Clinical, Cosmetic and Investigational Dentistry Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Single Nucleotide Polymorphisms (SNPs) of COL1A1 and COL11A1 in Class II Skeletal Malocclusion of Ethnic Javanese Patient This article was published in the following Dove Press journal: Clinical, Cosmetic and Investigational Dentistry I Gusti Aju Wahju Ardani1 Background: The prevalence of malocclusion cases in the orthodontic specialist clinic in Aulanni’am2 Airlangga University’s Dental Hospital in Surabaya, Indonesia, in 2014–2016 is fairly high, Indeswati Diyatri3 as 55.34% of the occurrences were identified as class II skeletal malocclusion. This type of skeletal malocclusion, which is usually recognized in adults, occurs as a result of variation 1Orthodontics Department, Faculty of Dental Medicine, Airlangga University, during growth and development. Lately, there have been many reports on gene polymorph- Surabaya, Indonesia; 2Biochemistry isms of COL1A1 and COL11A1, which are assumed to be associated with class II skeletal Department, Faculty of Veterinary malocclusion in Caucasians. Medicine, Brawijaya University, Malang, Indonesia; 3Oral Biology, Faculty of Purpose: This study aims to analyze the relationship between single nucleotide polymorph- Dental Medicine, Airlangga University, isms (SNPs) of COL1A1 and COL11A1 with class II skeletal malocclusion in Javanese Surabaya, Indonesia ethnic group patients with mandibular micrognathism. Materials and Methods: The diagnosis of class II skeletal malocclusion was established using the lateral cephalometric radiographs (ANB angle ≥4°) (n=50). DNA was extracted from the patient’s peripheral blood. After that, PCR, electrophoresis, and DNA sequencing were conducted on the extracted DNA based on COL1A1 and COL11A1 primers. Results: The SNPs in COL1A1 are c.20980G/A in 27 patients and c.20980G>A in 8 patients, whereas SNPs in COL11A1 are both c.134373C/A and c.134555C/T in 8 patients and both c.[134373A>C] and c.134582G>A in 10 patients. All samples show the deletion (c. [134227delA]) in COL11A1. Conclusion: SNPs in COL1A1 and COL11A1 have been found in class II skeletal mal- occlusion of Javanese ethnic group patients. Seventy percent of SNPs in COL1A1 occur in rs.2249492, whereas 36% of newly discovered SNPs appear in COL11A1. All samples also have deletion in COL11A1. Keywords: COL1A1, COL11A1, class II malocclusion, mandibular micrognathism, ethnic Javanese Introduction Class II skeletal malocclusion is defined as a distal relation of mandible to the maxilla and has both sagittal anomaly and vertical anomaly characteristics.1 Class II skeletal malocclusion with anteroposterior skeletal anomaly, which is shown by ANB angle 2 Correspondence: I Gusti Aju Wahju 6.42 ± 1.70°, presents maxillary and mandibular disharmony to the cranial base. Ardani Moreover, there are two divisions of class II skeletal malocclusion: division one Orthodontics Department, Faculty of Dental Medicine, Airlangga University, and division two. Dental characteristics of division one class II skeletal malocclu- Surabaya, Indonesia sion are maxillary protrusion, a deep bite, and a large overbite, whereas division Tel +623 15030255 Email [email protected] two is characterized by a deep bite with maxillary central incisors proclination and submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dentistry 2020:12 173–179 173 DovePress © 2020 Ardani et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. – http://doi.org/10.2147/CCIDE.S247729 php and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Ardani et al Dovepress lateral incisors retroclination or retroclination of all max- group population, which belongs to the Deutero – Malay illary incisors. However, most patients with class II skele- race who have special characteristics of class II skeletal tal malocclusion in Airlangga University Dental Hospital malocclusion with short mandibles. It also determined (AUDH) have special characteristics which make them whether the SNPs of COL1A1 and COL11A1, which is different from the typical class II skeletal malocclusions found in Caucasian’s class II skeletal malocclusion, are in Caucasians. Most patients have not only a deep bite, but also found in the Javanese population. This study aims to also protrusion of both maxilla and mandible, therefore the analyze the relationship between the SNPs of COL1A1 and overjet is not too large. Protrusion on maxilla only are COL11A1 with class II skeletal malocclusion