{"id":16280,"date":"2017-09-25T11:48:50","date_gmt":"2017-09-25T11:48:50","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=16280"},"modified":"2020-04-24T11:04:25","modified_gmt":"2020-04-24T11:04:25","slug":"msx1-gene-mutations-in-south-indian-population-a-genetic-research","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol10no3\/msx1-gene-mutations-in-south-indian-population-a-genetic-research\/","title":{"rendered":"MSX1 Gene Mutations in South Indian Population – A Genetic Research"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Cleft Lip and\u00a0 palate is considered as , among the most common birth defects to affect mankind. The Children affected\u00a0 with this, \u00a0are handicapped, for breast feeding, defective speech altered swallowing patterns, frequent \u00a0naso-bronchial infection, growth and development of maxillary arch defects \u00a0and overall \u00a0individual\u00a0 facial development, affecting the personality of the affected\u00a0 person.1,2<\/sup><\/p>\n

Non-Syndromic\u00a0 Cleft\u00a0 Lip and Palate<\/strong><\/p>\n

The most commonly proposed\u00a0 genetic model of a non-syndromic Cleft lip and Palate \u00a0is that of multifactorial threshold inheritance by (Carter.C.O 1969).4<\/sup>\u00a0 According to their model the occurrence of the disorder depended upon the cumulative \u00a0effects of several of \u00a0minor abnormal genes (polygenes) and also environmental factors. The accumulation of these minor and environmental \u00a0factors is tolerated by the developing fetus only \u00a0to a particular threshold point and beyond that there was a risk for malformation, the proposed (MF\/T) model.<\/p>\n

Homeo Box Genes<\/strong><\/p>\n

Recent research in the field of genetics\u00a0 has proved that there are an existence of a very large pool of genes, some of which seem to be highly conserved, and \u00a0possibly responsible for coding of transcription factors and are \u00a0involved directly in the regulation of down stream \u00a0genes, known as \u00a0homeobox genes.3<\/sup><\/p>\n

The word homeobox was originally found\u00a0 in the homeotic genes of the fruitfly` Drosophila Melanogaster\u2019, they are mainly responsible for specific \u00a0segment 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\u00a0 fly has \u00a0been transformed into another segment.<\/p>\n

MSX Genes (Muscle Segment Homeobox)<\/strong><\/p>\n

The MSX Genes found in \u00a0vertebrates, comprise of a small family of genes related to the drosophila fruit fly gene, muscle segment homeobox (MSH). The MSX genes are found \u00a0in some vertebrate specific tissues including the neural crest, bone and teeth and cranial sensory placodes. There are two \u00a0classes of MSX genes (muscle segment homeobox), the MSX1 and the MSX2 gene. A third class of MSX gene the (MSX3) has been identified \u00a0in the mouse.3<\/sup><\/p>\n

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<\/sup><\/p>\n

Contemporary research in cleft palate genetics is centered around western\u00a0 countries, the \u00a0design and study \u00a0the probable \u00a0genetic etiology of Indian non-syndromic cleft lip & palate patients, was needed for\u00a0 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 \u00a0field of prenatal genetic counseling.<\/p>\n

Aims and Objectives of the Study Were<\/strong><\/p>\n

The aim of study was to understand the role of Homeobox genes in the etiology \u00a0of \u00a0South \u00a0Indian Dravidian \u00a0non-syndromic CLP\u00a0 (cleft lip and palate)patients.<\/p>\n

Objectives of the Study Included<\/strong><\/p>\n

Amplify the chosen region of a Homeobox gene ( MSX1 exon 1) \u00a0from a South Indian cleft lip and palate patient sample.<\/p>\n

Perform a detailed mutation search.<\/p>\n

Discuss results and clinical significance of this study.<\/p>\n

Materials and Methodology<\/strong><\/p>\n

Case Sample Size<\/strong><\/p>\n

Twenty non syndromic \u00a0cleft lip and \u00a0palate patients were randomly selected for this study, the patient consent was obtained prior to the study \u00a0, \u00a0appropriate institutional review board approval was also \u00a0obtained. The study conformed to all the protocols as specified \u00a0in the Helsinki declaration for studies on humans.<\/p>\n

The Patients were selected on the following criteria ;<\/p>\n

South Indian Non-Syndromic cleft lip and palate patients .<\/p>\n

A Clinical evidence showing some tooth agenesis.<\/p>\n

Bilateral and unilateral cleft lip \/ palate patients were studied .<\/p>\n

Clinical history\u00a0 was taken ,case history and the\u00a0 diagnosis was recorded on specific patient records . A written consent \u00a0for the study was taken from the patients.<\/p>\n

