Hypervariable Region Polymorphism of mtDNA of Recurrent Oral Ulceration in Chinese

Background MtDNA haplogroups could have important implication for understanding of the relationship between the mutations of the mitochondrial genome and diseases. Distribution of a variety of diseases among these haplogroups showed that some of the mitochondrial haplogroups are predisposed to disease. To examine the susceptibility of mtDNA haplogroups to ROU, we sequenced the mtDNA HV1, HV2 and HV3 in Chinese ROU. Methodology/Principal Findings MtDNA haplogroups were analyzed in the 249 cases of ROU patients and the 237 cases of healthy controls respectively by means of primer extension analysis and DNA sequencing. Haplogroups G1 and H were found significantly more abundant in ROU patients than in healthy persons, while haplogroups D5 and R showed a trend toward a higher frequency in control as compared to those in patients. The distribution of C-stretch sequences polymorphism in mtDNA HV1, HV2 and HV3 regions was found in diversity. Conclusions/Significance For the first time, the relationship of mtDNA haplogroups and ROU in Chinese was investigated. Our results indicated that mtDNA haplogroups G1 and H might constitute a risk factor for ROU, which possibly increasing the susceptibility of ROU. Meanwhile, haplogroups D5 and R were indicated as protective factors for ROU. The polymorphisms of C-stretch sequences might being unstable and influence the mtDNA replication fidelity.


Introduction
Recurrent oral ulceration (ROU) is one of the most common oral mucosal diseases, affecting 25% of the population [1]. Recurrent oral ulceration may sometimes be atypical in that it clinically resembles recurrent aphthous stomatitis (RAS) but neither commences in childhood, fails to resolve with age, or is associated with other features not typically associated with classical RAS, such as fever [2][3][4][5][6][7]. However, the cause of ROU is still unclear so far. As a complex disease with significant genetic contribution, ROU may have a complicated etiology including both genetic and environmental factors. Under some circumstances, ROU patients may have some genetic tendencies or congenitally genetic abnormalities. When some environmental factors, such as microbial infection, stimulate the body, the pathological response may alternatively lead to the disease [8].
The human mitochondrial genome (mtDNA) is a small 16,569 bp molecule of double stranded DNA. The mtDNA encodes 13 protein subunits of multimeric oxidative phosphorylation, two rRNAs and all the tRNAs required for the translation of its mRNA [9]. It has non-coding regions at displacement loop region (D-loop) that contains three hypervariable segments (HV1, HV2 and HV3) with high polymorphism [10][11][12]. Most of the mutations observed in both mtDNA coding and non-coding regions have occurred on preexisting haplogroups and define the individual mtDNA types or haplotypes [13]. Haplogroups can have important implication for understanding of the relationship between the mutations of the mitochondrial genome and disease [14,15]. There is growing evidence that certain mtDNA clusters are associated with distinct disorders [16]. To examine the susceptibility of mtDNA haplogroups to ROU, we sequenced the mtDNA HV1, HV2 and HV3 in Chinese.

Results
The mtDNA haplogroups were analyzed in 249 ROU patients and 237 subjects. The typical clinical manifestations of ROU patients were shown in Fig. 1. Table 1 displayed the characteristics of the study populations, we found that the gender difference between ROU patients and healthy persons was significant (P,0.01). So considering the influence of gender difference, we did binary logistic regression in the data of MtDNA haplogroups and obtained the adjusted P-value and OR (95% CI). In Table 2, there were 16 types of haplogroups in both ROU patients and healthy persons including A, C, D, D5, F1, F, G1, H, L3, M, N, R, U5, W1, Y and Z. Twelve of them had no significance in statistics, while the other 4 types of haplogroups showed a significant difference between ROU patients and healthy persons, including D5 and G1 (P,0.05), H and R (P,0.01) (Fig. 2).

The Distribution of D5 and R in ROU Patients is Lower than those in Healthy Persons
Haplogroup of 5 of 249 ROU patients was grouped into D5, while 16 of 237 healthy persons belonged to this type in Table 2, and the difference was significant (P,0.05, OR = 0.310). In this study, there was a certain haplogroup attracting our attention, that was R. It was not distributed in ROU patients, but haplogroup of 42 of 237 ROU patients was grouped into R. Whether male or female, the difference was much significant (Male P = 3.046610 27 Female P = 5.804610 26 ). These results showed that the percentage of D5 and R in healthy persons was significantly higher than in ROU patients. In Fig. 3 this phenomenons could be obvious: the columnar curve of D5 and R in healthy persons could be more higher than in ROU patients.

