RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,KARNATAKA
BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the candidate and address(In block letters) / Dr. SHEETAL.B.S.
POST GRADUATE STUDENT , DEPARTMENT OF ORTHODONTICS AND DENTOFACIAL ORTHOPEDICS, A.M.E’S DENTAL COLLEGE AND HOSPITAL,BEJENGERE ROAD, RAICHUR. KARNATAKA-5804103.
2. / Name of the Institution / A.M.E’S DENTAL COLLEGE AND HOSPITAL, BEJENGERE ROAD, RAICHUR. KARNATAKA-5804103.
3. / Course of the study and subject / MASTER OF DENTAL SURGERY (MDS) IN ORTHODONTICS AND DENTOFACIAL ORTHOPEDICS.
4. / Date of admission to course / 15-05-2012
5. / Title of the topic:
SNP evaluation 799G>T variant of MSX1 gene in nonsyndromic oralcleft patients of North Karnataka by direct sequencing.
6. / Brief resume of the intended work
6.1 Need for the study:
Clefts of the lip and/or palate are among the most common birth defects world wide. It has a wide geographic distribution with average prevalence ranging from 1/300to 1/2500 live births. Approximately 2/3rd cases are not accompanied by other anamolies are called nonsyndromic.1 Nonsyndromic oralclefts is a complex trait determined by multiple interacting genetic and environmental factors.
Many genetic linkage and association analyses have provided evidence to support the involvement of several genes2 and chromosome regions in oralclefts. Among them loci at 1q32[IRF6],2p13[TGFa]4p16[MSX1],6p23-25,14q24[TGFb3],17q21[RARA],and 19q13[BCL3,TGB1]. The multiple genome scans and subsequent metaanalysis have significantly identified the important roles of MSX1 and IRF6.3
MSX1[muscle segment homeobox] gene is a member of MSX family,maps to 4p16.1 loci and spans 4.05kb. It has 2exons and 1intron.3 It plays a crucial role in programming of craniofacial morphogenesis, in the development of teeth and has been proposed to direct terminal cell differentiation.4
The knockout mice studies with MSX1 deletion have shown the development of oralclefts.4 Mutations of MSX1in the aetiology of oralclefts by sequencing is reported.3
The mutation and polymorphism of MSX1 have been shown to be diverse. One of the variation of MSX1is 799G>T.4
Hence this study with the use of Polymerase chain reaction followed by sequencing helps in understanding the aetiology of oralclefts so as to predict its occurrence and also to target at the molecular level for the correction of such problems in the future.
6.2 Review of literature
Tongkobpetch. S,Siriwa .P and Shotelersuk5 .V[2006] conducted mutation analysis of all coding regions of MSX1gene of 100Thai oralcleft patients and 162 control individuals of Thai population was done. Genomic DNA isolated from peripheral blood, amplified by PCR and direct sequencing done. A total of 8 variants identified. 6 were in the coding regions, including 4 nonsynonmous changes .101C>G[A34G],440C>A[P147Q] from exon1 and 799G>T[G267C],832C>T[P278S] from exon2. Mutations of MSX1 found in2% cases of oralclefts.
Jezewski .PA, Vieiera .I, Nishimura .C,et al3 [2003] conducted a genetic study comprising of complete sequencing of MSX1 gene in917oralcleft patients and 500 control subjects from European, Asian and native South American ancestry done.48 variant sites that included 3 small deletions, one rare single base insertion and 43 SNP were identified and mapped. These 43SNP’s comprised of 27 transitions and 16 transversions.At position 799,330 C>T,G110G a synonymous coding SNP was found significantly associated with Asian population.
Singh .VP and Dinesh .R8 [2012] a genetic study of 25 oralcleft patients and 25 controls subjects from India was done to test the gene variant MSX1 799[G>T] involved in the etiology of oralclefts.Genomic DNA extracted from blood sample. PCR was performed and digestion products were evaluated. There was a positive correlation between MSX1[799G>T] and oralclefts.
Butali .A,Mossey. PA,Jeweski .PA,et al 6[2011] conducted a study to investigate the role of gene variant interaction in the etiology of Oralclefts in Nigeria was done. DNA isolated from saliva of 118oralcleft cases with a family history of clefts and 166control subjects without history of clefts. Genotype association studies and direct sequencing of the cleft candidate genes:MSX1, IRF6, FOXE1,FGFR1,FGFR2, BMP4, MAFB. ABCA4, PAX7, and VAX1 and chromosome 8q region done. A missense mutation A34G in MSX1 was observed in nine cases and four hap map controls. The replicatrion of a mutation suggests a role for the MSX1 A34G variant in the etiology of oralclefts.
Ingersoll .RG,Hetmanski .J, et al7 [2010] conducted a study in four populations from Maryland, Korea, Singapore and Taiwan to know the association between genes and the oralclefts. Total 381 oralclefts cases selected. SNP selected for fine mapping in 2Mb region surrounding MSX1 on chromosome 4p16. 429 unique SNP selected. DNA samples prepared genotyped for SNP markers. Among 393 SNPs genotyped, 62 had an minor allele frequency score between 0.5 and 5% in one population. 54 SNP with minor allele frequency </= 0.05 in one or more population. Both individual marker and haplotypes of 2 to5 SNP’s showed several regions yielding statistical evidence for linkage and disequilibrium. This suggests MSX1, STK32B and EVC influence the risk of oralclefts.
