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The Genetic Etiology of Sporadic Microtia: A computational analysis of whole exome sequencing data from individuals with microtia Abbas T. Shaikh1,*, Iván E. Rodríguez2, Connor Lester3, Frederic Deleyiannis4, Tamim H. Shaikh1 1Department of Pediatrics, University of Colorado, Anschutz Medical Campus, Aurora, CO; 2Department of Surgery, University of Colorado, Anschutz Medical Campus, Aurora, CO; University of Colorado Denver 3 4 Anschutz Medical Campus Georgetown University School of Medicine, Washington, D.C.; UCHealth Medical Group, Colorado Springs, CO Introduction Methods: Data Analysis • Microtia Variant Categorization • Microtia is a congenital deformity of the outer ear[1] Somatic Mutation Germline Mutation • • Severity ranges from mild structural deformations to complete absence of the auricle (outer Model • SNVs or Indels Model • high, moderate, or low • ear), known as anotia[1] • Variants found in one or • high, moderate, or low • Variants found in both impact affected and unaffected • Sporadic microtia is an isolated occurrence of microtia with no associated syndromes or both affected tissues but affected and unaffected tissues deformities not in unaffected tissues Variant Filtering Common Mutation Variant Filtering • Gene function and Model • Gene function and pathway relevant to • Variants consistent pathway relevant to microtia across multiple patients microtia • Frequency of variant in with microtia • Frequency of variant in unaffected individuals unaffected individuals • Expression in tissues • Expression in tissues relevant to ear relevant to ear development development • Variants in genes that • Variants in genes that matched with microtia matched with microtia candidate gene list candidate gene list (204) Results Somatic Mutation Model Germline Mutation Model 37 high-priority candidate genes: 18 high-priority candidate genes: Image taken from Luquetti et al. 2012[1] • Etiology of Microtia ACTB, ALDOA, ALPK2, CDK13, CEBPD, CFL1, CLU, AKAP12, ATRX, CHD7, COL4A3, COL4A4, COL9A1, • Strong genetic contribution to the etiology of microtia, in addition to environmental causes[1-3] CNN2 COL1A1, CSNK1G2, DACT3, DEAF1, ESRP1, DCHS1, GPR98, KDM6A, LOXHD1, MCPH1, MUC6, FMNL2, GAS6, GNA12, GRHL2, HNRNPAB, MYO15A, PCNT, PLEC, SALL1, SIX1, TRIOBP • Patients can present with microtia as an isolated occurrence or as part of a syndrome (20– 60%)[2] HSPB1, KRT4, KRT10, MAF, MAP7D1, MARCKSL1, • Mutations in HOX, SIX, and EYA family genes have frequently been implicated in syndromic microtia[1] MUC3A, MUC6, NOTCH2, OGFR, POLE, RHOA, Genes previously strongly associated RNF187, SPARC, TCF25, TUBB6, UBB, VIM, YBX1 • However, the genetic etiology of sporadic, non-syndromic, microtia has remained elusive with microtia: Genes previously associated with DCHS1, SALL1, CHD7, SIX1, COL4A4 Rationale and Hypothesis microtia: MUC6 and UBB Genes associated with syndromes that impact ear development: • Microtia has a prevalence ranging from Wnt Signaling Pathway: GPR98, COL9A1, DCHS1, SALL1, KDM6A, 1-17.4 in every 10000 births[3] UBB, VIM, ALPK2, CSNK1G2, DACT3, TCF25, MYO15A, CHD7, SIX1, COL4A3, COL4A4 • Microtia places a significant and RHOA psychological and socioeconomic Townes-Brocks CHARGE SALL1 CHD7 burden on affected individuals and their MAPK/ERK Signaling Pathway GNA12, HSPB1, ESRP1, YBX1, CFL1, GAS6, families[1] NOTCH2, and COL1A1 • Identifying genes associated with sporadic microtia would provide the Notch Signaling Pathway first steps to developing methods to NOTCH2 and UBB control or treat microtia Genes associated with structural • Hypothesis: Sporadic microtia is caused deformities by mosaic, post-zygotic mutations in CLU, FMNL2, KRT4, KRT10, CNN2, VIM, [2] exons of genes, which are most likely to ACTB, COL1A1, TUBB6, and MAP7D1 Image taken from Cox et al. 2014 affect protein function Image taken from BioNinja