Indian Journal of Dental Research

ORIGINAL RESEARCH
Year
: 2019  |  Volume : 30  |  Issue : 2  |  Page : 185--190

Mutations in FGFR3 gene associated with maxillary retrognathism


Ravi M Subrahmanya1, Sreenivas V Prasad2, Rajendra B Prasad3, Subraya Mogra4, Veena Shetty5, Vamana Rao6,  
1 Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Nitte Deemed to Be University, Mangalore, Karnataka, India
2 Department of Oral and Maxillofacial Surgery, Abdul Aziz College of Dentistry, King Saud University of Health Sciences, Riyadh, KSA
3 Department of Oral and Maxillofacial Surgery, AB Shetty Memorial Institute of Dental Sciences, Mangalore, India
4 Department of Orthodontics, Manipal College of Dental Sciences, Mangalore, India
5 Department of Microbiology, KS Hegde Medical Academy, Mangalore, India
6 Department of Biotechnology, NMAM Institute of Engineering, Udupi, Karnataka, India

Correspondence Address:
Prof. Ravi M Subrahmanya
Department of Orthodontics and Dentofacial Orthopaedics, AB Shetty Memorial Institute of Dental Sciences, Nitte Deemed to Be University, Mangalore, Karnataka
India

Abstract

Context: Understanding the role of fibroblast growth factor receptor (FGFR) in the regulation of bone development and disease will ultimately lead to better prevention and treatment of related bone deformities and disorders. Aims: To evaluate the role of gene FGFR3 in individuals with retrognathic maxilla by polymerase chain reaction (PCR) technique at molecular level and evaluate the significance of the same. Settings and Design: Hospital based fundamental research involving individuals having maxillary retrognathism. Methodology: A total of 62 individuals (30M and32F) who were willing to take part in the study were selected from cephalometric measurements of N I A and the length PNS to ANS. The institution based basic genetic research study involved collection of fresh blood samples, DNA extraction, PCR analysis, and amplification using the specifically designed forward and reverse primers for targeting the commonly occurring mutations in FGFR3 gene. Further the products were sequenced to evaluate the presence of any novel mutations. Results: The targeted single-nucleotide polymorphisms, at position 1138 in exon 10 of the FGFR3 gene were not identified in the analyzed blood samples. The detailed sequencing of full gene revealed the presence of 2 novel mutations, Exon 3: A213G and Exon 3: A223A/G in one individual. Conclusions: The present study indicated 2 novel mutations in gene FGFR3 in individual with maxillary retrognathism. The genetic–environmental interactions might have played a significant role in the expression of retrognathic maxilla.



How to cite this article:
Subrahmanya RM, Prasad SV, Prasad RB, Mogra S, Shetty V, Rao V. Mutations in FGFR3 gene associated with maxillary retrognathism.Indian J Dent Res 2019;30:185-190


How to cite this URL:
Subrahmanya RM, Prasad SV, Prasad RB, Mogra S, Shetty V, Rao V. Mutations in FGFR3 gene associated with maxillary retrognathism. Indian J Dent Res [serial online] 2019 [cited 2019 Sep 19 ];30:185-190
Available from: http://www.ijdr.in/text.asp?2019/30/2/185/259211


Full Text

 Introduction



The mechanisms that control facial growth and development are poorly understood and are areas of great interest and research for Orthodontists and Maxillofacial Surgeons. As with all growth and development, there is interaction between genetic and environmental factors in the control of craniofacial growth and development. It is often difficult to distinguish the effects of hereditary and environmental factors. However, genetic control is undoubtedly significant in facial growth.[1]

It has been known that fibroblast growth factor (FGF) regulates proliferation and migration of osteoblastic cells and many different types of cells. The FGFs produce their effects in target cells by signaling through cell surface tyrosine kinase receptors (FGFR). However, the exact mechanism underlying their action has not been defined clearly.[2]

Several mutations in FGFs and FGFRs were reported in humans to cause different types of bone/osteo-genic disorders such as craniostenosis syndromes, chondroplasia syndromes, and syndromes with deregulated phosphate metabolism.[3]

It is reported that in addition to their role in bone development and genetic diseases, FGF signaling plays a significant role in the maintenance of adult bone fracture healing and homeostasis.[4]

Understanding the underlying molecular mechanism for the role of FGF/FGFR in the regulation of bone development and disease will ultimately lead to better prevention and treatment of FGF signaling related bone deformities and disorders.[3]

It was found that more than 97% of achondroplasia individuals DNA studied had mutation in FGFR3 gene at a single site (1138). This represented the most mutable single nucleotide reported in the human genome.[4],[5],[6] With this literature background, the present study is designed and planned.