Chemicals Used in the Study<\/strong><\/p>\n

Sodium Dode cyl Sulphate (SDS), Trizma base, Ethylene Diacetyl Tetra Acetate (EDTA), Glyceral, Formamide, Boric Acid and Dimethyl Suloxide (DMSO) were purchased\u00a0 from Sigma Chemical Co, USA.The dideoxy-nucleotide triphosphates\u00a0 were purchased\u00a0 from Pharmacia Biotech .Agarose procured \u00a0from FML bio – products, the \u00a0USA. Molecular weight (Gene Ruler, 100bp) purchased \u00a0from Genei Pvt Ltd, India.<\/p>\n

TAQ – Polymerase<\/p>\n

TAQ – Polymerase was procured\u00a0 from Perkin Elmer , and Pharmacia Biotec.<\/p>\n

Primers<\/p>\n

Primers\u00a0 for \u00a0MSX1 gene Exon 1\u00a0 FOR -5\u2019CGG \u00a0ACA TGA CTT C -3\u2019,MSX1 REV 5\u2019- GCC TGG GTT CTG \u00a0ACT AC -3\u2018<\/p>\n

Restriction Enzyme used<\/strong><\/p>\n

Mbo II<\/p>\n

Sourced from\u00a0\u00a0 an E Coli Strain that carries cloned Mbo II gene of the \u00a0Moraxella bovis.<\/p>\n

Reaction Buffer<\/strong><\/p>\n

10 nM MGCL2, 50 mM ,Dithiothreitol (ph 8.0 at 24\u00b0C) and incubated at 37\u00b0C.<\/p>\n

Storage Conditions<\/p>\n

10 nM TrisHcl (ph 7.8 ), EDTA, 2 nM Dithio Thresitol, 0.2 mM, \u00a050% Glycerol ,Stored at -20\u00b0C.<\/p>\n

5\u2019\u2026.GAAGA7\u2206\u2026..3\u2019<\/p>\n

3\u2019\u2026.CTTCT8\u2206\u2026\u2026\u2026.5\u2019<\/p>\n

Methodology <\/strong><\/p>\n

The venous \u00a0blood samples were taken \u00a0from south Indian non \u2013syndromic cleft lip and palate patients and\u00a0 were collected using \u00a0sterile 2 (ml)disposable syringes.<\/p>\n

1.5 ml of venous blood taken \u00a0from each patient, \u00a0was collected transferred to 2 ml polypropylene micro fuge tubes .<\/p>\n

The blood samples were stored in -20\u00b0c \u00a0freezer until the DNA isolation procedure.<\/p>\n

Extraction of Genomic DNA of the Patients<\/strong><\/p>\n

Genomic\u00a0 DNA can be taken by different methods. It is usually a simple procedure but great caution\u00a0 should be taken to a make sure no \u00a0contamination of DNA occurs from any other sources \u00a0like contaminated glass wares, plastic tubes and specific \u00a0buffers used for the\u00a0 DNA extraction. Polymerase Chain Reaction (PCR) \u00a0is a very effective and sensitive \u00a0method for\u00a0 DNA amplification, small amounts could cause\u00a0 contamination of the \u00a0DNA and would \u00a0be amplified ,and potentially\u00a0 alter \u00a0the results.<\/p>\n

Results of the Study<\/strong><\/p>\n

The genetic eitiology of non-syndromic cleft lip& palate has shown \u00a0the role of a major homeo box gene namely the \u00a0MSX1 in the development of Non \u2013Syndromic cleft lip and palate.7,8<\/sup>\u00a0The linkage of \u00a0genetic diseases is usually attributed to \u00a0defective gene (s),the presence of a specific mutation in a homeobox gene ( MSX1 gene)7<\/sup> would \u00a0prove its role in \u00a0South Indian non-syndromic cleft lip and palate patients.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1: The Genomic DNA isolates from blood samples.
\n<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Genomic DNA were taken \u00a0from the Peripheral lymphocytes ,of 25 \u00a0patient blood samples.<\/p>\n

The Genomic \u00a0DNA was isolated by Proteinase K and SDS digestion, followed by Phenol\/Isoamy alcohol extraction and .2M\/95% Ethanol precipitate.<\/p>\n

Further the precipitated genomic \u00a0DNA as\u00a0 suspended in MQ and was\u00a0 electrophoresed in a 0.8 agarose gel.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2: The PCR amplification of exon 1 region (MSX1) of the placental DNA samples.
\n<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The Placental DNA was used as control and was amplified with specific primers pairs .<\/p>\n

