Identifying Novel Genetic Causes of Cleft Palate Open Access
Robinson, Kelsey (Spring 2024)
Abstract
Cleft palate (CP) occurs in approximately 1 in 1700 live births per year and is one of the most common craniofacial birth defects. Although it is highly heritable, there are few known associated genetic risks. Therefore, we performed a comprehensive investigation in a trio-based cohort to evaluate both common and rare variant risks for CP using whole genome sequencing. We hypothesized there would be risk factors associated with any type of CP, as well as risks specific to CP subtypes (i.e., clefts affecting the hard and/or soft palate), proband sex, and based on the presence or absence of additional clinical features. We performed genome-wide association studies (GWAS) for 435 trios with any type of CP and stratified by subtype, identifying a genome-wide significant locus at 9q33.3 (rs7035976, p=4.24x10-8) associated with any cleft of the hard palate. When stratified by proband sex, we found distinct GWAS signals, prompting a genome-wide gene-by-sex interaction analysis. We identified 13 loci with significant interactions. Our top finding was within an intron of LTBP1 in the 2p33.3 locus (relative risk 3.37 (95% CI 2.04 - 5.56), p=1.93x10-8), for which we found a female-specific association between imputed genetically regulated gene expression for LTPBP1 and CP in cultured fibroblasts. We next evaluated de novo variants (DNs) and found global enrichment of protein-altering DNs and gene-specific enrichment for several genes, including three not previously associated with CP: PRKCI, POLR1F, and SCL25A41. We found subtype-specific enrichments including SATB2 and TGFBR2 in cleft hard and soft palate, and PRKCI for cleft soft palate. Moreover, PRKCI was specifically enriched in syndromic CP probands. We found identical DNs (N383S) in PRKCI in two individuals with identical phenotypes—Van der Woude syndrome (VWS) with lip pits and cleft soft palate—and a third individual with a Y136C DN with cleft soft palate and abnormal lip morphology. Functional testing of this variant using zebrafish embryos confirmed loss-of-function, implicating PRKCI as a novel gene for VWS. These findings expand our understanding of the genetic basis of CP and related syndromes, emphasizing the importance of considering sex-specific effects and performing subtype-specific analyses in genetic studies of CP.
Table of Contents
Chapter I: Introduction ..............................................................................................................1
Introduction .......................................................................................................................1
Background .......................................................................................................................2
Epidemiology ........................................................................................................2
Palatogenesis .........................................................................................................3
Classification.........................................................................................................5
Animal models ......................................................................................................6
Etiology .............................................................................................................................8
Environmental .......................................................................................................8
Epigenetics ..............................................................................................10
Genetics...............................................................................................................12
Cleft palate syndromes ............................................................................13
Common variants ....................................................................................15
Rare variants ...........................................................................................16
Emerging areas of investigation..........................................................................18
Conclusion ......................................................................................................................19
Figures.............................................................................................................................21
Tables ..............................................................................................................................24
Chapter II: Trio-based GWAS identifies novel associations and subtype-specific
risks for cleft palate ..................................................................................................................29
Abstract ...........................................................................................................................29
Introduction .....................................................................................................................30
Materials and methods ....................................................................................................32
Study population and phenotyping .....................................................................32
Sample preparation and whole genome sequencing ...........................................32
Quality control ....................................................................................................34
Principal component analysis .............................................................................34
Statistical analysis ...............................................................................................34
DECIPHER variants ...........................................................................................35
Animal studies and gene expression assays ........................................................36
Replication of previous published SNPs.............................................................37
Results .............................................................................................................................38
GWAS of any CP type ........................................................................................38
Subtype-specific GWAS .....................................................................................38
Expression during mouse palatogenesis and limb development.........................40
Attempted replication of previous published SNPs ............................................42
Discussion .......................................................................................................................42
Description of supplemental information .......................................................................47
Acknowledgements .........................................................................................................47
Declaration of interests ...................................................................................................48
Data availability ..............................................................................................................48
Web resources .................................................................................................................48
Figures.............................................................................................................................50
Tables .............................................................................................................................548
