Dissertation
The role of structural variation in cleft lip and palate
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2018
DOI: 10.17077/etd.phwkovkz
Abstract
<p>Clefts of the lip and/or palate (CL/P) are one of the most common birth defects in the world occurring about every 1 in 700 live births. Individuals with non-syndromic clefting (NSCL/P) account for about 70% of all cleft cases and exhibit a cleft only whereas syndromic occurrences (SCL/P) include additional cognitive or structural abnormalities. Linkage, genome-wide association, candidate gene, animal model, sequencing and copy number variant (CNV) analyses have been used to study CL/P and have established that it is a heterogeneous, complex disorder. However, the impact of identified sequence variants on protein structure and the contribution of structural genetic variation to CL/P remains poorly understood.</p>
<p>In our first analysis we reassessed the phenotype of a 30-year-old individual of SCL/P and noticed phenotypic overlap with Hartsfield syndrome, a rare syndrome resulting from sequence variants in Fibroblast growth factor 1 (FGFR1). We sequenced the coding region of FGFR1 and identified a novel, de novo variant. Due to the fact sequence variants in FGFR1 contribute to multiple syndromes encompassing a wide phenotypic spectrum, we performed an extensive literature search to record every published sequence variant of FGFR1 and mapped it to the protein structure by disease and phenotype. Although no statistically significant protein domain-phenotype correlations were identified, many regions neared significance. This work stresses the need for systematic, comprehensive phenotyping of patients and provides a method for assessing the impact of the location of sequence variants within the 3D structure of the protein.</p>
<p>Although rare and common CNVs have been identified in individuals with CL/P, prior to our work no large-scale studies of rare CNVs for the identification of novel clefting genes had been performed. For our second set of analyses, we conducted two such studies, first focusing on a smaller cohort of 140 individuals with NSCL/P from the Philippines to establish our informatic and functional validation pipeline. We used whole-genome tiling arrays to assess rare deletions overlapping genes not previously implicated in clefting, and identified one deletion overlapping Isthmin1 (ISM1) and a deletion just 3’ of the gene in a second affected individual. Functional validation of Ism1 in Xenopus laevis showed strong expression in structures necessary for craniofacial development, and morpholino and CRISPR/Cas9 knockdown of Ism1 resulted in a median cleft lip in some embryos, establishing ISM1 as a novel craniofacial patterning gene. We then expanded our study and assessed genomic CNVs in 1021 individuals with NSCL/P and 81 individuals with SCL/P, finding no differences in CNV number, load or burden between these groups. We also identified 8 putative clefting genes overlapped by deletions in two or more individuals but at a rare (< 1% frequency) in the cohort. Functional validation of these genes using CRISPR/Cas9 in zebrafish and Xenopus tropicalis is currently underway.</p>
<p>This work has identified a novel sequence variant leading to the diagnosis of Hartsfield syndrome in an individual with SCL/P, developed an innovative method for assessing the impact of sequence variation on protein structure, improved our understanding of the contribution of CNVs to SCL/P and NSCL/P and identified several putative novel clefting loci which may help explain a portion of the missing heritability of CL/P.</p>
Details
- Title: Subtitle
- The role of structural variation in cleft lip and palate
- Creators
- Lisa Ann Lansdon - University of Iowa
- Contributors
- John Manak (Advisor)Patrick John Breheny (Committee Member)Benjamin W. Darbro (Committee Member)Anne E. Kwitek (Committee Member)Jacob J. Michaelson (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Genetics
- Date degree season
- Spring 2018
- DOI
- 10.17077/etd.phwkovkz
- Publisher
- University of Iowa
- Number of pages
- xvii, 377 pages
- Copyright
- Copyright © 2018 Lisa Ann Lansdon
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Date submitted
- 09/05/2018
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 297-377).
- Public Abstract (ETD)
Clefts of the lip and/or palate (CL/P) are one of the most common birth defects in the world occurring every 1/1000 live births. Genetic approaches have shown small changes in DNA sequences contribute to clefting in individuals with or without additional clinical features. One such individual had CLP, diabetes, brain anomalies as well as hand and foot malformations. I noticed his features were most like Hartsfield syndrome—a diagnosis we confirmed by detecting a DNA variant in the FGFR1 gene. I also generated a list of all published DNA changes in FGFR1 and mapped them to the protein structure by disease and symptom to help doctors make future diagnoses of FGFR1-based disorders.
The remainder of my dissertation focused on determining the contribution of gains and losses of regions of the genome (known as copy number variants, or CNVs) to clefting by analyzing 1102 individuals with CL/P, identifying which of these changes they had in common, then functionally testing the variants in frogs. We identified 9 genes overlapped by CNVs which may be new causes of clefts when deleted. One gene we identified was ISM1. My experiments in frogs have shown ism1 is expressed in the developing face, and when I reduce the expression of the gene it results in craniofacial malformations (including a cleft-like lip) and decreases the expression of a known clefting gene, LHX8.
This work is the largest study of CNVs in individuals with CL/P to date and will serve as a valuable resource for years to come.
- Academic Unit
- Craniofacial Anomalies Research Center; Interdisciplinary Graduate Program in Genetics
- Record Identifier
- 9983777272302771
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