Get Permission Vaishnavi D, Kotian, and Daniel: Runx2 gene single nucleotide polymorphism in Class II and Class III malocclusions


Introduction

Genetics is a branch of biology deals with the mechanisms of inheritance and the causes of diversity in living beings. 1 The size, shape of the maxilla and mandible, morphology of teeth present, and soft tissue morphology are under genetic influences.2, 3

Growth and development is mainly the result of interaction between different genetic and environmental factors overtime.4, 5 Higher the genetic component, the lower the rate of a successful orthodontic treatment outcome.6, 7 The identification of the most vital genes and the biochemical action of these genes to a specific jaw discrepancy is the first approach essential for the search of a solution.8, 9

Malocclusions usually believed to have a negative interaction of hereditary and environmental factors.10, 11 From the beginning, the role of inheritance and environment as causes of malocclusion and dentofacial deformities have been the subject of great controversy. 12, 13, 14 During foetal development, the mandibular and maxillary growth is affected, resulting in skeletal malocclusion.15, 16, 17 The significant prognathic mandible in the Hapsburg Royal family's ancestry strongly suggests a genetic component in the inheritance of this craniofacial feature.18, 19, 20 One parent with a comparable phenotype was found in 1/3 of the affected Hapsburg family members with severe class III malocclusion.21, 22 Orthodontists must consider the genetic basis of a skeletal anomaly during the diagnosis and treatment planning.23, 24, 25 Genetic basis of skeletal anomaly can be identified using different methods like single gene sequencing, site specific mutation testing, gene panels, molecular testing, immunohistochemistry, microsatellite instability.26, 27

Runt – related transcription factor 2 ( RUNX2) plays an important role in osteoblast differentiation, tooth development and chondrocyte maturation; hence its involvement in the growth of the craniofacial area is crucial. 28, 29 Genetic studies therefore are important to know if this polymorphism affects different classes of malocclusion. 30

Aim and Objective

  1. To assess the prevalence of RUNX2 gene polymorphism in different classes of malocclusions mainly class II and class III malocclusion.

Source of data

Unstimulated salivary samples of 36 patients (18- 30 years of age group), comprising 18 with skeletal class II and 18 with skeletal class III were collected from a tertiary care hospital in Mangalore. Salivary DNA samples were collected and analyzed using Sanger sequencing. Digital tracing was performed on lateral cephalometric radiographs by using AutoCAD software for digitization to assess the anterio-posterior and vertical relationship of the maxillary and mandibular arch.

Ethics statement

This prospective study was approved by the Institutional Research Ethics committee of, AJIMS ( AJIEC/REV/271/2019)   

Inclusion criteria

  1. Patients with skeletal class II and class III malocclusions.

  2. Patients between 18- 30 years of age group.

Exclusion criteria

  1. Presence of systemic diseases.

  2. Presence of congenital deformities.

Materials and Methods

Cephalometric measurements: Skeletal class II and class III malocclusion cases were assessed using following parameters.

Steiner’s SNA angle: Class II> 82°, Class III <82°

Steiner’s SNB angle: Class II> 80°, Class III < 80°

ANB angle: Class II > 6°, Class III< 2°

Down’s A-B Plane angle: Class II <-4.6°, Class III >-4.6°

Wits appraisal (AoBo): Class II: BO is behind AO, Class III :BO is ahead of AO> 2mm

Dna extraction

DNA isolation is done with Favorgene kit which involves following steps:

  1. Transfer up to 200µl saliva to a micro centrifuge tube

  2. If RNA-free genomic DNA is required, add 10ul of 20 mg / ml RNase A. Mix thoroughly by vortexing and incubate at room temperature for 2 min

  3. Add 20 ul Proteinase K to the sample, and then add 200 ul FATG2 Buffer to the sample. Mix thoroughly by pulse-Vortexing. Incubate at 60°C for 30 min. Vortex occasionally during incubution.

