Mutation Screening Of MSX1 And IRF6 In Patients Non-syndromic Cleft Lip With Or Without Cleft Palate And Functional Analysis Of Mutant Protein

ObjectiveOrofacial cleft is the most common congenital developmental defects in clinical and the incidence rate is about one-thousandth to two thousandths.Patinet with cleft lip and/or palate are present facial appearance, functional and psychological and other issues, these patients often require sequential therapy, including surgery, orthodontics, language training and psychological treatment, thereby producing a series of family and social problems. Therefore, the study of the etiology of cleft lip and palate mechanism, not only helps to understand the occurrence and development of cleft lip and palate fundamentally, but also give the cause of clinical diagnosis and genetic counseling of great help, has a very important social significance. Depending on whether the merger changes in other organs, cleft lip and/or palate can be divided into syndromic cleft lip with or without cleft palate ?SCL/P? and non-syndromic cleft lip and palate ?NSCL/P?. Non-syndromic cleft lip and palate accounted for 70% of the total, including simple cleft lip ?cleft lip, CL?, cleft palate ?cleft palate, CP?, and cleft lip with cleft palate ?cleft lip and palate, CLP?. Genes that cause SCL/P tend to be more clear, mostly single-gene genetic diseases, risk factors, rather than the relatively complex syndrome cleft lip and palate, cleft lip and palate Non syndrome can better understand the pathogenesis of cleft lip and palate.In this study, we collected non-syndromic cleft lip and palate patients in Shandong Province and genomic DNA was extracted from peripheral blood leukocytes. Using molecular genetics to screening gene mutation in MSXl and IRF6 gene. Looking for MSX1 and IRF6 gene mutation carriers, mutation genotype of bioinformatics analysis, to explore the relationship between MSX1 and IRF6 gene mutation and NSCL/P.MethordsAt first, we collected case-control population data and DNA sample. Standard clinical cases were collected:2013-2015, Second Hospital of Shandong University in cleft lip and palate treatment of patients choose meet the following conditions:1) in patients with cleft lip and palate; 2) is not associated with other organ system abnormalities. On the basis of the patient or guardian consent on inclusion as an object of study. Collect normal selection criteria:1) no history of cleft lip and palate; 2) other organs no abnormalities. In himself or guardian consent on the basis of inclusion in the study.Experiments include:1) extracting the outer peripheral portion of the patient and family members of venous blood 2ml/person, normal human peripheral blood drawn another 208 copies, EDTA anticoagulant,-70? stored for use. Whole blood genomic DNA extraction kit genomic DNA; 2) polymerase chain reaction amplification and IRF6 gene MSX1 all exons, the product was directly sequenced; 3) extracted from human MSX1 and IRF6 protein homologous sequences from the NCBI-Homologene database, the human MSX1 protein ?NP₀02439.2? and nine vertebrate species Msx1 protein ?NP₀01182191.1, XP₀01118871.2, XP₅45946.2, NP₇77223.1, NP₀34965.2, NP₁12321.1, NP₉90819.1, NP₅71348.1 and NP₀01032329.1? for comparison, Homologous proteins extracted from human IRF6 NCBI-Homologene database. Human IRF6 protein ?NP₀06138.1? and 9 Irf6 vertebrate protein ?XP₅14168.2, XP₀01110321.1, XP₀05622392.1, NP₀01070402.1, NP₀58547.2, NP₀01102329.1, XP₄17990.4, NP₉56892.1 and NP₀01025493.1? for comparison, use ClustalW2 software to analyze the amino acid conservative; 4) extracted from the NCBI-Homologene database homologous sequence of human MSX1 protein, the use of these procedures ClustalX2.0.12 protein sequences were multiple comparisons, and use PsiPred 3.0 program 2D structure of the human MSX1 to predict; Homologous proteins extracted from human IRF6 NCBI-Homologene database. ClustalX2.0.12 program using these protein sequences were multiple comparisons, and use PredictProtein ?http://www.predictprotein? predict the 2D structure of the human IRF6 to predict; 5) use of these procedures ClustalX2.0.12 protein sequences were multiple comparisons. Using online website ?http://weblogo.berkeley.edu/logo.cgi? MSX1 protein sequence analysis and protein IRF6 conservative; 6) use of online software Phyre ?www.sbg.bio.ic.ac.uk/phyre/? the amino acid sequence of a conserved protein structure prediction and the known three-dimensional structure of the wild-type and mutant IRF6 protein, and use software PyMOL output image.ResultsCompleted MSX1 and IRF6 gene mutation screening in 54 cases of children with NSCL/P.In patient 13, We found a missense mutation in second exon of MSX1 gene ?c.492C>G; p.Cys164Trp?. Proband gender Male, aged 1 year old, simple type of cleft lip, phenotypically normal parents, no family history of cleft lip and palate. The genotype of his patients are normal. Mutations detected located 492 cDNA sequence encoding the amino acid cysteine ?Cys?, the presence of a patient in a locus heterozygous point mutation at nucleotide C into G, encoding the amino acid tryptophan into acid ?Trp?. Human MSX1 protein ?NP₀02439.2? and nine