https://ib.bioninja.com.au/standard-level/topic-3-genetics/33-meiosis/somatic-vs-germline-mutatio.html Transcription and Growth Factors: GRHL2, MAF, POLE, YBX1, HNRNPAB, Common Mutation Model Materials CEBPD, OGFR and ESRP1 No high-priority candidates identified Resources available for Study Discussion • Tissue samples from patients with microtia who had corrective surgery • Candidate genes identified under the Somatic Mutation Model • Wnt Signaling Pathway: involved in embryonic development through control of the • affected ear cartilage and skin formation of important tissues • unaffected rib cartilage • MAPK/ERK Signaling Pathway: involved in cell proliferation and survival • Whole exome sequencing (WES) data from • Notch Signaling Pathway: involved in cell fate decisions, cell communication, and regulation affected and unaffected tissues from 7 of the cytoskeleton microtia patients All Exons in the Genome = Exome • NOTCH2 has also been implicated in various facial deformities • Variant call files (VCFs) from the analysis of Image taken from: Maji, S. & Kanrar, S. SpliceCombo: A Hybrid Technique Efficiently Use • Genes identified as structural constituents suggest being directly involved structural for Principal Component Analysis of Splice Site Prediction. Ingénierie des systèmes d WES which provided: information 24, 67–75 (2019). deformities such as microtia and are important to auricular morphogenesis • single nucleotide variants (SNVs) and • Transcription and growth factors: Genes identified are important to control and regulate insertion-deletions (indels) the rate of development • location of mutations in exons of genes • Candidate genes identified under the Germline Mutation Model • type of mutation; missense, nonsense, • 5 genes strongly associated with microtia and 10 implicated in syndromes affecting ear splice site, frameshift development, suggests germline variants play a role in the etiology of microtia • impact of mutation on protein function • Proposed additive model in the etiology of sporadic microtia: Computational Resources and • Low effect, germline variants in microtia candidate genes combined with moderate to high effect somatic variants in other critical genes may together lead to an increased risk of Databases Image taken from Biology Dictionary https://biologydictionary.net/missense-mutation/ microtia in individual patients • Python and associated data analysis packages including Pandas and NumPy • This model needs to be validated in a larger sample • Genome Aggregation Database (gnomAD) - https://gnomad.broadinstitute.org/ • Future research would involve modeling the risk of having microtia based on a profile of • GeneCards – genecards.org/ and PathCards – pathcards.genecards.org/ observed genetic variants. This could help identify genes/variants that are most critical to the • Molecular Signatures Database - https://www.gsea-msigdb.org/ development of microtia • UCSC Genome Browser - https://genome.ucsc.edu/ • UniProt – uniport.org/ and Reactome – reactome.org/ Acknowledgements • Panther Classification System - http://www.pantherdb.org/ • Database for Annotation, Visualization and Integrated Discovery (DAVID) - https://david.ncifcrf.gov/ • Trips to Guatemala to provide Microtia repair at the Moore Pediatric Surgery Center were funded by the References John Lester Foundation in collaboration with Children's Hospital Colorado 1. Luquetti DV, Heike CL, Hing AV, Cunningham ML, Cox TC. Microtia: epidemiology and genetics. Am J Med Genet. 2012, 158A(1):124-39. • Funds for sequencing were provided by the 2. Cox T. C., Camci E. D., Vora S., Luquetti D. V. & Turner E. E. The genetics of auricular development and malformation: new findings in model systems driving future directions for microtia research. Eur J Med Genet. 2014, 57:394–401 Department of Surgery, University of Colorado 3. Alasti F, Van Camp G. Genetics of microtia and associated syndromes. J Med Genet. 2009, 46(6):361-9. Anschutz Medical Campus.