Aims and objectives

To evaluate the role of gene FGFR3 in individuals with retrognathic maxilla by PCR technique at molecular level and evaluate significance of the same.

Research hypothesis

Maxillary retrognathism is influenced by FGFR3 gene.

 Methodology



This is an institution based fundamental genomic research study. Institutional Ethics committee approval was obtained and a total of 62 individuals (age group of 18 to 32 years) who were willing to take part in the study were selected from lateral cephalometric measurements of N perpendicular to point A and the length of maxillary base represented by Anterior Nasal Spine to Posterior Nasal Spine (ANS-PNS).

Inclusion criteria

Individuals willing to take part in the study voluntarilyIndividuals clinically having retrognathic maxillaIndividuals with family history, where either a sibling or one of the parents had similar condition.

Exclusion criteria

Individuals having cleft/other craniofacial defectsIndividuals with H/O trauma either during or after birthIndividuals with H/O facial surgical treatment or orthodontic treatmentIndividuals with gross facial asymmetry and multiple missing teethIndividuals who are not willing to be part of the study

After obtaining clearance from Institutional Ethics Committee, each individual's informed written consent was obtained. Lateral cephalograms were made under standardized conditions for each individuals using Planmeca PM 2002 CC Proline (Planmeca, Finland) radiography machine. Each of the cephalograms was traced by the investigator up to an accuracy of 0.5 mm and 0.5 degrees. Cephalometric values of N-A (IIHP) and ANS-PNS were considered for confirming maxillary retrognathism [Figure 1].{Figure 1}

The male individuals had mean value of –5.416 mm (±1.889) for the cephalometric measurement of N-A (IIHP). The mean value for the measurement of ANS-PNS was 51.65 mm (±3.714). Female individuals had a mean value of –5.3125 mm (±1.899) for N-A (IIHP), and for ANS-PNS, the mean was 49.515 mm (±4.180).

Gene analysis

Blood samples were drawn from each of the consented individuals and were subjected to genetic evaluation.

DNA isolation

HiPura™ forensic sample genomic DNA purification kit was used to extract genomic DNA using 200 μl of blood mixed with 300 μl of lysis solution AL, 20 μl proteinase K (20mg/ml), and 20 μl of IMDTT in a micro-centrifuge tube. The mix is thoroughly mixed by pulse-vortexing for 10–15 s and incubated at 55° C for 1 hour; 300 μl of lysis solution C1 was added and again mixed thoroughly for 10–15 s and incubated at 70° C for 10 min. Then the mix was centrifuged at 12000 × g for 1 min at room temperature. The supernatant was transferred into another collection tube and mixed with 300 μl of ethanol (96–100%) and transferred to Hi Elute miniprep spin column placed in a 2 ml collection tube and centrifuged at 600 × g for 1 minute. The flow through was discarded, 700 μl wash solution was added, and centrifuged at 1200 × g for 1 minute. The DNA was eluted by adding 100 μl of elution buffer to the mini spin column placed in a fresh micro centrifuge tube and collected by centrifuging the column at 6500 × g for 1 min.

Polymerase chain reaction amplification

Standardization of PCR conditions for FGFR3 forward and reverse primers was carried out using a programmable gradient Thermo cycler (Corbett). The following sequences of primers were used FGFR3 F-5′-AGGAGCTGGTGGAGGCTGA-3′ and FGFR3 R-5′ GGAGATCTTGTGCACGGTGG-3′.

PCR was carried out in 5.0 μl of template DNA. IX assay buffer (10 mM Tris-HCl, Ph 9.0, 1.5 mMMgCl2, 50 mMKCl, 0.01% Gelatin), 100 μl of each of the four dNTP's, 10 picomoles of forward and reverse primers and 1.25 U of Taq DNA polymerase (Hi media) [Table 1]. Using the gradient function of the PCR machine, a gradient of 60°C to 68°C was set for determining the optimal annealing temperature of FGFR3 F/R primers. The annealing temperature of 68° C was considered for further evaluation, as a solid single band was observed at that annealing temperature [Table 2].{Table 1}{Table 2}

An initial denaturation step at 94° C for 12 min followed by 40 cycles of 30 s at 94° C, 30 s at 68° C, 30 s at 72° C, and a final elongation at 72° C for 10 min was used as the optimized PCR protocol [Table 3].{Table 3}

PCR products were detected using agarose gel electrophoresis 2.5% (w/v) agarose gel was prepared in IX TAE buffer. The molten gel was cooled to below 65°C and mixed with ethidium bromide to a final concentration of 0.5 μg/ml. It was then poured to gel mould and allowed to cool. 10 μl of products were mixed with 4 μl of 6X loading buffer, and then loaded in to the wells of agarose gel. Gene ruler 50 bp DNA ladder (thermo scientific) was used as a molecular weight marker. Gel electrophoresis was performed at 100–120 V, and the bands were visualized under UV trans-illuminator.

