The Analysis Of IRF6 Gene Of Health Persons And Patients Of Cleft Lip With Or Without Palate In Guangdong Province

BACKGROUND Congenital cleft lip and palate (CL/P) is one of the most common birth defects with the birth prevalence being highest in China(16.63/10000 live births).The disease could not only affect the appearance, nutrition, language, hearing, developmental and other physiological aspects of patients, but also has an impact on the psychological health of patients and gives a burden on society and family.Depending on whether the body associates with other congenital malformations, congenital cleft lip and palate often divides into syndromic cleft lip with or without palate (SCL/P) and non-syndromic cleft lip with or without palate (NSCL/P) . As a common birth defect, NSCL/P is very likely that both genetic and environmental factors contribute to the malformation.Genetic mechanism involves multiple genes, and its inheritance pattern is complicated. Searching for susceptible genes of CL/P could contribute to understanding of pathogenesis of CL/P and find out the genes that play a key role during development. All the work is an important consideration in genetic counseling.Interferon regulatory factor–6 (IRF6) gene is considered to be crucial to predisposition of CL/P. Irf6 is a key determinant of the keratinocyte proliferation-differentiation switch. Mutations in IRF6 underlie Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS), which are characterized by varying degrees of cleft lip, cleft palate, lip pits, skin folds, syndactyly and oral adhesions. Variation at IRF6 was responsible for 12 percent of the genetic contribution to cleft lip or palate and tripled the risk of recurrence in families that had already had one affected child.PURPOSE In order to find out the variations of IRF6 gene between normal human and CL/P groups in Guangdong population, and discuss the association between IRF6 SNPs and NSCL/P.METHODS1. DesignPart I: Sequence Analysis of the Complete Gene of IRF6 in Guangdong Province.Part II: The Analysis for the Coding Region of IRF6 Gene of Cleft Lip and/or Palate in Guangdong Province.Part III: Association between IRF6 SNPs and non-syndromic Cleft Lip and/or Palate in Guangdong Province.2. Samples2.1 normal human/control: Peripheral blood were collected from 13 unrelated health donors, including 7male and 6 female, from Guangdong Province after their consent.2.2 CL/P patient groups: Peripheral blood were collected from 42 CL/P patients and their parents, including 1 three nuclear family of VWS syndrome, 40 NSCL/P patients in Guangzhou Women and Children's Medical Center during the first half of 2009.3. Methods3.1 DNA extraction: Genomic DNA kit.3.2 Fragments amplification: Polymerase Chain Reaction(PCR).3.3 Genotyping: directly sequencing of PCR products.3.4 Statistical analysis: Hard-Weinberg Equilibrium(H-W);Case-Control Comparisons;Fisher exact method; odd ratio(OR)and 95% CI.RESULTS 1. IRF6 gene of Guangdong population is 25016~25046bp, contains 10 exons of total 2215bp. Exons 1,2, 10, and part of exon 3,9 are non-coding region. Coding sequence is 1404bbp.2. Submitted and accepted two IRF6 genes of Guangdong population to Genbank: HQ875393 and JF346417.3. 64 SNPs of IRF6 gene were found from 13 normal human in Guangdong Provence, two of which were located in coding region: c.459G>T and c.820G>A.4. Three mutations were found in CL/P patients from Guangdong Provence: 459G>T and 820G>A were detected both in controls and unaffected members in family. There were no differences in genetic frequency of 459G>T and.820G>A between NSCL/P group and control group(P >0.05).1234C>T was detected in Van der Woude syndrome patients only, and the mutation has been reported in VWS patients previously.5. There were significant differences in genetic frequency of rs2235375,rs2236909,rs2294408,rs2073487 between NSCL/P group and control group(P=0.044).6. The analytical result still could not confirm that rs2235375 GG, rs2236909 TT, rs2294408 CC, rs2073487 AA were protective factors, and also could not consider rs2235375 CC, rs2236909 CC, rs2294408 TT, rs2073487 GG as risk factors yet.CONCLUSION1. Submitted and accepted two IRF6 genes of Guangdong population to Genbank: HQ875393 and JF3464172. 64 SNPs of IRF6 gene were found from 13 normal human in Guangdong Provence, two of which were located in coding region: c.459G>T and c.820G>A.3. Three mutations were found in CL/P patients from Guangdong Provence: 459G>T, 820G>A , and 1234C>T .4. The 1234C>T mutation in exon 9 of IRF6 gene may be one of the risk factors of VWS patients in Guangdong Province.5. There were significant differences in genetic frequency of rs2235375,rs2236909,rs2294408,rs2073487 between NSCL/P group and control group(P=0.044).6. The analytical result still could not confirm that protective factors or risk factors exist among the genotypes of 13 tagging SNPs.7. There were still some differences between the results and other experimental data reported, which may be due to non-random, small size samples. The analysis of larger sample size is needed.