  4. Incubate at 70° C for 10 min

  5. Add 200 ul ethanol (96-100% to the sample mixture Mix thoroughly by pulse- vortexing

  6. Briefly spin the tube to remove drops from the inside of the lid

  7. Place a FATG Mini Column in a Collection Tube. Transfer the mixture (including any precipitate) carefully to the FATG Mini Column. Centrifuge at full speed(18,000 xg) for 1 min then plaçe the FATG Mini Column to a new Collection Tube.

  8. Add 400 ul WI Buffer to the FATG Mini Column. Centrifuge at full speed for 1 min then discard flow-through. Make sure that ethanol has been added into WI Buffer when first open. 

  9. Add 750 ul Wash Buffer to the FATG Mini Column. Centrifuge at full speed for 1 min then discard flow-through. Make sure that ethanol has been added into Wash Buffer when first open.

  10. Centrifuge at full speed for an additional 3 min to dry the column.This step will remove the residual liquid.

  11. Add 100 ul of preheated Elution Buffer to the membrane of the FATG Mini Column. Stand the FATG Mini Column for 3 min. For effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely. If less sample to be used, reduce the elution volume to 50 ul to increase DNA concentration and do not elute the DNA using less than suggested volume (50 ul). It will lower the final yield.

  12. Centrifuge at full speed for 2 min to elute DNA.

Additional requirement: RNase A, 96- 100% ethanol, PBS Hint: Set dry or water baths: 60° C for step 3 and 70 °C for step 4.

Polymerase chain reaction

Amplification of RUNX2 gene was performed from the extracted DNA (n=27) using 2X PCR Master Mix (SMOBio,Hsinchu City, Taiwan). The primer sets (Eurofins Genomics India Pvt. Ltd., Bangalore) used for amplification were:

rs6930053 RUNX2 Forward: 5’-GTATGTCATTTCTGTACTTTCG-3’

rs6930053 RUNX2 Reverse : 5’-GTGCTATTTCCTGTCCTTATC-3’   

The reaction mixture contained 0.2µM of each primer per sample and the rest of reaction mixture was prepared as per the kit protocol. Typically one reaction contained the following ingredients:

0.5µM of ech primer, 2mM Mgcl2, 0.2 mM of dNTP, 200 mg of gDNA, 1.75U of DNA polymerase.

Standard amplification conditions are: 95 degree for 4min followed by 35 cycles and final extension for 10 min at 72 degree Celsius.

The PCR products (639bp) were visualised in 1% Agarose gel and subsequently sent for sequencing.

Gene sequencing

The RUNX2 PCR amplified gene was sent to Eurofins Genomics Pvt. Ltd., Bangalore for Sanger sequencing with forward primers.

Sample size estimation

To detect a difference of 50% of RUNX2 gene in class II and class III malocclusions using 95% confidence interval and 90% power. The sample size estimated for the study is 18 saliva samples of each class II and class III.

n = [ z1-α + z 1-β ]2 p1 q1 +p2q2/( the difference in proportions)2

Sampling technique

Convenience sampling will be adopted.

Results

When polymorphism of 36 samples comprised of 18 Class II and 18 Class III were assessed, the following results were obtained.

Table 1

Prevalence of RUNX2 polymorphism in different classes of malocclusions using Chi square test.

Polymorphism in malocclusion

Malocclusion

Total

Class II

Class III

Absent

Count

17

3

20

%

94.4%

16.7%

55.6%

Present

Count

1

15

16

%

5.6%

83.3%

44.4%

Total

Count

18

18

36

%

100.0%

100.0%

100.0%

. x2=22.05 p<0.001 vhs

Chart 1

Comparison and association of polymorphism in different classes of malocclusions.

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SNP’s are found in 44.4% of the total sample

83.3% Class III malocclusion and 5.6% Class II showed RUNX2 gene polymorphism in the population. Chi Square test which indicated that the results are statistically significant in Class III malocclusion for RUNX2 single nucleotide polymorphism (p<0.001)

Figure 1

Genotypic analysis shows the following results:

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/09c74785-7a22-44a1-817a-573a7a71e5a9image1.jpeg

There is alteration in the nucleotidic sequence where in T is replaced by C in the mutant sample.

