twitch, the frequency of twitch can be increased or decreased, the course of disease is more than 1 year; Onset before the age of 18; exclude some drugs or medical diseases caused by.
Exclusion criteria: The patients with intracranial infection, epilepsy, intracranial hemorrhage, hepatolenticular degeneration, rheumatic chorea,or with the in ammation of eyes, ears and throat were excluded.
Control population: The children from normal primary school children were recruited as the control group.
Detection of COL27A1 (rs4979356, rs4979357 and rs7868992) TD patients' and Control children's whole blood samples were collected and stored at -20℃ until analysis.
Genomic DNA was prepared from peripheral blood by using the whole blood genomic DNA mini kit (SIMGEN, Hangzhou, China). The COL27A1 gene sequence fragment including the SNP locus was ampli ed by PCR, and then sent to the sequencing company for Sanger sequencing and then analyzed to determine the genotype. Primer sequences were as follows: upstream primer 5'-AGACAGGCTGCCTAGTGT-3' and downstream primer 5'-GATAGCGTCATTGAACTCC-3'. Polymerase chain reactions were performed in a total volume of 30 µl, containing 50-100 ng of genomic DNA, 0.6 µl of each 10 µM primer (Table 1)     Statistical Analysis SPSS21.0 software package was used to manage all the data, and the measurement data was expressed as mean standard deviation(`x ± s). Comparisons between two or more population rates were tested by χ 2 .
The level of statistical signi cance was set at p < 0.05.  Fig. 1. Children in the TTD group ranged in age from 3 to 12 years old, with an average age of 7.58 ± 2.402 years old and a male to female ratio of 5.4:1. In the CTD group, the children ranged in age from 5 to 14 years old, with an average age of 8.72 ± 2.312 years old and a male to female ratio of 2.75:1. The age range of children in TS group was 4-15 years old, with an average age of 8.85 ± 2.720 years old, and the ratio of male to female was 7.5:1.

Clinical manifestations of three groups of children
The  Table 1.
Comparison of genotype frequency and gene frequency of COL27A1 rs4979356, rs4979357 and rs7868992 between TD children and control group The genotype frequency and gene frequency of COL27A1 rs4979356, rs4979357 and rs7868992 in the case group and the control group showed no statistically signi cant difference (P > 0.05). See Table 1    (46.0%). Genotype TC distribution in TS group was higher than that in TTD + CTD group (χ 2 = 10.814, P = 0.004) and control group (χ 2 = 13.618, P = 0.001), with statistically signi cant difference (P < 0.05). There was no signi cant difference in allele frequency between groups (P > 0.05). See Table 3 and Table 4 for details. frequency G129 (64.5%), a71 (35.5%); genotype GA in TS group was higher than that in TTD group + CTD group (χ 2 = 8.034, P = 0.018) and control group χ 2 = 6.695, P = 0.035), the difference was statistically signi cant (P < 0.05). The distribution of G allele frequency in CTD group was higher than that in TS group, the difference was statistically signi cant (χ 2 = 5.013, P = 0.025), and there was no signi cant difference in other allele frequencies between the two groups (P > 0.05). See Tables 3 and 4  The pathogenesis of TD is unknown, which is believed to be closely related to genetic factors, neurobiochemical and metabolic factors, and environmental factors, among which the study of genetic factors in TS has attracted much attention. Studies have found that most of TS patients had a family history of tic, and most of TS patients were from both parents, and their rst-degree relatives had a signi cantly higher risk of disease than the normal population [10][11][12]. Other studies suggested that the prevalence rate of identical twins was 53% ~ 56%, compared with 8% for fraternal twins [13]. In recent years, studies on TS genetic characteristics mainly include genome-wide linkage studies, candidate associated genes studies, chromosome aberration studies, copy number variation, epigenetic studies of TS, and total exome sequencing studies, but the exact etiology of TD is still unclear [14,15].
In recent years, genome-wide association studies (GWAS) have discovered the genetic association recognition of many common complex traits [16]. This non-modular genetic testing approach has not only improved people's understanding of the pathophysiology of many diseases, but also improved drug treatment strategies [17]. The rst GWAS was performed in TS cases by Scharf and colleagues in 2013 [2]. They found the strongest association with rs7868992 in collagen XXVII typeα1 gene (COL27A1) in european-sourced TS patient samples. In Shandong province of China, Shiguo Liu et al applied haplotype relative risk (HRR) and transmission imbalance test (TDT), and used PCR-guided sequencing of COL27A1 (rs4979356, rs4979357 and rs7868992) to understand the genetic distribution of the three SNPS in TS [7]. The results suggested that rs4979356 was associated with the etiology of TS development, and allele C and genotype C(+) of rs4979357 were also risk factors for TS, while TDT results of rs7868992 showed no statistically signi cant signi cance. The results of linkage disequilibrium test con rmed that the three SNPS were inherited independently. Therefore, the difference of genetic spectrum between Chinese and European people may be the reason for the insigni cant TDT results of rs7868992.
In this study, 111 children with TD showed no signi cant difference in genotype frequency and gene frequency of rs4979356, rs4979357 and rs78689923 SNPS in COL27A1 compared with children in the control group. The results showed that the distribution of genotype CG, genotype TC and genotype GA of rs4979356, rs7868992 were signi cantly higher in the TS group than in the TTD + CTD group and the control group. These results suggest that genotype CG of rs4979356, genotype TC of rs4979357 and genotype GA of rs7868992 are high risk factors for TS in children in Guangdong province. The results of this study were consistent with the strong correlation of COL27A1 rs7868992 in TS in the European population reported by Scharf, and slightly different from the results of Shiguo Liu et al [2,7]. In addition, the allele frequency of rs7868992 was signi cantly higher in the CTD group than in the TS group, and there was no statistically signi cant difference between the other allele frequency groups. The G allele frequency of rs7868992 is only higher in CTD, and its results and signi cance need to be veri ed and observed in a larger sample size.

Conclusion
The genotype frequency of rs4979356, rs4979357 and rs7868992 of COL27A1 gene was related to the onset of TS. Whether the three SNPS of COL27A1 gene can be included in routine screening to assist TD therapy and assess prognosis, and the role of COL27A1 in the pathogenesis of TS still needed to be veri ed by expanding the sample size and expanding the ethnic range.