species of vertebrates ?P.troglodytes, M.mulatta, C.lupus, B.taurus, M.musculus, R.norvegicus, G.gallus, D.rerio and X.tropicalis? of Msxl protein ?NP₀01182191.1, XP₀01118871.2, XP₅45946.2, NP₇77223.1, NP₀34965.2, NP₁12321.1, NP₉90819.1, NP₅71348.1 and NP₀01032329.1? were compared, the results showed 164 points highly conserved amino acid Cys in the process of biological evolution, suggesting that the mutation site may lead to changes in amino acid functions. Dimensional protein structure analysis,164 Cys in highly conserved residues, located conservative 2D fragments. The residue mutations affect human MSX1 protein function. Wherein, p.Cysl64Trp this mutation replaces cysteine tryptophan, cysteine is an amino acid containing a hydroxyl group in the side chain, this mutation may eliminate 164Cys and electrostatically neighboring residues by charged residues group into a neutral residue, the charged nature of the change will seriously affect the activity of the protein; hydrophilic group in addition to the cysteine and tryptophan is a hydrophobic group, hydrophilic and hydrophobic nature of the changes will affect the activity of the protein. Weblogo MSX1 protein analysis showed that only the first 164 Stack C ?Cys?, indicating that the site is highly conserved, suggesting that we mutation site may lead to changes in protein function.Completed IRF6 gene sequencing of 54 patients with NSCL/P, we found an IRF6 gene nonsense mutation in one case of sporadic infants. Study No.# 40, male gender, age 1.5 years, simplex palate. Sporadic cases in children is not obtained biological parents DNA, family history is unknown. The sequencing results with normal genomic sequence and the control group IRF6 sequence alignment, display a IRF6 gene exon 9 of nonsense mutations that can lead to the termination of IRF6 protein in position 440 ?c.1320C>G; p.Tyr440X?. Human IRF6 protein ?NP₀06138.1? and nine vertebrate species ?P.troglodytes, M.mulatta, C.lupus, B.taurus, M.musculus, R.norvegicus, G.gallus, D.rerio and X. tropicalis? of Irf6 protein ?XP₅14168.2, XP₀01110321.1, XP₀05622392.1, NP₀01070402.1, NP₀58547.2, NP₀01102329.1, XP₄17990.4, NP₉56892.1 and NP₀01025493.1? were compared, the results showed 440 amino acid sequence point after the highly conserved, suggesting that the change to a larger influence protein function. Protein secondary structure prediction shows that the truncated protein secondary structure changes, suggesting that the mutation in the future have an impact on protein function. Weblogo IRF6 protein analysis showed that only the first 440 Stack Y ?Tyr?, indicating that the site is highly conserved, suggesting that we mutation site may lead to changes in protein function. Three-dimensional protein structure prediction displayed mutant protein three-dimensional structure of the truncated IRF6 changes, and reduce the formation of a-helix, had an impact on protein function.ConclusionsThe clinical study collected NSCL/P patients in Shandong region. From the detection of pathogenic mutations in the gene start, molecular etiology studies to explore the correlation between genotype and NSCL/P. The first part of this study studied the association between NSCL/P and MSX1 gene mutation, using bioinformatics analysis to clarify the correlation between MSX1 gene mutation and clinical phenotype. The secpnd part of this study studied the association between NSCL/P and IRF6 gene mutation, using bioinformatics analysis to clarify the correlation between IRF6 gene mutation and clinical phenotype. Above research results are as follows:First, through the MSX1 gene screening test for NSCL/P patients with non-syndromic clinical collection, we found a possible missense mutations. The site bioinformatic analysis showed that the site is highly conserved during evolution, mutations may affect protein function. Currently no reports on MSX1 mutations can lead to NSCL/P, the present study is the first discovery of a MSX1 gene mutation in Chinese population with NSCL/P patients.Second, through the IRF6 gene screening test for NSCL/P patients with non-syndromic clinical collection, we found a possible nonsense mutations. The site bioinformatic analysis showed that the site is highly conserved during evolution, mutations may affect protein function.Research SignificanceNon-syndromic cleft lip and palate disease genes study has been a hot and difficult research in this field. In this study, we colledted sample in patients with NSCL/P and found novel gene mutaions in Chinese population. Moreover, we analised mutant protein to further verify association between the mutant protein and NSCL/P.This study of clinical cases collected in non-syndromic cleft lip and palate disease gene mutation screening detected non-syndromic cleft lip and a new disease gene mutations in Chinese population. This study was first discovered two new mutations on the gene MSX1 and IRF6, and bioinformatics analysis conducted. Not only can deepen the understanding of these diseases, but also conducive to more in-depth understanding of the structure and function of genes involved in pathogenesis. Provide clues to improve the level of clinical prevention and treatment of such diseases.