For rs6930053, the ancestral allele is T, allele C is mutant.(Figure 2)

Figure 2

Agarose gel picture

https://s3-us-west-2.amazonaws.com/typeset-prod-media-server/09c74785-7a22-44a1-817a-573a7a71e5a9image2.png

Electrophoresis gel shows a band of amplified dnaat 639 bp which would indicate the dna extraction is successful.(Figure 3)

Results of this study can be summarized as follows

  1. RUNX2 gene polymorphism (rs6930053) in skeletal Class II malocclusion was 5.6% and in skeletal Class III malocclusion was 83.3% from the samples obtained from a tertiary care hospital in Mangaluru.

  2. RUNX2 gene polymorphism was statistically significant in skeletal Class III malocclusion when compared to skeletal Class II malocclusion.

Discussion

Skeletal malocclusion is generally predisposed by genetic and environmental factors as it’s etiology. In this study RUNX2 gene mutation has been assessed to identify it’s relationship with the malocclusion.

The RUNX2 genes are found on chromosomes 6p21 and 17 in humans and mice, respectively. 31, 32

This gene encodes a nuclear protein with a Runt DNA-binding domain that belongs to the RUNX transcription factor family. 33, 34 This protein is required for osteoblast development and skeletal morphogenesis, and it serves as a scaffold for nucleic acids and regulatory factors involved in the expression of skeletal genes. 35

Bones are formed through one of two ossification processes: (i) intramembranous or (ii) endochondral ossification.36

Both endochondral and intramembranous mechanisms are used in the ossification of the skull37 both procedures entail the conversion of pre-existing mesenchymal tissue into bone tissue. Intramembranous ossification is the direct conversion of mesenchymal tissue to bone and endochondral ossification is a process by which a cartilage intermediate is formed initially which will be replaced by bone cells. 38, 39

RUNX2 is involved in the differentiation of chondrocytes into hypertrophic chondrocytes and further differentiation processes, such as ossification.40

The TMJ's general structure is similar to that of most synovial joints, but the cartilage that caps the mandibular condyle (mandibular condylar cartilage or MCC) is a secondary cartilage, that indicates the morphogenesis of the TMJ and its components begins after the mandible has formed and analogous joints in the limbs have formed. 41 Primary cartilages of the limbs are formed by the interplay of the mesenchyme and epithelium, whereas secondary cartilages form in response to local biomechanical stimuli. RUNX2 is responsible for osteogenic response, as evidenced by the presence of mRNA for osteogenic lineage markers (e.g., collagen, RUNX2, and Osterix) in mandibular condylar cartilage. 42, 43

The skeletal form had a strong correlation to the presence of type II collagen fibres in the masseter muscle. In both the sagittal and vertical dimensions, these correlations have substantial implications for facial structure. Mechanism through which RUNX2 may function in the sagittal dimension is through its effect on condylar development,44 and periosteal activation of osteoblast gene expression.

Cleidocranial dysplasia (CCD), an autosomal-dominant heritable skeletal disorder characterised by open or delayed closure of calvarial sutures, hypoplastic or aplastic clavicles, and supernumerary teeth, is caused by RUNX2 mutations in humans. 45

An allele is one of two or more versions of a gene. A gene's allele is one of two or more variants. For each gene, an individual inherits two alleles, one from each parent. The individual is homozygous for that gene if the two alleles are same. The individual is heterozygous if the alleles are different. Though the term allele was originally intended to describe gene variation, it is now also used to denote variation in non-coding DNA sequences.45

The two members of a pair of alleles separate during gamete production, according to Mendelian law of segregation, which is one of the Mendelian Laws of Inheritance. As a result, each gamete only has one member of each gene pair.

Figure 3

Seggregation of parent chromosomes

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Mendelian law of dominance when applied says that “It is stated that one factor in pair of traits dominates while the other remains suppressed in inheritance unless the two factors in the pair are recessive. In the next generation of parents who are pure for contrasting traits, there will be only one type of trait.42, 43

While most genes exist in two allele forms, some have multiple alleles for a trait.44 Likewise, there is polygenic inheritance where in the traits are determined by more than one gene.45

Alteration in even one non coding sequence would result in a definitive phenotypic variation. Identification of the mutant allele in one generation can significantly predict the probability of transfer of this allele to the future generations based on the laws of genetics.