Gene sequencing

The PCR products were gel purified, and they were sequenced to study the single-nucleotide polymorphisms (SNPs) responsible for the malformation (compared with the previous studies).

Sequencing was performed from Sanger's method. The purified products were subjected to bio applied biosystem (ABI) incorporated kit, and the genetic analyzer was used to analyze the data.

Steps followed

The genomic DNA was extracted using gDNA extraction kit (RKN09/10)Primers were designed and synthesized for the Gene FGFR3PCR was standardized, and products were gel purifiedThe PCR products were sequenced and analyzed for SNP.

 Results



The most commonly occurring SNPs in FGFR 3 gene has been identified in relation to 1138 in exon 10 of the gene FGFR3. These mutations result in particular amino acid substitution (G380R). The mutation in nucleotide 1138 of gene FGFR 3 has been reported to be among the highly mutable single nucleotides occurring in humans.[5] In this study, the mutations SNPs at nucleotide 1138 were analyzed in the selected individuals with retrognathic maxilla.

The targeted SNPs, at 1138 in exon 10 of the gene FGFR3 were not identified in the analyzed blood samples collected from the selected individuals in our study. The absence of these SNPs suggested that the most commonly occurring SNPs in FGFR3 gene (G1138A and G1138C in exon 10) cannot be held responsible for retrognathic maxilla.

PCR products were then sequenced to evaluate the presence of SNPs between the levels of 150-200 bp. No significance findings could be observed [Figure 2]. Further sequencing of the full gene was considered, and the entire process of gene analysis including primer designing and synthesis, PCR amplification and sequencing were repeated before subjecting to full sequencing of the gene.{Figure 2}

The sequencing of full gene revealed the presence of 2 mutations which are registered at NCBI with following accession numbers as specified above

[Figure 3] and [Table 4].{Figure 3}{Table 4}

 Discussion



Appropriate diagnosis of the etiology is very important for the successful treatment of any orthodontic problem. It is now understood that both genetics and environmental factors play a significant role in the etiology of skeletal defects. Effects of genetics on the dento-facial characteristics have been stated in recent studies in genetic science.[6]

Skeletal malocclusion is one of the areas where the role of genetics has been studied extensively as one of the major etiological factors. The concept that the facial form is predominantly a product of the person's hereditary has been concurred, and many studies have reported facial appearance to have a familial tendency.[6],[7]

The role of genetic factors and their interactions with environmental factors need to be assessed, and how it affects the facial growth is of crucial importance in orthodontic clinical practice.[8] It is now established that the growth and development of facial skeletal structures are under the control of genetic as well as environmental factors. Recently, there has been significant progress in the field of genetics and its application in clinical orthodontics. Although most of the dento-facial and cranio facial anomalies are of polygenic nature, the human genome project has made it possible to identify the inherited growth and development related dento-facial conditions. However, more studies are suggested to clearly identify the genes responsible for specific variability in facial skeletal characteristics. With the enormous progress in the science, the genetic correction of these dento-facial anomalies and malocclusions may become the order of the day in the future.[6]

The relative influence of genetics and environmental factors in the etiology of malocclusion has been a matter for discussion, debate, and controversy in the orthodontic literature. This paper reviews the literature and summarizes the evidence for the influence of genetics in dental anomalies and malocclusion. While phenotype is inevitably the result of both genetic and environmental factors, there is irrefutable evidence for a significant genetic influence in many dental and occlusal variables. The influence of genetics, however, varies according to the trait under consideration and in general remains poorly understood. Some insight into the genetic mechanisms involved in craniofacial morphogenesis at the molecular level in the embryo assists our appreciation of the role of genetics not only in the etiology of craniofacial abnormalities but also in the regulation of maxillary, mandibular, and tooth morphology. In polygenic multi-factorial systems, there is an additional factor, environmental modification. Such is the nature of the etiology of many craniofacial malformations and of malocclusion.[5],[9]

