Cleft lip and palate:Examining variations on the ZEB1 gene

Jingwen ZhangThomas Worthington High School ‘14Research Institute: Murray Lab, University of Iowa

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.

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

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-cadherinα-cateninγ-catenin

CollagenSmooth muscle actinFibronectin

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.

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 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.

Results: Total variants found

Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9

Iowa 31,610,234 (A/T)

rs7918614 (D64D)

31,784,572 (T/C) (4x)

rs2839664 (A/G) (24x)

-------- 31,815,588 (C/G)

rs12217419 (G91R) (G/A)

rs41289011 (A/G) (8x)

Phil 31,610,282 (T/A)31,610,320 (G/T)31,610,515 (C/G)31,610,638 (C/T)31,610,687 (A/T)

31,750,166 (R87A) (A/G)

rs41289011 (A/G) (6x)

-------- rs220060 (A/G)

31,816,261 (A/G)

rs7918614 (D64D) (10x)

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

Results: Minor allele count

rs7918614 rs7918614 rs41289011 rs12217419 rs2839664 rs2200600

5

10

15

20

25

30 Minor allele count of known SNPs

CL/P casesControl

Results: SNP minor allele ct. significance

Pop. SNP p-valuePhil rs7918614 0.01

Iowa rs7918614 0.22Iowa rs41289011 0.40Iowa rs12217419 0.88Iowa rs2839664 0.03Phil rs220060 0.35

Results: Total variants found

Exon 1 Exon 2 Exon 3 Exon 4 Exon 6 Exon 9

Iowa 31,610,234 (A/T)

rs7918614 (D64D)

31,784,572 (T/C) (4x)

rs2839664 (A/G) (24x)

-------- 31,815,588 (C/G)

rs12217419 (G91R) (G/A)

rs41289011 (A/G) (8x)

Phil 31,610,282 (T/A)31,610,320 (G/T)31,610,515 (C/G)31,610,638 (C/T)31,610,687 (A/T)

31,750,166 (R87A) (A/G)

rs41289011 (A/G) (6x)

-------- rs220060 (A/G)

31,816,261 (A/G)

rs7918614 (D64D) (10x)

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.

Results: Variant on exon 2

R87 R87

A G A

G87 87

G G AExon 2 Exon 3

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.

Results: New variations MAF

31610234

31610282

31610320

31610515

31610638

31610687

31784572

31815588

31816261

317501660.000

0.005

0.010

0.015

0.020

0.025

New SNP MAF

MAF

Explanation

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

Results: Orthologous conservation

Human

Rhesus

MouseDog

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

SNP conservation

Rhesus

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.

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

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

Acknowledgements

Dr. Jeff MurrayFor giving me the opportunity and resources for this research project

Maria A. MansillaFor mentoring me throughout the experiment and analysis processes

Elizabeth LeslieFor helping to answer my technical questions

The Murray LabFor their welcome and support