Cleft lip and palate:Examining variations on the ZEB1 gene                  Jingwen Zhang                  Thomas Worthing...
Cleft lip and palate: Background   Cleft lip with or without cleft palate (CL/P) is a common birth defect   Affects 0.5-...
Cleft lip and palate: Background                    5-7% CL/P cases syndromic, or                     mendelian, caused b...
ZEB1: Background   Zinc finger E-box-binding homeobox 1   Gene on chromosome 10   Plays vital role in epithelial-to-mes...
ZEB1 in present experiment   ZEB1: candidate gene for involvement in CL/P development   Look for variations within the g...
MethodsExperiment: DNA from CL/P cases in the Philippines and Iowa (studied separately) Polymerase chain reactions (PCR)...
Results: Total variants found       Exon 1       Exon 2         Exon 3         Exon 4        Exon 6     Exon 9Iowa   31,61...
Explanation   13 new mutations found on 10 locations in gene. Indicated by position    numbers, as they have not been doc...
Results: Minor allele count30                 Minor allele count of known SNPs25201510                                    ...
Results: SNP minor allele ct. significance  Pop.          SNP       p-value   Phil     rs7918614      0.01  Iowa      rs79...
Results: Total variants found       Exon 1       Exon 2         Exon 3         Exon 4        Exon 6     Exon 9Iowa   31,61...
Explanation   Compared the Minor Allele Counts of known SNPs in cases studied to    the minor allele counts from the cont...
Results: Variant on exon 2         A                   G         A         R87                     R87Exon 2   G          ...
Explanation   Only one new mutation was found in a coding section of the gene.    Changes arginine to glycine.   Amino a...
Results: New variations MAF0.025        New SNP MAF0.0200.0150.010                              MAF0.0050.000
Explanation   Minor Allele Frequencies of new SNPs found.   New; most are very rare.
Results: Orthologous conservation                                  SNP conservation      Human      Rhesus      Mouse     ...
Explanation   Shows orthological conservation (conservation between species) of the    locations of the gene in which new...
Summary of results   13 new variants – 10 locations   1 new SNP in coding region split across 2 exons   7 locations of ...
Future research   Research non-coding SNPs – TF binding sites and gene expression   Explore possible link between freque...
AcknowledgementsDr. Jeff Murray    For giving me the opportunity and resources for this research projectMaria A. Mansilla ...
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Cleft lip and palate: Examining variations on ZEB1 gene

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  • 13 new variants were found in 10 different locations on the ZEB1 gene. 7 locations of known SNPs were also found.
  • 13 new variants were found in 10 different locations on the ZEB1 gene. 7 locations of known SNPs were also found.
  • Cleft lip and palate: Examining variations on ZEB1 gene