in Javanese rarely found. The results of previous studies showed that ethnic group patients with mandibular micrognathism. class II skeletal malocclusion in AUDH increased ANB which, for the most part, was not caused by the increased Materials and Methods SNA (82.34 ± 3.49°). It was due to the decreased SNB This research was conducted at the Airlangga University (75.91 ± 3.77°). Based on McNamara analysis, class II Dental Hospital in Surabaya, Indonesia, from April 2015 skeletal malocclusion cases with a short mandible (107.87 to April 2016. This study was approved by the Ethical ± 8.55 mm) was found more often than the exceeded Clearance Committee of the Faculty of Dental Medicine, maxillary length.3,4 This result is consistent with class II Airlangga University with the reference number 059/ skeletal malocclusion characteristics in the Brazilian popu- HRECC.FODM/V/2018. The inclusion sample criteria of lation, whose mandibular length is 107.87 ± 8.54 mm.5 this study were Javanese men and women, aged between However, studies on other ethnic groups show different 15–35 years old, who had never had any previous ortho- results, such as in French Canadian, Italian, and Iraqi dontic treatment and all permanent teeth had erupted populations.6–8 except for the third molar. Fifty patients fulfilled the inclu- Meanwhile, the patterns and direction of skeletal sion criteria and agreed to become samples in this study. growth can be identified by examining the profile photo, All participants, and a parent/legal guardian of participants cephalometric radiograph, and cast model. However, class under the age of 18 years provided written informed con- II skeletal malocclusion cases are mostly diagnosed once sent, and that this study was conducted in accordance with patients have reached adulthood. The late diagnosis of the Declaration of Helsinki. skeletal malocclusion limits treatment options to camou- Class II skeletal malocclusion was determined using the flage orthodontic treatment or orthognathic surgery. On the digital cephalometric tracing (OrthoVision - 2017, South other hand, an earlier diagnosis enables any growth mod- Korea). Anatomical landmarks (such as: N, S, A, B, Ba, ification treatment to be performed on children or teen- Gn, Co) were determined manually using the cephalometric agers, which provides a shorter treatment duration and analysis software (Figure 1). DNA extraction was done using relatively more stable result compared to those diagnosed QiaAmp Blood Mini Kit (Qiagen), as instructed by standard later as adults. Dental and skeletal malocclusion that left blood-DNA extraction protocol. DNA amplification was untreated can lead to temporomandibular disorder (TMD) completed using the PCR technique with a pair of primers. such as TMJ arthritis, which can affect oral health related Primers COL1A1_F: 5ʹ-GCCTCCCAGAACATCACCTA to quality of life (OHRQoL) in adolescents.9 -3ʹ and COL1A1_R: 5ʹ-TGGGATGGAGGGAGTTTACA Nowadays, many studies found that polymorphism/ -3ʹ were used for COL1A1, both with a target of 456 bp. gene variation also can be the cause of malocclusion.10– Primers COL11A1_F: 5ʹ-TGTGTGCCTGTATTCCCAGA 12 Previous studies stated that the development of maloc- -3ʹ and COL11A1_R: 5ʹ-GAGGAC CCTGCATGAGAA-3ʹ clusion was affected by human evolution and some genetic were used for COL11A1, both with a target of 415 bp. GoTaq factors, including the muscle fibers plasticity and hetero- Green Master Mix (Promega) was utilized as PCR Mix. geneity of both jaw skeletal morphology and masticatory A total of 35 cycles of PCR (Thermocycler SensoQuest muscle, which can be inherited.13 GmbH) were performed, starting with Pre-Denaturation The existence of genetic variations in skeletal malocclu- (94°C) for 4 minutes, Denaturation (94°C) for 30 seconds, sion can be investigated through the detection of single Annealing (55°C) for 30 seconds, Extension (72°C) for nucleotide polymorphisms (SNPs). Therefore, this study 1 minute and Final Extension (72°C) for 7 minutes. raised the question of whether genetics can cause variations Positive results would be shown at the location of 456 bp in class II skeletal malocclusions in the Javanese ethnic for COL1A1 and 415 bp for COL11A1. 174 submit your manuscript | www.dovepress.com Clinical, Cosmetic and Investigational Dentistry 2020:12 DovePress Dovepress Ardani et al electrophoresis and amplified using PCR and 2% agarose electrophoresis, which led to the formation of a single band of