With the recent advances in developmental genetics, craniofacial biology is on the threshold of research and discovery that has already affected scientific understanding of normal and abnormal craniofacial growth. The human genome is now mapped in its entirety. Basic scientists and clinicians will now be able to search for and identify the specific gene factors that cause significant craniofacial dysmorphologies. It is now very clear that there are a number of genetically encoded regulatory factors that have profound effects on the morphogenesis and pre-natal development of craniofacial complex.[10]

Growth factor such as FGF regulates the proliferation and migration of osteoblastic cells and many other varieties of cell types. These factors produce their effects in target cells by signaling through the tyrosine kinase receptors on the cell surface (FGFR). However, the exact mechanism underlying their action has not been defined clearly.[3] The FGFs produce their effects in target cells by diseases such as chondroplasia syndromes, cranio stenosis syndromes, and syndromes with deregulated phosphate metabolism have been linked to mutations in FGFs and FGFRs.[4] Research has shown that the FGFR3 protein has a major role in the development and maintenance of bone and brain tissue and that the gene limits the formation of bone from cartilage and thereby regulates bone growth.

Hypochondroplasia and thanatophoric dysplasia are 2 skeletal dysplasias with resemblance to achondroplasia. Additionally, FGFR3 is reported to be the prime gene involved.[6]

The extensive literature is available regarding the genetic control of the dento-facial characteristics and their abnormalities. However, the evidences provided by most of these studies are not conclusive. It is reported that the mechanical modulations and hereditary factors have a common pathway of genes. Bioengineering, Quantitative Biology and Genetics have brought several new insight into growth and development that may lead to innovative ideas for treatments of dental malocclusion, dento-facial and craniofacial deformities.[11]

The present study is an institution based fundamental research involving the analysis of gene FGFR3 in selected individuals with retrognathic maxilla. The gene analysis involved collection of fresh blood samples, DNA extraction and followed with PCR analysis, and amplification using the specifically designed forward and reverse primers. The PCR products were sequenced to evaluate the responsible SNPs.

The novel SNPs found in this study belong to a female individual having severe retrognathic maxilla. She belongs to a socioeconomically backward class having strong genetic and environmental influences. The understanding of the interaction of genetic and environmental factors that influence the treatment response of our patient is essential to the practice of orthodontics.[12],[13] The study on influence of genetic and environmental factors on growth and development of dento-facial complex reported that though the control of growth and development of craniofacial complex is multifactorial in nature, the dominant role is played by the genetic factors.[14]

Significant improvements in understanding the genetic basis of craniofacial development have taken place in the last 2 decades. To a large extent, this has helped to identify the gene variants associated with various dento-facial deformities. The fundamental concepts of orthodontic treatments have undergone significant changes resulting in better emphasis on precision orthodontics.[15],[16]

The present study has been successful in identifying 2 novel mutations in the gene FGFR3 in individuals with maxillary retrognathism. Further study is being contemplated with these mutations in focus in individuals having retrognathic maxilla with strong genetic background and possible influence of environmental factors. Studies are required to conclusively establish that these mutations are indeed responsible for the maxillary morphology in different racial populations. The exact nature of the effect of these mutations also could be studied as also the role of FGFR3 genes on other skeletal structures. The role of other genes in the morphology and the facial structures also need to be analyzed for conclusive evidences.

 Summary and Conclusions



This is an institution based fundamental genomic research that involved the analysis of gene FGFR3 in selected individuals with maxillary retrognathism. The gene analysis involved collection of fresh blood samples, DNA extraction, and PCR analysis using the specifically designed forward and reverse primers. The PCR products were amplified and subjected to electrophoresis followed by detail sequencing to evaluate the responsible SNPs.

The targeted SNPs, G1138A, and G1138C in exon 10 of the FGFR3 gene were not identified in the analyzed blood samples collected from the selected individuals in this study.

Further sequencing of the full gene was considered, and the entire process of gene analysis including primer designing and synthesis, PCR amplification, and evaluation were repeated before subjecting to full sequencing of the gene. On evaluation by full sequencing of the gene revealed the presence following 2 novel mutations, Exon 3: A213G and Exon 3: A223A/G.

These novel SNPs found in this study belong to a female individual having severe retrognathic maxilla which indicates that the gene FGFR3 had a significant influence over the maxillary morphology in that individual. The genetic–environmental interactions might have had significant role in the expression of retrognathic maxilla.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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