Insights into the Molecular Basis of Craniofacial Development

  • Register
    • Regular Member - Free!
    • Early Career Member - Free!
    • Resident/Clinical Fellow Member - Free!
    • Postdoctoral Fellow Member - Free!
    • Graduate Student Member - Free!
    • Undergraduate Student Member - Free!
    • Emeritus Member - Free!
    • Life Member - Free!
    • Trainee Member - Free!

    Continue

Platform sessions are abstract driven sessions with 6 talks per session. These talks are 10 minutes in length and are cross-topical in nature to represent the broad discipline our field of genetics and genomics represent. After each talk, there will be a 5-minute Q&A with each speaker. For information on each individual session, please view the "Details" tab. 

Recorded session from the 2021 virtual meeting.

Key:

Complete
Failed
Available
Locked
Video
Open to view video.
Open to view video.
You must register to access.

HDAC9 structural variants disrupting TWIST1 transcriptional regulation lead to craniofacial and limb malformations

Structural variants (SVs) such as insertions, deletions duplications, translocations and inversions, are associated with human disorders. SVs can affect protein coding sequences as well as gene regulatory elements. However, SVs disrupting protein coding sequences that also function as cis regulatory elements remain largely uncharacterized. Here, we show that craniosynostosis patients with SVs containing the Histone deacetylase 9 (HDAC9) protein coding sequence are associated with disruption of TWIST1 regulatory elements that reside within HDAC9 sequence. Using epigenetic marks and in vivo enhancer assays, we characterized six craniofacial TWIST1 enhancers located in the TWIST1-HDAC9 locus. Based on SVs within the HDAC9-TWIST1 locus, we defined the 3' HDAC9 sequence (~500Kb) as a critical Twist1 regulatory region. By deleting Twist1 enhancers within the Hdac9 protein coding sequence in mice (eTw5-7Del/Del), we showed that Twist1 expression was decreased, resulting in smaller sized and asymmetric skull and polydactyly. Furthermore, deletion of a Ctcf site (CtcfDel/Del) within the Hdac9 protein coding sequence, disrupted Twist1 enhancer-promoter interactions and altered Twist1 expression which led to deformed skull and hindlimb polydactyly, resembling Twist1+/- mouse phenotype. Deletions of Twist1 regulatory elements altered the distinct anterior\posterior expression patterns of Shh pathway genes, including Hand2 and Alx4. Using UMI-4C, we demonstrated that both enhancers and Ctcf site regions interact with Twist1 promoter region. These interactions are depended on the presence of both regulatory regions, indicating a specific chromatin conformation of Hdac9 in regulating Twist1 expression. Finally, a large inversion of the entire Hdac9 sequence (Hdac9INV/+) that does not disrupt Hdac9 expression but rather repositions Twist1 regulatory elements showed a decrease in Twist1 expression that led to subtle craniofacial phenotype and hindlimb polydactyly. Thus, our study elucidated essential components of TWIST1 transcriptional machinery that reside within the HDAC9 sequence, suggesting that SVs, encompassing protein coding sequence, such as HDAC9, could lead to a phenotype that is not attributed to its protein function but rather to a disruption of the transcriptional regulation of a nearby gene, such as TWIST1. 

Ramon Y Birnbaum
Ben Gurion University

Kate Wilson Oxford Centre for Genomic Medicine, Oxford University Hospitals NHS Foundation TrustDe-Novo Mutations identified in Nonsyndromic Cleft lip/Palate Families from Africa