6.3 Objectives of the study
1. To investigate the role of MSX1 single nucleotide polymorphism [SNP] 799G>T variant in the etiology of oralclefts patients in North Karnataka by sequencing 484 base pairs in the region of 799
2. To investigate the role of other single nucleotide polymorphism[SNP] in the etiology of oralcleft patients of North Karnataka in the region of 799G>T by direct sequencing.
7. / Materials and Methods
7.1 Source of data
This genetic study will be conducted on the people of North Karnataka residency having non- syndromic cleft lip with or without cleft palate reported to A.M.E’s DENTAL COLLEGE AND HOSPITAL, RAICHUR.
7.2 Method of selection of data
INCLUSION CRITERIA OF PATIENTS :
1. A Patient having non syndromic cleft lip with or without cleft palate.
2. Patient having North Karnataka residency history of his and his ancestors.
EXCLUSION CRITERIA OF PATIENTS :
1. Patient not having North Karnataka residency history of his and his ancestors.
2. Patient having any positive family history of syndromes related to cleft lip and palate.
3. Patients having any positive family history of infection to mother during carriage phase.
4. Patient’s mother having any positive history of drug medication that can have teratogenic effect as cleft lip with or without palate.
5. Patient having any history of blood dyscriasis.
INCLUSION CRITERIA OF CONTROLS :
1. Subjects having North Karnataka residency history of his and his ancestors.
2. Subjects should not possess family history of cleft lip with or without palate and any syndromes related to cleft lip with or without palate.
EXCLUSION CRITERIA OF CONTROLS :
1. Subjects not having North Karnataka residency history of his and his ancestors.
2. Subjects that possess family history of cleft lip with or without palate and any syndromes related to cleft lip with or without palate.
SAMPLE SIZE AND DESIGN:
Study will consist of 15 Non syndromic cleft lip with or without cleft palate Patients and 15 Control subjects.
METHODOLOGY:
Informed written consent will be taken from the respective subject prior to the study
A study will be conducted on 15 Non syndromic oralcleft Patients and 15 Control subjects. As preoperative evaluations, all patients will be screened for the presence of associated anomalies or syndromes , and only those determined to have non syndromic cleft lip with or without cleft palate will be included in the study. Venous blood samples of 2ml will be obtained from both patients and controls. Genomic DNA will be extracted from the blood of the patients and control subjects by using phenol chloroform extraction protocol1 . Once the isolated genomic DNA precipitate is obtained, it will be used as a template for the polymerase chain reaction test. Precipitated genomic DNA will be amplified using the polymerase chain reaction (PCR) with primers having the base pair sequence G267C-R -“CAGGAAACAGCTATGACCCTGGAAGGGGCCAGAGGCTC” 2F - “GGCTGATCATGCTCCAATGC”, giving a PCR product of 484 bases pairs (bp)5. The PCR product (484 [bp]) that will be obtained will be run on 1.5% agarose gel containing ethidium bromide to assess the initial amplified PCR products. Bidirectional sequencing of DNA of 484 base pairs will be done by automatic sequencer.
The data will be tabulated and calculated by using Chi-square test.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals?
Yes
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes
8. / List of references
1. Morkuniene A, Steponaviciute D, Ambrozaityte L,Utkus A, Linkeviciane L, Kucinskas V. TGFA,TGFB3, GABRB3, RARA and BCL3 loci associated with nonsyndromic orofacial clefts? A Lithuanian study. Biologija 2007;18: 1–6.2.
2. Vieira AR, Avila JR, Daack-Hirsch S, Dragan E, Felix TM, Rahimov F, et al Medical sequencing of candidate genes for nonsyndromic cleft lip and palate. Journal pgen PLoS Genet 2005;1(6):651–659.
3. Jezewski PA, Vieira AR, Nishimura C, Ludwig B, Johnson M,’Brien SEO, et al. Complete sequencing shows a role for MSX1 in non-syndromiccleft lip and palate. J Med Genet 2003;40:399–407.
4. Lace B, Vasijeva I, Dundure I, Barkane I, Akota I,Krumina A. Mutation analysis of the MSX1 gene exons and intron in patients a nonsyndromic cleftlip and palate.. Stomatologija 2006;8: 21–24.
5. Tongkobpetch S, Siriwan P, Shotelersuk V. MSX1mutations contribute to non syndromic cleft lip in a Thai population. Jr Hum Genet 2006;51:671–676.
6. Butali A, Mossey PA,Adeyemo WL, Jezewski PA,Onwuamah CK,Ogunlewe MO,et al. Genetic studies in the Nigerian population implicate a MSX1 mutation in complex oral facial clefting disorders. Cleft Palate Craniofacial J,2011; 48(6):646-653.
7. Ingersoll RG, Hetmanski J, Park JW,Fallin MD, McIntash I,Chou YHW,et al. Association between genes of chromosome 4p16 and nonsyndromic oral cleft in four population. .Eur J. Human Genetics 2010;18:726-732
8. Singh VP, Dinesh R, Association of MSX1 799G>T variant with nonsyndromic cleftlip/palate in south Indian adolescents patients. .International journal of Paediatric dentistry 2012; 22(3): 228-231.