    1. 1. Cleft lip and palate:Examining variations on the ZEB1 gene Jingwen Zhang Thomas Worthington High School ‘14 Research Institute: Murray Lab, University of Iowa
    2. 2. Cleft lip and palate: Background Cleft lip with or without cleft palate (CL/P) is a common birth defect Affects 0.5-2.5% of live births CL/P cases have high morbidity rates due to feeding difficulties, speech impairment, surgical and dental care, etc. Develops in a variety of phenotypes Dixon MJ, Marazita ML, Beaty TH, Murray JC. 2011. Cleft lip and palate: synthesizing genetic and environmental influences. Nat Rev Genet 12(3):167-178.
    3. 3. Cleft lip and palate: Background  5-7% CL/P cases syndromic, or mendelian, caused by specific chromosomal malformation patterns  Can affect parts of body other than palate and lip  Van der Woude Syndrome caused by mutations on IRF6  Most cases non-syndromic, caused by variety of genetic and environmental factors interacting to onset CL/P
    4. 4. ZEB1: Background Zinc finger E-box-binding homeobox 1 Gene on chromosome 10 Plays vital role in epithelial-to-mesenchymal transition (EMT) Expression of ZEB1 decreases expression of epithelial markers such as E- cadherin, increases mesenchymal markers E-cadherin Collagen α-catenin Smooth muscle actin γ-catenin Fibronectin
    5. 5. ZEB1 in present experiment ZEB1: candidate gene for involvement in CL/P development Look for variations within the gene that may be related to CL/P Find mutations that merit further investigation into ZEB1 gene as potentially involved in CL/P Predicted: That new variations relating to CL/P would be found in ZEB1 That information regarding previously known SNPs will also be found to be significant and aid future research in CL/P.
    6. 6. MethodsExperiment: DNA from CL/P cases in the Philippines and Iowa (studied separately) Polymerase chain reactions (PCR), amplified exons and sections of nearby introns; gel electrophoresis Send plates away for sequencingAnalysis: Analyze clear, successfully sequenced reads on Phred and Phrap Consed server Look for variations from control genome (obtained from NCBI dbSNP) Check for novelty of SNP mutation on UCSC Genome Browser, Exome Variant Server, 1000 Genomes Browser. Assess predicted damage of missense mutations on PolyPhen-2.
    7. 7. Results: Total variants found Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9Iowa 31,610,234 rs7918614 31,784,572 rs2839664 -------- 31,815,588 (A/T) (D64D) (T/C) (4x) (A/G) (24x) (C/G) rs12217419 (G91R) (G/A) rs41289011 (A/G) (8x)Phil 31,610,282 31,750,166 rs41289011 -------- rs220060 31,816,261 (T/A) (R87A) (A/G) (A/G) (6x) (A/G) (A/G) 31,610,320 rs7918614 (G/T) (D64D) (10x) 31,610,515 (C/G) 31,610,638 (C/T) 31,610,687 (A/T)
    8. 8. Explanation 13 new mutations found on 10 locations in gene. Indicated by position numbers, as they have not been documented in browsers/servers yet. Known SNPs found in 7 locations in gene. Indicated by rs numbers. Also indicated: Base change, amino acid change (if any), number of appearances of mutation
    9. 9. Results: Minor allele count30 Minor allele count of known SNPs25201510 CL/P cases Control50 rs7918614 rs7918614 rs41289011 rs12217419 rs2839664 rs220060
    10. 10. Results: SNP minor allele ct. significance Pop. SNP p-value Phil rs7918614 0.01 Iowa rs7918614 0.22 Iowa rs41289011 0.40 Iowa rs12217419 0.88 Iowa rs2839664 0.03 Phil rs220060 0.35
    11. 11. Results: Total variants found Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9Iowa 31,610,234 rs7918614 31,784,572 rs2839664 -------- 31,815,588 (A/T) (D64D) (T/C) (4x) (A/G) (24x) (C/G) rs12217419 (G91R) (G/A) rs41289011 (A/G) (8x)Phil 31,610,282 31,750,166 rs41289011 -------- rs220060 31,816,261 (T/A) (R87A) (A/G) (A/G) (6x) (A/G) (A/G) 31,610,320 rs7918614 (G/T) (D64D) (10x) 31,610,515 (C/G) 31,610,638 (C/T) 31,610,687 (A/T)
    12. 12. Explanation Compared the Minor Allele Counts of known SNPs in cases studied to the minor allele counts from the control population (control data obtained from NCBI dbSNP). In several of these SNPs, the minor allele counts of cases were significantly higher (p<0.05). One of these SNPs is located in the Iowan population, the other in the Filipino population, suggesting that CL/P in these isolated populations may have developed with different mechanisms.
    13. 13. Results: Variant on exon 2 A G A R87 R87Exon 2 G G A Exon 3 G87 87
    14. 14. Explanation Only one new mutation was found in a coding section of the gene. Changes arginine to glycine. Amino acid is split over exons 2 and 3 Predicted by PolyPhen-2 to be benign, but could possibly affect splicing.
    15. 15. Results: New variations MAF0.025 New SNP MAF0.0200.0150.010 MAF0.0050.000
    16. 16. Explanation Minor Allele Frequencies of new SNPs found. New; most are very rare.
    17. 17. Results: Orthologous conservation SNP conservation Human Rhesus Mouse Dog Elephant Opossum Chicken X_tropicalis ZebrafishConserved in: 5/7 7/7 5/5 6/8 8/8 8/9 2/2 6/7 6/9
    18. 18. Explanation Shows orthological conservation (conservation between species) of the locations of the gene in which new SNPs were found. Green = conserved; Orange = not conserved; Gray = no data given Orthological conservation important in speciation and evolutionary studies; the more conserved a region is through more species, the more importance it may have.
    19. 19. Summary of results 13 new variants – 10 locations 1 new SNP in coding region split across 2 exons 7 locations of known SNPs Higher minor allele count of known SNPs (rs7918614, rs2839664) in CL/P cases than in unaffected controls. – Statistically significant (p<0.05) – rs2839664 significant in Iowa pop., rs7918614 significant in Filipino pop. Some new non-coding variations located in conserved regions of introns
    20. 20. Future research Research non-coding SNPs – TF binding sites and gene expression Explore possible link between frequently recurring known SNPs and development of CL/P Research CL/P in a geographical context, exploring how isolated pathways developed independently Examine interaction of new genetic factors with environmental factors and how the two work together to cause CL/P Broaden scope of experiment
    21. 21. AcknowledgementsDr. Jeff Murray For giving me the opportunity and resources for this research projectMaria A. Mansilla For mentoring me throughout the experiment and analysis processesElizabeth Leslie For helping to answer my technical questionsThe Murray Lab For their welcome and support

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