Background: Despite successes in the investigation of de novo mutations (DNMs) in the etiology of some birth defects (autism, congenital heart defects), only a limited number have been reported for nonsyndromic cleft lip with or without cleft palate (NSCL/P), the most common craniofacial birth defect. To identify high impact DNMs controlling risk of NSCL/P, we conducted whole genome sequencing (WGS) analyses of case-parent trios from an understudied population.
Method: A total of 150 nsCL/P African case-parent trios were sequenced for this study. Each trio comprises an affected child (with nsCL/P) and unaffected parents and were recruited from Ghana and Nigeria. Saliva samples were collected from these individuals and their genomes were sequenced from extracted DNA. After quality control, we screened the genomes of the remaining 130 trios for high impact DNMs possibly contributing to risk of nsCL/P. We used bioinformatic prediction tools to identify those mutations predicted to damage and affect the protein structures and functions.
Results: We identified 110 potential pathogenic DNMs. These include novel loss of function (LOF) variants in TTN, MINK1 and ARHGAP10 genes; and missense variants in DHRS3, TULP4, SHH, TP63, FKBP10, ACAN, RECQL4 and KMT2D. These variants are predicted to be damaging and are among the most deleterious (top 1% ) mutations in the human genome. Experimental evidence in published works showed TTN, SHH, TP63, FKBP10, ACAN, RECQL4 and KMT2D genes are involved in facial development and are involved in the etiology of syndromic CL/P. While DHRS3, SHH and TP63 contribute to the risk of nsCL/P. Interestingly, our SHH de novo variant p.Ser362Leu has been reported to cause holoprosencephaly 3 (HPE3), a syndromic form of CL/P. Damaging mutations in the DHRS3 gene affects retinoic acid signaling during embryogenesis which causes cleft palate. Association studies have identified TULP4 as a potential cleft candidate gene, while ARHGAP10 interacts with CTNNB1 to control WNT signaling. MINK1 plays a role in cell-cell adhesion and migration, and causes abnormal tooth morphogenesis in mice. Our gene-set enrichment analysis identified additional genes that are important in palatal development. These include DLX6 and EPHB2 and they both harbored novel damaging DNMs.
Conclusion: Our WGS adds to the available data on Africa population (a historically underrepresented group in genetics study) and has identified novel pathogenic de novo variants that may contribute to the developmental pathogenesis of NSCL/P. These findings demonstrate the power of WGS analysis of trios for discovering potential pathogenic variants. 

Waheed Awotoye
Iowa Institute for Oral Health Research, University of Iowa

Identification of novel molecular pathways in syndromic orofacial clefting

Background: Syndromic orofacial clefting (OC) accounts for 30% of cleft lip and/or palate. An updated review of molecular pathways associated with syndromic OC is unavailable. The Deciphering Developmental Disorders (DDD) study provides a source of quality data to assemble this information. The Genomics England PanelApp is a publicly available database of curated virtual gene panels and is a valuable reference tool for genes associated with syndromic OC.
Aim: To investigate molecular pathways associated with syndromic OC by reviewing the results of exome sequencing (ES) and exon-arrayCGH in a large cohort of patients with syndromic OC.
Methods: Patients with the HPO terms ‘cleft’ and ‘bifid uvula’ were identified through a Complementary Analysis Project within the DDD study. Possible diagnostic variants were identified by automated variant filtering and manual review. Single nucleotide variants (SNVs) within known disease-causing genes and copy number variants (CNVs) were classified according to the ACMG guidelines, the ACGS Best Practice Guidelines and consensus opinion. Functional analyses of identified genes were performed within STRING, Cytoscape and MCODE. Associated phenotypes were explored using the International Mouse Phenotyping Consortium. Gene expression analyses were performed within GENE2FUNC.
Results: 603/13612 (4.4%) patients were identified of whom 453/603 (75.1%) had trio ES. Pathogenic (P) or likely pathogenic (LP) variants were identified for 220/603 (36.5%) patients in 124 known disease-causing genes with SATB2 the most common (16/220, 7.3%). 23/220 (10.5%) patients had a P or LP CNV of partial or full contribution to their phenotype. 35/124 genes fulfilled criteria to be added to the PanelApp ‘Clefting’ panel, increasing the size of the current panel by 23.8%. Gene ontology and pathway analyses identified novel molecular networks for syndromic OC which were distinct from those in non-syndromic OC. Gene expression analyses and investigation of knockout phenotypes also showed a distinction between syndromic and non-syndromic OC. Pathway and expression analyses showed an enrichment of genes associated with intellectual disability (FDR=2.8x10-33), RNA metabolism (FDR<3.5x10-21), transcription (FDR<2.3x10-20) and chromatin organisation (FDR=1.03x10-11).
Conclusion: This study demonstrates the utility of ES and CNV analysis for patients with syndromic OC and increases the diagnostic rate for this patient cohort. It also highlights novel molecular pathways specific to syndromic OC and enhances our understanding of lip and palate development. 

RERE deficiencycontributes to the development of orofacial clefts in humans and mice

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7, and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH), but have not been shown to cause orofacial clefting. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate, and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox;Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of cleft palate in individuals with proximal 1p36 deletions and NEDBEH, and that RERE expression in cranial neural crest cells and their derivatives is required for normal palatal development. 