The Distribution of G1 and H in ROU Patients is Higher than those in Healthy Persons
In Table 2, haplogroup was grouped into G1 in 11 of 249 ROU patients and 2 of 237 healthy persons (P,0.05, OR = 0.049). Haplogroup was grouped into H in 86 of 249 ROU patients and 16 of 237 healthy persons (P = 2.336610 211 , OR = 7.047). The distribution characteristics of haplogroup H in ROU patients and the healthy persons were similar to those of G1. The distributions showed that the percentage of G1 and H in ROU patients was significantly higher than the healthy persons respectively. In Fig. 3 the columnar curve changes of G1 and H in ROU patients significantly increased as compared with those in healthy persons.
The Polymorphism of C-stretch in mtDNA HV1, HV2 and HV3 Regions of Healthy Persons In nt 16184-16193, nt 303-315 and nt 568-573, there were 42 types of C-stretch sequence patterns as shown in Table 3. Of these, 65 cases were CCCCCTCCCC-CCCCCCCCTCCCCCC-CCCCCC with the highest frequency (27.43%) of C-stretch nucleotide sequence (Fig. 4a,b,c), and 1 case (0.42%) showed the same structure as the rCRS.
The Polymorphism of C-stretch in mtDNA HV1, HV2 and HV3 Regions of ROU Patients The C-stretch sequence patterns for nt 16184-16193, nt 303-315 and nt 568-573 were composed of 26 types as shown in Table 4. The pattern with the highest frequency was CCCCCTCCCC-CCCCCCCCTCCCCCC-CCCCCCC with 104 cases (41.77%) (Fig. 4a,c,d) and this was followed by CCCCCTCCCC-CCCCCCCTCCCCCC-CCCCCCCC with 77 cases (30.92%) (Fig. 4a,b,c). There were no case that indicated the same structure as the rCRS.

Discussion
This concerted action that the mutations of genomic DNA play an important role in the disease is complex, but mtDNA offers the possibility of approaching the problems from better defined perspective. MtDNA has already been fully sequenced [17] and many subsequent publications have revealed polymorphic sites, haplogroups and haplotypes.
Distribution of various disease groups among these haplogroups showed that some of the mitochondrial haplogroups are predisposed to disease [18]. The researchers found some haplogroups had a significantly higher occurrence in Leber's hereditary optic neuropathy (LHON) patients suggested that they are risk factors [19][20][21]. All the seven probands and their maternal relatives of Noonan syndrome (NS) were clustered under a major haplogroup R, suggesting that these haplogroups may influence NS in South Indian populations [22]. Mitochondrial haplogroup H was associated with early onset of myocardial infarction (MI) in male smokers [23]. For breast and esophageal cancer, haplogroup N was a risk factor because of mitochondrial DNA G10398A polymorphism [24]. Substitutions in the D-loop may be part of a haplotype with mutations elsewhere in the mtDNA. The hypothesis is that on their own some polymorphisms are selectively neutral, but in specific combinations they act in a synergistic, deleterious manner with established pathogenic mtDNA mutations to increase the risk of disease expression or to produce a more severe clinical outcome. The rich variability in HV1, HV2 and HV3, compared with the relatively constant constellation within the gene regions, provides useful criteria for pathogenetic studies [25].    We screened HV1, HV2 and HV3 to assess correlation between the ROU patients and the healthy persons. This was the first study to trace mtDNA H variants in ROU patients of Chinese population. Our results showed that haplogroups G1 and H were significantly more abundant in ROU patients (P = 0.049 for haplogroups G1 and P = 2.336610 211 for haplogroup H) ( Table 2). Thus, mtDNA haplogroups G1 and H might constitute a risk factor for ROU. These haplogroups might increase the incidence of ROU disease. On the other hand, haplogroups D5 and R were significantly more abundant in normal subjects (P = 0.025 for haplogroup D5, and Male P = 3.046610 27 Female P = 5.804610 26 for haplogroup R), suggesting that they might be protective factors for ROU.
In the process of mtDNA haplogroups to research, we found an interesting phenomenon-the generation of C-stretch. It was mainly produced by the mechanism of polymerase's copying slippage and on the regulation of nuclear code factors to mitochondria sequences. In the process, polymerase's copying slippage could not only produce the sequence's length polymorphism, can also cause the length heterogeneity [26]. Some scholars reported t the polymorphism of C-stretch was maternally inherited showing a similar distribution along maternal lineage and the pattern of the length heteroplasmy seems to be maintained in an individual and it was regenerated de novo by replication slippage following each cell division [27]. Along with the increase of the poly[C] number, the polymorphism of C-stretch became being unstable and influence the mtDNA replication fidelity [28].
Clinical characteristics and pathogenesis of ROU patients are rather complicated. Our results reveal association of haplogroups G1, H, D5 and R with ROU for the first time. This work may be aspiring for further studies on haplogroups in this disease, and may shed new light on the molecular pathogenesis of ROU.

