Human Genetics

Meniere disease (MD) is a polygenic condition defined by episodes of vertigo associated with sensorineural hearing loss and tinnitus. Genetic studies in familial MD in East Asian population are limited and the potential MD genes remain to be established in non-Finnish European populations. By exome sequencing and rare variant analysis, we search for existing and novel genes associated with MD in a South Korean cohort of 16 MD individuals with bilateral sensorineural hearing loss. We have found one individual with two rare missense variants in the OTOP2 gene, a new candidate gene for MD and three heterozygous variants in the MYO7A gene, supporting the hypothesis of biallelic inheritance. Protein modelling was conducted on three rare missense variants in OTOP2 to further elucidate functional consequences. The structural and functional implications inferred from these models suggest a likely pathogenic role, providing additional insights into the molecular mechanisms underlying MD.

Sensorineural hearing loss (SNHL) is one of the most common sensory disorders, predominantly driven by monogenic causes with a higher Mendelian contribution. Although ELMOD3 variants have been implicated in both autosomal dominant (DFNA81) and autosomal recessive (DFNB88) hereditary deafness, only a single DFNA81 family has been reported, leaving the pathogenic role of the dominant allele largely unexplored. Through targeted panel sequencing, we herein identified a novel heterozygous ELMOD3 variant (c.640G>A; p.Gly214Ser) that co-segregates with autosomal dominant hearing loss in a Korean family. Molecular modeling and structure analysis indicates that replacing glycine at residue 214 with serine introduces spatial clashes with adjacent Ala160 and Cys162, thereby disrupting intermolecular interactions and compromising protein stability. Consistent with this, stability assays revealed a rapid degradation rate for the mutant protein. Furthermore, the ability to localize with F-actin in mutant protein was disrupted compared to the wild-type protein. Based on functional assays, the p.Gly214Ser variant demonstrated the functional pathogenicity and was classified as likely pathogenic according to the ACMG guideline for hearing loss. Collectively, these findings provide confirmatory insights into ELMOD3-associated DFNA81, potentially through a dominant-negative mechanism.

PurposeThe transcription factor TBX2 plays a critical role in inner hair cells development in mice. Yet, the link between TBX2 malfunction and human hearing-related disorders remains unexplored. MethodsLinkage analysis combined with whole genome sequencing was applied to identify the causative gene in two autosomal dominant Chinese families characterized by late-onset progressive sensorineural hearing loss and incomplete penetrance of horizontal oscillatory nystagmus. Functional evaluation of TBX2 variants was performed through protein expression, localization, and transcriptional activity analysis in vitro, phenotypic analysis and mechanism study in knockout mice model in vivo. ResultsMultipoint parametric linkage analysis of Family 1 revealed a maximum LOD score of 3.01 on chromosome 17q23.2. Whole genome sequencing identified distinct TBX2 variants, c.977delA (p.Asp326Alafs*42) and c.987delC (p.Ala330Argfs*38) in each family, co-segregating with hearing loss. These variants resulted in premature termination and the generation of a new peptide segment, reducing transcriptional activity. Further, heterozygous Tbx2 knockout mice exhibited late-onset progressive hearing loss, along with ectopic expression of Prestin in IHCs and a gradual decrease in expression from P7 to P42. ConclusionOur findings indicate that heterozygous TBX2 frameshift variants are the genetic cause of late-onset progressive hearing loss and incomplete penetrance of nystagmus. The heterozygous Tbx2 knockout mouse model mirrored the human hearing loss phenotype, further validating TBX2s role in auditory function. These insights enhance our understanding of TBX2 in the auditory system, providing valuable information for molecular diagnostics and genetic counseling in related hearing disorders.

