Deepak C, Ramanathan A. MSX1 Gene Mutations in South Indian Population - A Genetic Research. Biomed Pharmacol J 2017;10(3).
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MSX1 Gene Mutations in South Indian Population - A Genetic Research

C. Deepak1 and Arvind Ramanathan2

1Department of Orthodontics, Bharath University Chennai, India

2Sree Balaji Dental College and Hospital Bharath University Chennai, India.

Corresponding Author E-mail: drcdeepak1307@gmail.com

DOI : http://dx.doi.org/10.13005/bpj/1207

Abstract:

To understand the role of a homeobox  gene (MSX1)  in South  Indian non-Syndromic cleft palate patients. A cross sectional study was made   of non-Syndromic cleft lip and palate patients. The study was conducted at  an Orthodontic Department of a Post graduate  teaching hospital. Twenty five  patients (South Indian ) were included in the study,the  inclusion criteria consisted of ,non-syndromic oral clefting, cl/p,and absence of a tooth or teeth ( partial tooth agenesis). Clinical examination Case Summary,  and radiographic evidence of missing teeth were taken .Blood samples were taken after a written consent, and were collected in 1.5 ml polypropylene microfuge tubes , transferred to the genetic  laboratory in a box of  dry ice. Genomic DNA was isolated for all patients and controls and exon 1 site (MSX1) was sent for polymerase chain reaction  amplification. The PCR (polymerase chain reaction) product was further sent  for  restriction enzyme analysis with 0.5 units of MboII. A Mutations search done in the exon 1 region of  the MSX1 gene was  reported in a study done by Vandenboogard et al.15, a dutch family showing non –syndromic oral  facial cleft lip and palate , this research study was done to find  if any  mutation of a homeobox gene (MSX1 exon 1 region) could have been  involved in the  Indian non-syndromic oral clefting. There was a perfect digestion of  the enzyme, thus confirming that there was a  absence of mutation in all the patient samples. Conclusion: The finding of  absence of mutation in all  the selected patient samples correlated with those reported for a predominately Caucasian  population (Lidral et al.,1998),but they not correrate with  those reported from a study  of a Dutch Family(vanden Boogerd et al., 2000).The findings from this study are very  significant as they establish that a mutation of MSX1 gene could have a diverse genetic involvement .As this study was done in the coding region of the selected gene it  can be further expanded to include all the non-coding regions.

Keywords:

South Indian  Non-Syndromic clefting; Homeobox gene; MSX1 exon 1  Gene; Poly merase chain reaction; Mutations

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Introduction

Cleft Lip and  palate is considered as , among the most common birth defects to affect mankind. The Children affected  with this,  are handicapped, for breast feeding, defective speech altered swallowing patterns, frequent  naso-bronchial infection, growth and development of maxillary arch defects  and overall  individual  facial development, affecting the personality of the affected  person.1,2

Non-Syndromic  Cleft  Lip and Palate

The most commonly proposed  genetic model of a non-syndromic Cleft lip and Palate  is that of multifactorial threshold inheritance by (Carter.C.O 1969)4 . According to their model the occurrence of the disorder depended upon the cumulative  effects of several of  minor abnormal genes (polygenes) and also environmental factors. The accumulation of these minor and environmental  factors is tolerated by the developing fetus only  to a particular threshold point and beyond that there was a risk for malformation, the proposed (MF/T) model.

Homeo Box Genes

Recent research in the field of genetics  has proved that there are an existence of a very large pool of genes, some of which seem to be highly conserved, and  possibly responsible for coding of transcription factors and are  involved directly in the regulation of down stream  genes, known as  homeobox genes.3

The word homeobox was originally found  in the homeotic genes of the fruitfly` Drosophila Melanogaster’, they are mainly responsible for specific  segment identity in the developing fruit fly. Homeotic genes as they are known, usually exhibit a specific feature called co-linearity,and their spatial arrangement which is along the chromosome, seems to be in the same order as their patterns of expressions. Several mutations in these homeotic genes have lead to bizarre transformations where one segment of the fruit  fly has  been transformed into another segment.

MSX Genes (Muscle Segment Homeobox)

The MSX Genes found in  vertebrates, comprise of a small family of genes related to the drosophila fruit fly gene, muscle segment homeobox (MSH). The MSX genes are found  in some vertebrate specific tissues including the neural crest, bone and teeth and cranial sensory placodes. There are two  classes of MSX genes (muscle segment homeobox), the MSX1 and the MSX2 gene. A third class of MSX gene the (MSX3) has been identified  in the mouse.3

Direct evidence involving the function of MSX genes (muscle segment homeobox) at the tissue level, relates to mutations. Experiments with MSX1 deleted mice failed to form some teeth and have shown several craniofacial abnormalities, which included the absence of the alveolar bones of both the jaws and several abnormalities in parietal,nasal membrane bones, frontal, malleus of the ear and cleft lip and palate.3

Contemporary research in cleft palate genetics is centered around western  countries, the  design and study  the probable  genetic etiology of Indian non-syndromic cleft lip & palate patients, was needed for  an indepth understanding of non syndromic genetics of clefting will help the clinicians to understand the development of cleft lip and palate, and also develop the  field of prenatal genetic counseling.