Materials
The 249 cases of ROU patients were diagnosed by School of Stomatology Fourth Military Medical University. The diagnostic criteria [29] was shown in Table 5. Of them, 122 were male and 127 were female, aged from 8 to 87 years old. The control group included 237 healthy volunteers, of whom 145 were male and 92 were female, aged from 10 to 79 years old. The healthy persons never had a history of oral diseases. All participants included 74 elderly people above the age of 60 year, of whom 63 were bachelor degree and fully understood the content of the consent when they signed informed written consent. Of them, 11 were other qualification and we particularly All participants didn't exist in the following situation: participants who declined to participate or otherwise did not participate were eligible for treatment (if applicable) and were not disadvantaged in any other way by not participating in the study.

DNA Extraction
0.5 mL of venous blood was dealt with EDTA-Na 2 anticoagulation in both of the groups, and then DNA was extracted with the RelaxGene Blood DNA System of Tiangen (catalogue number: DP319-02). The quality of the extracted DNA was tested with NanoDrop 2000 (Thermo Scientific).

Primer Design
Referencing Yao [30] et al, through the primer 6.0 software, the primer was designed. And then it's specificity was detected via NCBI. The primer was shown in Table 6. The Beijing AUGCT DNA-SYN Biotechnology Co., LTD synthesized the primers and purified by PAGE.

PCR and Sequencing
PCR system was 25 ml, including 1 m L of DNA template, 12.5 m L of PCR Mix(100 mM Kcl, 20 mM Tris-Hcl, 3 mM Mgcl 2 , 400 mM dNTP, 0.1U/ml Taq DNA polymerases), 1 mL of 10 pmol the upstream primer, 1 mL of 10 pmol the downstream primer, the added ultrapure water to 25 mL. The research used the 2720 thermal cycler PCR instrument (AB company), circulation conditions: 95uC for 2 min followed by 35 cycles of 95uC 30 s, 60uC 30 s, 72uC 30 s, and a final extension for 5 min at 72uC. After preserved at 4uC. the 1.5% of agarose gel electrophoresis inspection was conducted, The sequence's length by the Beijing  Table 3. C-stretch sequences and frequencies of mtDNA HV1, HV2 and HV3 regions in healthy persons.

Data Analysis
Referencing the revised Cambridge Reference Sequence (rCRS, NC_012920) [16], the sequences of HV1, HV2 and HV3 were 960 bp in all. The BioEdit software comparised the sequences, and then DNASP 5.0 software corrected them. The statistics of the polymorphic sites were conducted through Sequencher 4.1.4 software and the mtDNA haplogroups were categorized on the haplogrep (http://haplogrep.uibk.ac.at/). The experimental data were processed through SSPS 13.0 software. On the one hand, two test group (ROU patients and healthy persons) was applied chi-square test in everyone Haplogroup. The values of chi-square test were selected through difference of expected count and total number. On the other hand, in order to reduce errors we compared gender diversity between ROU patients and healthy persons. If the difference was significant, we did binary logistic regression and obtained the adjusted P-value and OR (95% CI).Pvalues below 0.05 were considered statistically significant.