Bum-Jun Kim
Molecular & Human Genetics, Baylor College Medicine

Single cell transcriptomics-directed investigation of de novo mutations and rare inherited genetic variants in cleft lip and palate

Cleft lip and palate (CL/P) is one of the most common congenital anomalies. The etiology of CL/P is complex with both environmental and genetic risk factors. While previous studies have identified several CL/P-associated genes or regions, only a fraction of all cases can be clearly attributed to specific genes. Additional genetic causes may be due to rare inherited variants (RIVs) or de novo mutations (DNMs) in simplex CL/P cases. To investigate this further, we performed single cell RNA sequencing on epithelial cells of the lambdoidal junction (λ) from gestational day (E)10.5 wildtype mouse embryos at the point of upper lip fusion. We identified six cell clusters, and using the top 150 differentially expressed genes from each, we carried out targeted evaluation of both DNMs and RIVs in whole genome sequences from 756 CL/P case-parent trios of Asian, Latino, and European ancestry. For each cluster we analyzed enrichment of nonsynonymous, loss-of-function (LOF), and protein altering (nonsynonymous + LOF) DNMs. Of these, the olfactory epithelium cluster was enriched for protein altering (p=0.005) and the periderm cluster was enriched for nonsynonymous variants (p=0.005). We then evaluated exonic RIVs as defined by a minor allele frequency of <0.05% in gnomAD. A total of 2,976 variants were identified, with subsequent filtering for gene and variant specific constraint resulting in 445 variants of interest in 109 genes. Among these were several CL/P risk associated genes, including IRF6, TFAP2A, and GRHL3, all of which contained both DNMs and RIVs, suggesting other genes identified via this method may also be significant in risk for CL/P. Although all clusters were evaluated, the λ-fusion effector cluster was of specific interest given its critical role in prominence fusion during normal craniofacial development. This cluster harbored variants of interest in 31 genes, a higher percentage than other clusters (23% vs. 9-19%). Further, gene ontology revealed a group of 14 genes enriched for terms related to transcription and, interestingly, both negative regulation of epithelial cell proliferation (FDR 0.015) and positive regulation of mesenchymal cell proliferation (FDR 0.049). Among this group was transcription factor ZFHX3, which contained the most variants including loss-of-function DNMs and RIVs; thus, it remains of high interest for novel CL/P risk association. This investigation illustrates the benefit of integrating genomic technologies to prioritize and identify novel genetic associations with risk to CL/P. Continued evaluation focused on gene interactions and pathways represented by these variants may further elucidate CL/P etiology. 

Kelsey Robinson
Emory University

A novel 3 MB deletion on 6p24 removes distant neural-crest enhancers controlling TFAP2A resulting in mild Branchiooculofacial syndrome in a multiplex family with orofacial clefting

In a previous effort to characterize large copy number variants as genetic risk factors for orofacial clefting (OFC) following a genome-wide association study, we identified a novel 3 Mb deletion on 6p24 in three affected members of the same family from Colombia. The reported pedigree had a dominant inheritance pattern and included 5 individuals with OFC. All affected family members carried the deletion, which was inherited from the proband’s unaffected grandmother. Supporting the pathogenicity of this deletion, exome sequencing of this family found no segregating single nucleotide variants in OFC candidate genes. The 3Mb deleted region included 12 genes, none of which are strong OFC candidates and a 840 kb gene desert. We observed that the 3’ breakpoint of this deletion occurred within a large non-coding region directly adjacent to the important developmental transcription factor TFAP2A. TFAP2A loss of function is one of the primary genes associated with Branchiooculofacial syndrome (BFOS) that includes OFC in conjunction with branchial and ocular abnormalities. Affected members of this family display OFC, broad nasal root, and slight hypotelorism characteristic of BFOS. Cell type-specific enhancers in the genomic region directly flanking TFAP2A have been previously implicated in a subset of BOFS patients, however these enhancers remain intact in this family.We hypothesized that this deletion contains additional regulatory elements of TFAP2A, resulting in attenuated expression of this gene and a milder form of BOFS. To address this hypothesis and predict enhancer-gene interactions at this region, we used an activity by contact model (ABC-Enhancer) to integrate ChIP-seq based chromatin state annotations, chromosome conformation (Hi-C), and gene expression (RNA-Seq) from primary human craniofacial tissues and a culture model of cranial neural crest cells (CNCCs). We identified closely clustered, strong enhancer states that were predicted to have interactions with TFAP2A over a distance of greater than two megabases. To validate these predictions in vivo, we used CRISPR-Cas9 in human embryonic stem-cells to create homozyguous deletions of a 25kb region encompassing the strong enhancer segments. We differentiated these lines to CNCCs and compared gene expression and proliferative capacity to wildtype. We found this region is essential for cell viability during CNCC differentiation and differential gene expression analysis revealed substantial effects on TFAP2A expression. In summary, we identified a region within the inherited deletion that regulates TFAP2A gene expression and could contribute to the mild BOFS phenotype in this family. 

Tara Yankee
Department of Genetics and Genome Sciences, UConn Health Graduate Program in Genetics and Developmental Biology