Oculo-auriculo-vertebral spectrum (OAVS) is a complex developmental disorder of the first and second pharangeal arches characterized by asymmetric ear anomalies, hemifacial microsomia, ocular and vertebral defects. Congenital heart defects (CHDs), in 5-58% of patients, include ventricular septal defects and Tetralogy of Fallot (TOF), Coarctation of the Aorta (COA), and Transposition of the Great Arteries (TGA). OAVS is relatively rare, (1 -5,600 to 45,000 or 3.8 per 100000 births), and molecular diagnosis is complicated because of incomplete penetrance and clinical expressivity, alongside genetic heterogeneity. Most cases are sporadic but autosomal dominant (AD) cases, have been reported, which indicates a genetic component. Craniofacial (CF) abnormalities have severe physical and psychological consequences for patients, but OAVS aetiology is poorly understood, and better genetic diagnosis and treatments are needed. Our hypothesis was that OAVS is heterogeneous and genome-wide screening would uncover causal genes involved in important CF pathways, such as retinoic acid (RA) signalling. The aim was to identify and characterize novel genes associated with OAVS using microarray screening (aCGH) for copy number variants (CNVs), whole exome sequencing (WES) and Sanger sequencing (SS) of candidate genes in phenotyped patient cohorts, where known environmental causes of CF defects had been excluded. In silico bioinformatics analyses, alongside in vitro mutagenesis cell assays would be used to identify the consequences of variants on gene and protein expression and function. aCGH screening (15 OAVS, 47 CHD cases), identified 308 mainly benign CNVs (49 in OAVS, 182 in CHD). One overlapping pathogenic deletion in one OAVS and CHD patient (15q11.2 deletion/ TUBGCP5 CYFIP1, NIPA2, NIPA1) and 2 CNVs (22q11.2 deletion/ CRKL; 8p23.1 duplication/ GATA4) in 2 CHD patients, are known CHD loci and explain the cardiac phenotypes, but not OAVS. We also implicated four genes (SARS, ANGPT2, TBX6, and ZMYM5 duplications) in TGA and COA. In our cohort of 71 OAVS patients, 24 had WES, and we found variants for 4 AD families (9 affected, in which one has a sporadic variant). There are still 15 unexplained cases, and 47 patients have not had WES. Retinoic acid (RA) metabolising enzyme, CYP26A1, has a crucial role in head morphogenesis and is important for maintaining RA homeostasis during embryogenesis, and CYP26A1 variants (p.Arg418Ter, p.Ser132Leu and p.Ala245Val) were identified as a novel cause of OAVS. These pathogenic variants segregated with disease in 3 AD families (7/71, 10% of patients screened) and functional analysis using quantitative expression analyses (RT-qPCR and Western blotting) and in vitro protein cell assays (Cignal RARE-Luc reporter assays) in HEK293 cell lines, showed that mRNA expression was not affected but protein expression and RA clearing enzyme function were affected, making CYP26A1 a strong gene for OAVS. One patient has 2 loss-of-function variants: sporadic variant in AQR1 (p.Gln1374Ter) and an inherited variant in MED14 (Tyr927Ter) from affected mother. AQR1 and MED14 are also good candidates through their role in RA signalling and stem cell maintenance/vertebrate embryogenesis, respectively. Functional studies (gene and protein expression and activity in cell assays) are required to confirm the effects of the variants. Targeted engineering of the variants in animal models could be future work to determine if they cause OAVS phenotypes. There are still more causal genes to be identified in 62 OAVS patients, and WGS should aid this, prioritising genes involved in RA signalling for analysis. Ten genes have now been linked to OAVS (MYT1, ZYG11B, VWA1, ZIC3, EYA3, SF3B2, AMIGO2, YPEL1, OTX2, CRKL) in the literature and we identified an additional 3 genes, CYP26A1, AQR1 and MED14. Four genes (MYT1, ZYG11B, CYP26A1 and AQR1) appear to cause craniofacial abnormalities through their effect on RA signalling, highlighting this as an important pathway in OAVS. The outcome of this study will improve genetic testing and genetic counselling for OAVS patients and their families and help develop therapeutic interventions.
| Date of Award | 18 Oct 2022 |
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| Original language | English |
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| Awarding Institution | - The University of Manchester
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| Supervisor | Mayada Tassabehji (Main Supervisor) & Gillian Rice (Co Supervisor) |
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