Aims and Objectives of the Study Were

The aim of study was to understand the role of Homeobox genes in the etiology  of  South  Indian Dravidian  non-syndromic CLP  (cleft lip and palate)patients.

Objectives of the Study Included

Amplify the chosen region of a Homeobox gene ( MSX1 exon 1)  from a South Indian cleft lip and palate patient sample.

Perform a detailed mutation search.

Discuss results and clinical significance of this study.

Materials and Methodology

Case Sample Size

Twenty non syndromic  cleft lip and  palate patients were randomly selected for this study, the patient consent was obtained prior to the study  ,  appropriate institutional review board approval was also  obtained. The study conformed to all the protocols as specified  in the Helsinki declaration for studies on humans.

The Patients were selected on the following criteria ;

South Indian Non-Syndromic cleft lip and palate patients .

A Clinical evidence showing some tooth agenesis.

Bilateral and unilateral cleft lip / palate patients were studied .

Clinical history  was taken ,case history and the  diagnosis was recorded on specific patient records . A written consent  for the study was taken from the patients.

Chemicals Used in the Study

Sodium Dode cyl Sulphate (SDS), Trizma base, Ethylene Diacetyl Tetra Acetate (EDTA), Glyceral, Formamide, Boric Acid and Dimethyl Suloxide (DMSO) were purchased  from Sigma Chemical Co, USA.The dideoxy-nucleotide triphosphates  were purchased  from Pharmacia Biotech .Agarose procured  from FML bio – products, the  USA. Molecular weight (Gene Ruler, 100bp) purchased  from Genei Pvt Ltd, India.

TAQ – Polymerase

TAQ – Polymerase was procured  from Perkin Elmer , and Pharmacia Biotec.

Primers

Primers  for  MSX1 gene Exon 1  FOR -5’CGG  ACA TGA CTT C -3’,MSX1 REV 5’- GCC TGG GTT CTG  ACT AC -3‘

Restriction Enzyme used

Mbo II

Sourced from   an E Coli Strain that carries cloned Mbo II gene of the  Moraxella bovis.

Reaction Buffer

10 nM MGCL2, 50 mM ,Dithiothreitol (ph 8.0 at 24°C) and incubated at 37°C.

Storage Conditions

10 nM TrisHcl (ph 7.8 ), EDTA, 2 nM Dithio Thresitol, 0.2 mM,  50% Glycerol ,Stored at -20°C.

5’….GAAGA7∆…..3’

3’….CTTCT8∆……….5’

Methodology

The venous  blood samples were taken  from south Indian non –syndromic cleft lip and palate patients and  were collected using  sterile 2 (ml)disposable syringes.

1.5 ml of venous blood taken  from each patient,  was collected transferred to 2 ml polypropylene micro fuge tubes .

The blood samples were stored in -20°c  freezer until the DNA isolation procedure.

Extraction of Genomic DNA of the Patients

Genomic  DNA can be taken by different methods. It is usually a simple procedure but great caution  should be taken to a make sure no  contamination of DNA occurs from any other sources  like contaminated glass wares, plastic tubes and specific  buffers used for the  DNA extraction. Polymerase Chain Reaction (PCR)  is a very effective and sensitive  method for  DNA amplification, small amounts could cause  contamination of the  DNA and would  be amplified ,and potentially  alter  the results.

Results of the Study

The genetic eitiology of non-syndromic cleft lip& palate has shown  the role of a major homeo box gene namely the  MSX1 in the development of Non –Syndromic cleft lip and palate 7,8 .The linkage of  genetic diseases is usually attributed to  defective gene (s),the presence of a specific mutation in a homeobox gene ( MSX1 gene)7 would  prove its role in  South Indian non-syndromic cleft lip and palate patients.

 Figure 1: The Genomic DNA isolates from blood samples. Figure 1: The Genomic DNA isolates from blood samples.

Click here to View figure

 

Genomic DNA were taken  from the Peripheral lymphocytes ,of 25  patient blood samples.

The Genomic  DNA was isolated by Proteinase K and SDS digestion, followed by Phenol/Isoamy alcohol extraction and .2M/95% Ethanol precipitate.

Further the precipitated genomic  DNA as  suspended in MQ and was  electrophoresed in a 0.8 agarose gel.