This study was conducted to determine whether there was an association of class II and class III skeletal malocclusion and the gene RUNX2 in Mangalore population. The genomic DNA was extracted from unstimulated saliva of 18 class II and 18 class III patients based on the skeletal parameters.

In the present study, it is observed on forward gene sequencing that the nucleotide T has been replaced by C in the non coding sequence which has resulted in the phenotypic Class III malocclusion in the patients.

SNP’s are found in 44.44 % of the total sample.

In previous studies conducted by Mokhtar KIet alsignificant association of class II malocclusion and the gene RUNX2 in Malaysian population was found where as 83.3% Class III malocclusion and 5.6%Class II showed RUNX2 gene mutation in population.

Sample 13 was taken from a cleft patient with Class III malocclusion, Sample 14 andsample23 patient hadhypodontia. This indicates that RUNX 2 plays a vital role in the development of craniofacial skeleton and dentition, thus opening up new horizons for future studies in this direction.

Sample 25 whose sample had a mutation when her family tree was traced, it was found that patient’s other sibling and mother had similar phenotype. However the patient’s prognathism was more severe in comparison to her other family members. This would suggest the severity of the skeletal malocclusion can increase in the subsequent generation constantly. This tells us how important it is to identify a genetic malocclusion and plan treatment accordingly with all the measures available. Sample 11 and sample 18 were dead during the transportation for gene sequencing.

It is difficult to control skeletal malocclusion due to genetic causes by the conventional orthodontic treatment. Orthodontic treatment coupled with orthognathic surgery was the only resort to overcome this problem. That’s why it is important to diagnose early these skeletal malocclusions to carry out definitive treatment.

The results of this study enable us to identify a genetic malocclusion which would aid in accurate diagnosis and treatment planning, thus providing appropriate treatment. So it is always better to determine the cause first than the effects. Genetic analysis aids in planning definitive treatment and reduces the treatment duration for the patient. Knowing the exact treatment that has to be carried out right after the patient enters the clinic in an early age would resolve the problem early without worsening the malocclusion, as this would be cost effective and treatment effective.

Orthopedic and functional appliances can be used to treat malocclusions of genetic origin( skeletal discrepencies) when detected in growing period except in extreme cases where surgical intervention is needed after the growth completion. Genetic testing would give information regarding the need of treatment for a child and age at which treatment can be begun to control a skeletal malocclusion from becoming a more severe one.

This is a preliminary study done on a smaller sample, we need a larger sample size to confirm our findings. Also forward and reverse sequencing give confirmatory results for the sample.

Conclusions

The study concluded that:

  1. RUNX2 gene polymorphism (rs6930053) in skeletal Class II malocclusion was 5.6% and in skeletal Class III malocclusion was 83.3% from the samples obtained from a tertiary care hospital in Mangalore.

  2. RUNX2 gene polymorphism was statistically significant in skeletal Class III malocclusion when compared to skeletal Class II malocclusion.

  3. This is a preliminary study done on a smaller sample, we need a larger sample size to confirm our findings.

Source of Funding

None.

Conflict of Interest

None.

References

1 

TK Nayak SN Sahoo SB Nanda S Pattanaik N Mohammad P Panigrahi The Basic Genetics of MalocclusionIndian Journal of Public Health201891225036

2 

AH Muhamad N Watted Genetics and OrthodonticsInt J Appl Dent Sci20195338490

3 

S Gangane Human Genetics2ndChurchill LivingstoneNew Delhi2000

4 

NE Morton Genetic epidemiologyAnn Rev Genet199327523810.1146/annurev.ge.27.120193.002515

5 

DS Carlson Evolving concepts of heredity and genetics in orthodonticsAm J Orthod Dentofacial Orthop2015148692238

6 

C Nishio N Huynh Skeletal malocclusion and genetic expression: An evidence based reviewJ Dent Sleep Med2016325763