Lane 1: \u00a0Molecular Weight marker (100 bp ruler )<\/p>\n

Lane 2: 482 bp product of exon1 of MSX1\u00a0 amplified from \u00a0DNA.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: Restriction\u00a0 Analysis of EXON1 (MSX1) PCR product of \u00a0DNA samples.
\n<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

15 \u00b5l \u00a0of PCR product of EXON1 (MSX1) of\u00a0 DNA was digested using\u00a0 2 units of Mbo11 in a 50 \u00b5l \u00a0at 37 \u00b0c . The next \u00a0day ,DNA from \u00a0the reaction was precipitated. by addition of 2.5 volumes of\u00a0 0.1M Sodium Acetate. The \u00a0DNA was suspended in 10\u00b5l Mq and left in \u00a02% agarose gel. The restriction digestion resulted in two products of sizes 322 bpand 160 bp respectively.<\/p>\n

The\u00a0 band corresponding to the\u00a0 region between 300 bp and 400 bp marker and the \u00a0band corresponding to the region between 200\u00a0 bp and 100 bp marker confirms a perfect digestion, and\u00a0 the confirms \u00a0result.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 4: The PCR amplification of exon 1 region of MSX1 \u00a0of the patients.
\n<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The\u00a0 DNA were amplified using \u00a0specific primer pairs , and an aliquot of 10\u00b5l was electrophoresed in 2% agarose gel .<\/p>\n

Lane 1: MW (Molecular Weight marker 100 bp ruler )<\/p>\n

Lane 2-16 : 482 bp product of \u00a0Exon1 was amplified from the\u00a0 peripheral \u00a0DNA.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 5: Restriction enzyme\u00a0 Digestion Analysis \u00a0EXON1 PCR product of DNA sample.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

A 10 \u00b5l aliquot of PCR product of EXON1 \u00a0MSX1 from the \u00a0\u00a0DNA\u00a0 isolated from CL\/P patients \u00a0was reacted\u00a0 with 2 units of MboII, in a 20 \u00b5l reaction, at 37 \u00b0c overnight.<\/p>\n

A band corresponding to the regions\u00a0 between 300 bp and 400 bp markers \u00a0confirmed the perfect digestion ,and hence the \u00a0product. However \u00a0since the band corresponding to the regions\u00a0 between 200 bp and 100 bp markers is not clearly\u00a0 visible in this picture,\u00a0 because of a\u00a0 higher DNA \u00a0concentration.<\/p>\n

Lane 1: Molecular Weight Marker (100 bp ruler )<\/p>\n

Lane 2: Undigested \u00a0( Control) placental DNA<\/p>\n

Lane 3-17: The digested patient CL\/P samples.<\/p>\n

Discussion<\/strong><\/p>\n

Non-Syndromic cleft lip \u00a0and palate is considered a common congenital anomaly with \u00a0medical, \u00a0social , phycological and economic ramifications.7<\/sup><\/p>\n

Non-syndromic cleft lip and palate has always been \u00a0thought to have a multifactorial etiology . It has been reported in literature that with \u00a0a significant increase in both \u00a0Dental and Dermatoglyphic asymmetries \u00a0within individuals with cleft lip\/palate.<\/p>\n

Recent studies have given a \u00a0direct evidence to the role of the MSX1 gene \u00a0(earlier \u00a0known as the Hox1), in the normal development and \u00a0formation of craniofacial structures including \u00a0the \u00a0tooth formation.3<\/sup><\/p>\n

The Homeobox genes are \u00a0\u00a0considered \u00a0as the master genes , controlling ,induction , patterning, and programmed cell death \u00a0during \u00a0the\u00a0 development of the craniofacial complex.3<\/sup><\/p>\n

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<\/sup><\/p>\n

Those of \u00a0special interest on development of craniofacial region include the MSX1,MSX2,OTX,GSC,and Shh.5,6,7,12<\/sup><\/p>\n

Genetic Mutation in MSX1 gene for South Indian population was reported by VekannaS Prasad and Venkatesh Shivani.16\u00a0<\/sup>The authors reported a mutation (414 to T).<\/p>\n

Our study of EXON 1 region of MSX1 gene did not reveal any mutation of the clp samples.
\nThis establishes the genetic diversity of MSX1 mutation.<\/p>\n

Furthermore studies may\u00a0 be designed to search for mutations in the non coding regions of MSX1 gene.<\/p>\n

