Document Type


Date of Degree

Fall 2012

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Murray, Jeffrey C

First Committee Member

Cornell, Robert

Second Committee Member

Bassuk, Alex

Third Committee Member

Burns, Trudy

Fourth Committee Member

Kwitek, Anne


Orofacial clefts are a heterogeneous group of craniofacial malformations that affect the lip and/or palate and represent the most common craniofacial birth defect in humans. In 30% of patients the cleft is accompanied by additional physical or cognitive abnormalities. Hundreds of these clefting syndromes have been described, many of which have Mendelian inheritance patterns. The most common of these is Van der Woude syndrome (VWS), caused by mutations in the transcription factor IRF6 (Kondo et al. 2002). The other 70% of patients lack additional features and are considered nonsyndromic. The etiology of nonsyndromic clefts is complex and involves the combined action of multiple genetic variants interacting with environmental factors.

A common approach for identifying genetic risk factor for complex disorders such as nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the genome wide association study (GWAS). We pursued a locus on 1p22 shown to be associated with NSCL/P by Beaty et al. (2010). Through a combination of expression studies in a mouse model and mutation screening in NSCL/P patients, we identified ARHGAP29 as a novel gene for NSCL/P and the likely etiologic gene at this locus. We identified eight rare variants in NSCL/P patients absent in controls including a nonsense and a frameshift mutation. These rare variants are reminiscent of previous resequencing studies that reported rare coding mutations in 20 different candidate genes for NSCL/P. We reviewed these variants and compared them with variants found in over 7000 exomes from the 1000 Genomes Project (1kGP) and NHLBI Exome Sequencing Project (ESP) to identify the variants and genes most likely to contain etiologic rare variants. We found good support for a role for rare variants in NSCL/P, particularly for MSX1 and genes of the FGF signaling pathway.

We next performed several studies to understand the genetic architecture of syndromic forms of clefting, focusing on VWS and popliteal pterygium syndrome (PPS), which is allelic to VWS. We compiled all of the nearly 300 published IRF6 mutations and compared the distribution of these mutations with IRF6 variants obtained from the 1kGP and ESP exomes. We found that mutations causing VWS were significantly over-represented in the DNA-binding domain and for the most part were absent from control exomes, indicating that they are likely to be truly causative for VWS or PPS. These mutations in VWS and PPS only account for 70% of VWS and 97% of PPS. We next hypothesized that mutations in RIPK4, which causes an autosomal recessive pterygia syndrome, could underlie the remaining VWS and/or PPS cases. We found novel homozygous mutations in RIPK4 in two PPS patients. This result has significant clinical ramifications, as counseling of recurrence risk is very different for PPS patients whose disease is caused by dominant IRF6 mutations compared to recessive RIPK4 mutations.

Finally, to understand the variable expressivity of VWS and PPS we performed an association study to identify genetic modifiers. We also looked for genotype-phenotype correlations between the type and location of IRF6 mutations. Although we did not find strong evidence that the candidate genes we selected from GWAS of NSCL/P or other clefting syndromes are modifiers of the VWS or PPS phenotypes, several marginal associations suggest that members of the IRF6 gene regulatory network could act as modifiers. Finally, we found evidence of a larger genotype-phenotype correlation by demonstrating that mutation-negative VWS families have a deficiency of cleft lip phenotypes. Together this work has advanced our understanding of the genetic basis of this diverse set of cleft lip and palate disorders, informing both the biology of craniofacial development and the clinical care of patients affected by these disorders.


Cleft lip and/or palate, Complex trait, craniofacial, rare variant, Van der Woude syndrome


xvii, 219 pages


Includes bibliographical references (pages 183-219).


Copyright 2012 Elizabeth Jane Leslie

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