7 

TM Schroeder ED Jensen JJ Westendorf Runx2: A master organizer of gene transcription in developing and maturing osteoblastsBirth Defects Res C Embryo Today200575321325

8 

RM Cruz JK Hartsfield G Falcão-Alencar DL Koller RW Pereira J Mah Exclusion of Class III malocclusion candidate loci in Brazilian familiesJ Dent Res2011901012025

9 

WD Lin SP Lin CH Wang Y Tsai CP Chen FJ Tsai RUNX2 mutations in Taiwanese patients with cleidocranial dysplasiaGenet Mol Biol20113422014

10 

Y Li C Ge JP Long DL Begun JA Rodriguez SA Goldstein Biomechanical stimulation of osteoblast gene expression requires phosphorylation of the RUNX2 transcription factorJ Bone Miner Res2012276126374

11 

M Ghergie D Festila I Lupan O Popescu B Kelemen Testing the association between orthodonthic classes I, II, III and SNPs (rs731236, rs8004560, rs731236) in a Romanian clinical sampleAnn Rom Soc Cell Biol20131824351

12 

L Morford TJ Coles K Stewart D Fardo K Kula J Hartsfield Association Analysis of RUNX2/3 SNPs with Class-II-Division-2-Malocclusion (CII/D2) nd Concurrent-Tooth-Agenesis IADR/AADR/CADR General Session and Exhibition2013

13 

KE Lee F Seymen J Ko M Yildirim EB Tuna K Gencay RUNX2 mutations in cleidocranial dysplasiaGenet Mol Res2013124456774

14 

D Festila M Ghergie A Muntean Genetic Implications in Class II Subdivision 2 Malocclusion in Two Siblings- Case ReportMed Sci20143715

15 

H Desh SL Gray MJ Horton G Raoul AM Rowlerson J Ferri Molecular motor MYO1C, acetyltransferase KAT6B and osteogenetic transcription factor RUNX2 expression in human masseter muscle contributes to development of malocclusionArch Oral Biol20145966017

16 

Y Qian Y Zhang B Wei M Zhang J Yang C Leng A novel Alu-mediated microdeletion in the RUNX2 gene in a Chinese patient with cleidocranial dysplasiaJ Genet201897113743

17 

T Takarada R Nakazato A Tsuchikane K Fujikawa T Iezaki Y Yoneda Genetic analysis of Runx2 function during intramembranous ossification Development201614322118

18 

F Jazaldi ED Handayani YN Damayanti AT Sarwono BM Soegiharto N Soedarsono The LEPR Q223R polymorphism as a potential bioindicator of class II malocclusionJ Int Dent Med Res20169351

19 

FD Bir N Dinçkan Y Güven F Baş U Altunoğlu SS Kuvvetli Cleidocranial dysplasia: Clinical, endocrinologic and molecular findings in 15 patients from 11 familiesEur J Med Genet20176031638

20 

NV Grupioni NB Stafuzza AB Carvajal AM Ibelli JO Peixoto MC Ledur Association of RUNX2 and TNFSF11 genes with production traits in a paternal broiler lineGenet Mol Res201716110.4238/gmr16019443ALICE

21 

A Doraczynska-Kowalik KH Nelke W Pawlak MM Sasiadek H Gerber Genetic Factors Involved in Mandibular PrognathisJ Craniofac Surg201728542231

22 

AM Buo RE Tomlinson ER Eidelman M Chason JP Stains Connexin43 and Runx2 interact to affect cortical bone geometry, skeletal development, and osteoblast and osteoclast functionJ Bone Miner Res2017328172738

23 

CV Cruz CT Mattos JC Maia JM Granjeiro MF Reis JN Mucha Genetic polymorphisms underlying the skeletal Class III phenotypeAm J Orthod Dentofacial Orthop201715147007

24 

KI Mokhtar NA Bakar AF Kharuddin RUNX2 Single nucleotide polymorphism (rs6930053) in Class II malocclusion patients: A preliminary studyAsian J Med Biomed2018