The exogenous application of TGFB3 as part of \u00a0anti-Scarring therapy following surgical correction of the cleft , may be beneficial to those individuals.11 ,13,14\u00a0<\/sup>The study \u00a0done on a \u00a0Dutch family,reported by (VandenBoogard,et al,2000)15<\/sup> with\u00a0 mutation in \u00a012 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.<\/p>\n

A positive finding in the form of a mutation would help to understand a genetic abnormality in the \u00a0Indian CLP patients. Such a finding is very important to understand the development of clefting, and for therapeutic possibilities in these unfortunate patients.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

There is no conflict of interest.<\/p>\n

Funding Source<\/strong><\/p>\n

The Authors would like to thank Bharath University for the Funding of the study.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Niswander J. D. A. Development `noise\u2019 and congenital malformation. Genetics Research.<\/em> 196;10:313.<\/li>\n
  2. \u00a0Niswander J. D. A.\u00a0 Oral clefts in the American Indian.\u00a0\u00a0Public Health Rep.<\/em> 1967;82:807.
    \n    CrossRef<\/a><\/li>\n
  3. Lidral A. C\u00a0 and\u00a0 Reising B. C. The Role of MSX1 in Human tooth agenesis. J Dent Res.<\/em> 2002;81(4):274-27.
    \n    CrossRef<\/a><\/li>\n
  4. Carter C. O. Genetics of Common Disorders.\u00a0Br.Med. Bul.<\/em> 1969;25:52.
    \n    CrossRef<\/a><\/li>\n
  5. Hu D.,Helms A. J. The role of Sonic hedgehog in normal and abnormal craniofacial morphogenesis. Development<\/em>. 1999;126:4873-4884.<\/li>\n
  6. Evans R. M. The steroid and thyroid hormone receptor super family. Sceince.<\/em> 1988;240;887-885.
    \n    CrossRef<\/a><\/li>\n
  7. Furgeson M. N. J.\u00a0 A hole in the head. Nature genetics<\/em>. 2000;24:330-331.
    \n    CrossRef<\/a><\/li>\n
  8. Lakshmi N. J. R.Genetic,cytogenetic and teratologic studies of cleft lip and cleft palate;Phd Thesis,University of Madras. 1988.<\/li>\n
  9. Lidral A. C.,Romitti\u00a0 P. A.,Basart A. M.\u00a0 Association of MSX1 and TGFB3 with non \u2013syndromic clefting in humans. Am J Hum .Genet.<\/em> 1998;63:557-568.
    \n    CrossRef<\/a><\/li>\n
  10. Phan M.,Conte F., Khandelwal K. D., Ockeloen C. W.,Bartzela T., Kleefstra T.,Van Bokhoven H. Rubini M.,\u00a0 Zhou H and Carels C. E. L. Tooth agenesis and orofacial clefting genetic brothers in arms ? Human Genet<\/em>. 2016;135(12):1299-1327.
    \n    CrossRef<\/a><\/li>\n
  11. Proetzel G.,Mackenzie R. The role of TGFB3 in the Development of craniofacial region. Nature Genetics.<\/em> 1996;11:409-414.
    \n    CrossRef<\/a><\/li>\n
  12. Satokata I.,MAAS R. Msx1 deficient mice exihibit cleft\u00a0 palate and abnormalities of abnormalities of craniofacial development. Nature genetics.<\/em> 1994;6:348-355.
    \n    CrossRef<\/a><\/li>\n
  13. Shah M.,Recombinant T. g FB3-A therapeutic possibility. J.Cell.Science.<\/em> 1995;108:985-1002.<\/li>\n
  14. Taya Y. Exogenous TGFB3 in palatal fusion defects. Development<\/em>. 1999;126;3869-3879.<\/li>\n
  15. Boogard V.,Dorland M. J. H.,Beemer F. A. M. MSX1 mutation is associated with orofacial clefting and tooth agenesis in Humans(letter). Nature Genet.<\/em> 2000;24:342-343.
    \n    CrossRef<\/a><\/li>\n
  16. Prasad S. V., Shivani V. Genetic significance of muscle segment homeo box1 gene in South Indian population for cleft lip and palate.\u00a0Indian Journal of Human genetics.<\/em> 2012;18(3):332-339.
    \n    CrossRef<\/a><\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    Introduction Cleft Lip and\u00a0 palate is considered as , among […]<\/p>\n","protected":false},"author":9,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[51],"tags":[],"class_list":["post-16280","post","type-post","status-publish","format-standard","hentry","category-vol10no3"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/16280","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/9"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=16280"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/16280\/revisions"}],"predecessor-version":[{"id":32728,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/16280\/revisions\/32728"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=16280"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=16280"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=16280"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}