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. 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. 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. 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. 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. Methods
Experiment:
 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 sequencing
Analysis:
 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. Results: Total variants found
Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9
Iowa 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. 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. Results: Minor allele count
30 Minor allele count of known SNPs
25
20
15
10 CL/P cases
Control
5
0
rs7918614 rs7918614 rs41289011 rs12217419 rs2839664 rs220060

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. Results: Total variants found
Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9
Iowa 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. 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. Results: Variant on exon 2
A G A
R87 R87
Exon 2 G G A Exon 3
G87 87

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. Results: New variations MAF
0.025 New SNP MAF
0.020
0.015
0.010
MAF
0.005
0.000

16. Explanation
 Minor Allele Frequencies of new SNPs found.
 New; most are very rare.

17. Results: Orthologous conservation
SNP conservation
Human
Rhesus
Mouse
Dog
Elephant
Opossum
Chicken
X_tropicalis
Zebrafish
Conserved in: 5/7 7/7 5/5 6/8 8/8 8/9 2/2 6/7 6/9

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. 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. 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. Acknowledgements
Dr. Jeff Murray
For giving me the opportunity and resources for this research project
Maria A. Mansilla
For mentoring me throughout the experiment and analysis processes
Elizabeth Leslie
For helping to answer my technical questions
The Murray Lab
For their welcome and support