25 

MM Saad NA Abdrahman KI Mokhtar N Abu Bakar AF Kharuddin WR Taib W R Preliminary study of PAX9 single nucleotide polymorphism (rs8004560) in patients with Class II skeletal base malocclusion contributed by mandibular retrognatismArch Orofacial Sci2018131128

26 

D Ma X Wang J Guo J Zhang T Cai Identification of a novel mutation of RUNX2 in a family with supernumerary teeth and craniofacial dysplasia by whole-exome sequencing: a case report and literature reviewMedicine201897329710.1097/MD.0000000000011328

27 

T Zhang J Wu X Zhao F Hou T Ma H Wang Whole-exome sequencing identification of a novel splicing mutation of RUNX2 in a Chinese family with cleidocranial dysplasiaArch Oral Biol2019100495610.1016/j.archoralbio.2019.02.005

28 

Q Jiang L Mei Y Zou Q Ding RD Cannon H Chen Genetic polymorphisms in FGFR2 underlie skeletal malocclusionJ Dent Res2019981213407

29 

Y Shirai K Kawabe I Tosa S Tsukamoto D Yamada T Takarada Runx2 function in cells of neural crest origin during intramembranous ossificationBiochem Biophys Res Commun20195094102833

30 

EC Kuechler CL Reis J Carelli R Scariot P Nelson-Filho RD Coletta Potential interactions among single nucleotide polymorphisms in bone-and cartilage-related genes in skeletal malocclusionsOrthod Craniofac Res202124227787

31 

T Aonuma N Tamamura T Fukunaga Y Sakai N Takeshita S Shigemi Delayed tooth movement in Runx2+/− mice associated with mTORC2 in stretch-induced bone formationBone Rep20201210028510.1016/j.bonr.2020.100285

32 

A Jaruga E Hordyjewska G Kandzierski P Tylzanowski Cleidocranial dysplasia and RUNX2-clinical phenotype-genotype correlationClin Genet2016905393402

33 

RUNX2 RUNX family transcription factor 2 [ Homo sapiens (human) ]2016https://www.ncbi.nlm.nih.gov/gene/860

34 

G Breeland MA Sinkler RG Menezes Embryology, Bone OssificationTreasure Island (FL): StatPearls Publishing2020

35 

C Zhang Transcriptional regulation of bone formation by the osteoblast-specific transcription factor OsxJ Orthop Surg Res201053710.1186/1749-799X-5-37

36 

SF Gilbert Osteogenesis: The Development of BonesDevelopmental Biology6thSunderland (MA): Sinauer Associates2000

37 

IGAW Ardani AM Aulanni I Diyatri Single Nucleotide Polymorphisms (SNPs) of COL1A1 and COL11A1 in Class II Skeletal Malocclusion of Ethnic Javanese PatientClin Cosmet Investig Dent202012173910.2147/CCIDE.S247729

38 

S Shibata N Suda S Yoda H Fukuoka K Ohyama Y Yamashita Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel's cartilageAnat Embryol (Berl)2004208227380

39 

T Kanno T Takahashi W Ariyoshi T Tsujisawa M Haga T Nishihara Tensile Mechanical Strain Up-Regulates Runx2 and Osteogenic Factor Expression in Human Periosteal Cells: Implications for Distraction OsteogenesisJ Oral Maxillofac Surg2005634499504

40 

TA Brown Mutation, repair and recombinationGenomes2ndWiley-Liss2002

41 

JD Watson Molecular biology of the genePearson Education India2004

42 

PV Oltramari-Navarro RR Almeida AC Conti RD Navarro MR Almeida LS Fernandes Early treatment protocol for skeletal Class III malocclusionBraz Dent J201324216773

43 

N Siddique H Raza S Ahmed Z Khurshid MS Zafar Gene therapy: A paradigm shift in dentistryGenes20167119810.3390/genes7110098

44 

PW Whiting Multiple alleles in complementary sex determination of HabrobraconGenetics194328536582

45 

K Mather Polygenic inheritance and natural selectionBiological Rev19431813264



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Article History

Received : 22-08-2024

Accepted : 11-09-2024


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https://doi.org/10.18231/j.jds.2024.025


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