Supplemental material ....................................................................................................58
Supplemental figures ..........................................................................................58
Supplemental tables ............................................................................................61
Chapter III: Genome-wide study of gene-by-sex interactions identifies risks for
cleft palate .................................................................................................................................72
Abstract ...........................................................................................................................73
Introduction .....................................................................................................................73
Methods...........................................................................................................................75
Study population and phenotyping .....................................................................76
Sample preparation and whole genome sequencing ...........................................76
Quality control ....................................................................................................77
Statistical analysis ...............................................................................................77
Genetically regulated gene expression imputation and association testing ........78
Rare variants .......................................................................................................79
Results .............................................................................................................................80
Discussion .......................................................................................................................81
Figures.............................................................................................................................86
Tables .............................................................................................................................88
Supplemental data ...........................................................................................................93
Chapter IV: Cleft palate probands are significantly enriched for protein-altering de novo
variants ......................................................................................................................................98
Abstract ...........................................................................................................................99
Introduction ...................................................................................................................100
Methods.........................................................................................................................102
Study cohort ......................................................................................................102
Whole genome sequencing ...............................................................................103
Identification of de novo variants .....................................................................103
Variant annotation .............................................................................................104
De novo enrichment ..........................................................................................105
Enrichment analysis and creation of gene sets .................................................105
Results ...........................................................................................................................106
Exome-wide analyses........................................................................................106
Gene-specific analyses ......................................................................................108
Gene set enrichments ........................................................................................110
Discussion .....................................................................................................................112
Figures...........................................................................................................................117
Tables ...........................................................................................................................123
Supplemental data .........................................................................................................125
Chapter V: Rare variants in PRKCI cause Van der Woude syndrome and
other features of peridermopathy..........................................................................................184
Abstract .........................................................................................................................185
Introduction ...................................................................................................................186
Methods.........................................................................................................................1889
Study cohort ......................................................................................................188
Sample preparation and whole genome sequencing .........................................189
Variant filtering and annotation ........................................................................190
DN enrichment ..................................................................................................191
Functional validation in zebrafish .....................................................................191
Results ...........................................................................................................................192
Discussion .....................................................................................................................194
Figures...........................................................................................................................199
Tables ...........................................................................................................................202
Chapter VI: Discussion...........................................................................................................205
Summary of Results ......................................................................................................205
Interpretation and implications .....................................................................................209
Study limitations ...........................................................................................................211
Future directions ...........................................................................................................213
Conclusion ....................................................................................................................214
Chapter VII: References ........................................................................................................215
List of Figures
Figure 1.1: Human palatal development .....................................................................................21
Figure 1.2: Correlation between human and mouse palates .......................................................22
Figure 1.3: Phenotypic heterogeneity in orofacial clefts ............................................................23
Figure 2.1: Genome-wide significant locus at 9q33.3 spans craniofacially-expressed gene