Figure 2: The PCR amplification of exon 1 region (MSX1 ) of the placental DNA samples. Figure 2: The PCR amplification of exon 1 region (MSX1) of the placental DNA samples.

Click here to View figure

 

The Placental DNA was used as control and was amplified with specific primers pairs .

Lane 1:  Molecular Weight marker (100 bp ruler )

Lane 2: 482 bp product of exon1 of MSX1  amplified from  DNA.

Figure 3: Restriction Analysis of EXON1 (MSX1) PCR product of DNA samples. Figure 3: Restriction  Analysis of EXON1 (MSX1) PCR product of  DNA samples.

Click here to View figure

 

15 µl  of PCR product of EXON1 (MSX1) of  DNA was digested using  2 units of Mbo11 in a 50 µl  at 37 °c . The next  day ,DNA from  the reaction was precipitated. by addition of 2.5 volumes of  0.1M Sodium Acetate. The  DNA was suspended in 10µl Mq and left in  2% agarose gel. The restriction digestion resulted in two products of sizes 322 bpand 160 bp respectively.

The  band corresponding to the  region between 300 bp and 400 bp marker and the  band corresponding to the region between 200  bp and 100 bp marker confirms a perfect digestion, and  the confirms  result.

Figure 4: The PCR amplification of exon 1 region of MSX1 of the patients. Figure 4: The PCR amplification of exon 1 region of MSX1  of the patients.

Click here to View figure

 

The  DNA were amplified using  specific primer pairs , and an aliquot of 10µl was electrophoresed in 2% agarose gel .

Lane 1: MW (Molecular Weight marker 100 bp ruler )

Lane 2-16 : 482 bp product of  Exon1 was amplified from the  peripheral  DNA.

Figure 5: Restriction enzyme Digestion Analysis EXON1 PCR product of DNA sample. Figure 5: Restriction enzyme  Digestion Analysis  EXON1 PCR product of DNA sample.

 

Click here to View figure

 

A 10 µl aliquot of PCR product of EXON1  MSX1 from the   DNA  isolated from CL/P patients  was reacted  with 2 units of MboII, in a 20 µl reaction, at 37 °c overnight.

A band corresponding to the regions  between 300 bp and 400 bp markers  confirmed the perfect digestion ,and hence the  product. However  since the band corresponding to the regions  between 200 bp and 100 bp markers is not clearly  visible in this picture,  because of a  higher DNA  concentration.

Lane 1: Molecular Weight Marker (100 bp ruler )

Lane 2: Undigested  ( Control) placental DNA

Lane 3-17: The digested patient CL/P samples.

Discussion

Non-Syndromic cleft lip  and palate is considered a common congenital anomaly with  medical,  social , phycological and economic ramifications.7

Non-syndromic cleft lip and palate has always been  thought to have a multifactorial etiology . It has been reported in literature that with  a significant increase in both  Dental and Dermatoglyphic asymmetries  within individuals with cleft lip/palate.

Recent studies have given a  direct evidence to the role of the MSX1 gene  (earlier  known as the Hox1), in the normal development and  formation of craniofacial structures including  the  tooth formation3.

The Homeobox genes are   considered  as the master genes , controlling ,induction , patterning, and programmed cell death  during  the  development of the craniofacial complex.3

In a systematic review done by M. Phan et al, where the authors thoroughly investigated the contemporary literature in an attempt to identify the genomic loci and genes which contributed to syndromic or non syndromic clefting and tooth agenesis . A total of 26 candidate genes were found of which MSX1 was one candidate gene .10

Those of  special interest on development of craniofacial region include the MSX1,MSX2,OTX,GSC,and Shh.5,6,7,12

Genetic Mutation in MSX1 gene for South Indian population was reported by VekannaS Prasad and Venkatesh Shivani16. The authors reported a mutation (414 to T).

Our study of EXON 1 region of MSX1 gene did not reveal any mutation of the clp samples.
This establishes the genetic diversity of MSX1 mutation.

Furthermore studies may  be designed to search for mutations in the non coding regions of MSX1 gene.

The exogenous application of TGFB3 as part of  anti-Scarring therapy following surgical correction of the cleft , may be beneficial to those individuals  11 ,13,14. The study  done on a  Dutch family,reported by (VandenBoogard,et al,2000)15 with  mutation in  12 affected members in a family, prompted an interest to conduct a similar study in the context of South Indian Non-Syndromic patients, aimed to understand the genetic abnormality in such cases.

A positive finding in the form of a mutation would help to understand a genetic abnormality in the  Indian CLP patients. Such a finding is very important to understand the development of clefting, and for therapeutic possibilities in these unfortunate patients.

Conflict of Interest

There is no conflict of interest.

Funding Source

The Authors would like to thank Bharath University for the Funding of the study.

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