ANGPTL2 and a craniofacial super enhancer ...........................................................50
Figure 2.2: Regional association plots illustrate differences between groups ............................51
Figure 2.3: Comparison of odds ratios for any suggestive loci demonstrates subtype-specific
effects ........................................................................................................................52
Figure 2.4. Angptl2 expression during mouse palate and limb development .............................53
Figure 3.1: Miami plot highlighting suggestive SNPs in females and males .............................86
Figure 3.2: Relative risks for males and females demonstrating GxS effect SNPs ....................86
Figure 3.3: Comparing relative risks with 95% confidence intervals .........................................87
Figure 4.1: Exome-wide enrichment for CP ............................................................................117
Figure 4.2: Gene-specific enrichment for CP ..........................................................................118
Figure 4.3: Gene-specific enrichment for protein-altering variants by CP subtype .................119
Figure 4.4: Gene set enrichment analysis with ToppFun .........................................................120
Figure 4.5: Enrichment for CP in a specific set of OFC-associated genes as determined by
denovolyzeR ...........................................................................................................121
Figure 4.6: Enrichment for CP in a set of marker genes from the secondary mouse palate at
E15.5 ......................................................................................................................122
Figure 5.1: Expression of PRKCI in embryogenesis ................................................................202
Figure 5.2: Structure and distribution of PRKCI variants ........................................................203
Figure 5.3: Representative zebrafish embryo assays for functional and non-functional PRKCI
variants ....................................................................................................................204
List of Tables
Table 1.1: Common syndromes featuring cleft palate ................................................................24
Table 1.2: Genes associated with cleft palate .............................................................................26
Table 2.1: Suggestive and significant loci from any CP type and subtype-specific GWAS ......54
Table 2.2: Credible sets from fine-mapping and probability for inclusion of listed SNPs .........55
Table 2.3: Previous published OFC-associated SNPs with evidence of replication in the
current study...............................................................................................................56
Table 3.1: Loci of suggestive significance from sex stratified TDTs .........................................88
Table 3.2: Loci of suggestive significance in the LRT 2df .........................................................89
Table 3.3: Tissues and genes with significant association between imputed gene expression
and phenotype ............................................................................................................92
Table 4.1: Subtype enrichment for protein-altering DNs in palatal osteogenesis marker
genes .......................................................................................................................123
Table 4.2: Overlap in CPSeq genes with protein-altering DNs and DDD genes with P/LP
DNs ..........................................................................................................................124
Table 5.1: DNs and rare variants in PRKCI .............................................................................199
List of Supplemental Figures
Figure 1.1: Human palatal development .....................................................................................21
Figure S2.1: Principal Components for Genetic Ancestry..........................................................58
Figure S2.2: Asian-Ancestry Specific Analysis..........................................................................59
Figure S2.3: Any Cleft Palate Manhattan & QQplots. ...............................................................60
Figure S2.3: Soft Cleft Palate Manhattan & QQplots.................................................................60
Figure S2.3: Hard Cleft Palate Manhattan & QQplots. ..............................................................60
Figure S3.1: Manhattan and qq plots for male probands ............................................................93
Figure S3.2: Manhattan and qq plots for female probands .........................................................93
Figure S3.3: Manhattan and qq plots for all probands ................................................................93
Figure S3.4: Manhattan and qq plots for LRT2df probands .......................................................94
Figure S3.5: Manhattan and qq plots for GxS probands .............................................................94
Figure S3.6: Post hoc power calculations ...................................................................................95
Figure S4.1: Distribution and frequency of de novo variants ...................................................125
Figure S4.2: Enrichment of genes associated with the OFC-gene panel ..................................125
Figure S4.3: Enrichment of genes associated with the secondary palate at E15.5 ...................126
List of Supplemental Tables
Table 2.1: CPSeq Cohort Description .........................................................................................61
Table S2.2: Angptl2 gene-specific primers .................................................................................61
Table S2.2: RT-qPCR primers ....................................................................................................61
Table S2.4: All SNPs tested for replication ................................................................................62
Table S3.1: Rare variants in LTBP1 ...........................................................................................96
Table S4.1 Breakdown of CP subtypes by proband sex and self-reported race .......................127
Table S4.2 Features of syndromic CP probands .......................................................................127
Table S4.3 All de novo variants in CP probands ......................................................................129
Table S4.4 Expected versus observed DNs in males versus females with respect to
prevalence differences ............................................................................................149
Table S4.5 Global DN enrichment for all categories ................................................................150
Table S4.6: Protein altering gene-specific DN enrichment in probands with and without
syndromes and/or PRS .........................................................................................152
Table S4.7: Protein altering gene-specific DN enrichment in probands by subtype ................165
Table S4.8: Gene set enrichment terms ...................................................................................173
Table S4.9: Enrichment in OFC-gene panel ............................................................................173
Table S4.10: Enrichment in snRNAseq marker genes..............................................................178
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