Congenital heart defects (CHD) are structural defects of the heart affecting approximately 1% of newborns. CHDs exhibit a complex inheritance pattern. While genetic factors are known to play an important role in the development of CHD, relatively few variants have been discovered so far and very few genome-wide association studies (GWAS) have been conducted. We performed a GWAS of general CHD and five CHD subgroups in FinnGen followed by functional fine-mapping through eQTL analysis in the GTEx database, and target validation in human induced pluripotent stem cell - derived cardiomyocytes (hiPS-CM) from CHD patients. We discovered that the MYL4-KPNB1 locus (rs11570508, beta = 0.24, P = 1.2x10-11) was associated with the general CHD group. An additional four variants were significantly associated with the different CHD subgroups. Two of these, rs1342740627 associated with left ventricular outflow tract obstruction defects and rs1293973611 associated with septal defects, were Finnish population enriched. The variant rs11570508 associated with the expression of MYL4 (normalized expression score (NES) = 0.1, P = 0.0017, in the atrial appendage of the heart) and KPNB1 (NES = -0.037, P = 0.039, in the left ventricle of the heart). Furthermore, lower expression levels of both genes were observed in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) from CHD patients compared to healthy controls. Together, the results demonstrate KPNB1 and MYL4 as in a potential genetic risk loci associated with the development of CHD.

Primary ciliopathies are a group of inherited developmental disorders resulting from defects in the primary cilium. Mutations in CEP290 (Centrosomal protein of 290kDa) are the most frequent cause of recessive ciliopathies (incidence up to 1:15,000). Pathogenic variants span the full length of this large (93.2kb) 54 exon gene, causing phenotypes ranging from isolated inherited retinal dystrophies (IRDs; Leber Congenital Amaurosis, LCA) to a pleiotropic range of severe syndromic multi-organ ciliopathies affecting retina, kidney and brain. Most pathogenic CEP290 variants are predicted null (37% nonsense, 42% frameshift), but there is no clear genotype-phenotype association. Almost half (26/53) of the coding exons in CEP290 are in-phase "skiptic" (or skippable) exons. Variants located in skiptic exons could be removed from CEP290 transcripts by skipping the exon, and nonsense-associated altered splicing (NAS) has been proposed as a mechanism that attenuates the pathogenicity of nonsense or frameshift CEP290 variants. Here, we have used in silico bioinformatic techniques to study the propensity of CEP290 skiptic exons for NAS. We then used CRISPR-Cas9 technology to model CEP290 frameshift mutations in induced pluripotent stem cells (iPSCs) and analysed their effects on splicing and ciliogenesis. We identified exon 36, a hotspot for LCA mutations, as a strong candidate for NAS that we confirmed in mutant iPSCs that exhibited sequence-specific exon skipping. Exon 36 skipping did not affect ciliogenesis, in contrast to a larger frameshift mutant that significantly decreased cilia size and incidence in iPSCs. We suggest that sequence-specific NAS provides the molecular basis of genetic pleiotropy for CEP290-related disorders.

DNA sequencing of patients with rare disorders has been highly successful in identifying "causal variants" for numerous conditions. However, there are many reports of healthy individuals who harbor these deleterious variants, leading to the concept of incomplete penetrance and doubt about the utility of genetic testing in clinical practice and population screening. As the deleterious variants are rare, the penetrance of these variants in the population is largely unknown. We analyzed the genetic and clinical data from 486,956 participants of the Taiwan Precision Medicine Initiative (TPMI) to determine the risk difference between those with and without deleterious variants. In all, we analyzed 292 disease-relevant variants and their clinical outcomes to assess their association. We found that only 15 variants show a risk difference exceeding 5% between those with or without the variants. In essence, 87.3% of deleterious variants exhibit minimal risk differences, suggesting a limited impact on the individual and population levels. Our analysis revealed increasing trends with age in six cardiovascular and degenerative diseases and bell-shaped trends in two cancers. Additionally, we identified three clinical outcomes exhibiting a dose-response relationship with the number of deleterious variants. Our findings show that large-scale testing of deleterious variants found in the literature is not warranted, except for those exhibiting large disease risk differences.

Familial Meniere Disease (FMD) is a rare polygenic disorder of the inner ear. Mutations in the connexin gene family, which encodes gap junction proteins, can also cause hearing loss, but their role in FMD is largely unknown. Here, we found an enrichment of rare missense variants in the GJD3 gene when comparing allelic frequencies in FMD (N=94) with the Spanish reference population (OR=3.9[1.92-7.91], FDR=2.36E-03). In the GJD3 sequence, we identified a rare haplotype (TGAGT) composed of two missense, two synonymous, and one downstream variants. This haplotype was found in five individuals with FMD, segregating in three unrelated families with a total of ten individuals; and in another eight Meniere Disease individuals. GJD3 encodes the gap junction protein delta 3, also known as human connexin 31.9 (CX31.9). The protein model predicted that the NP_689343.3:p.(His175Tyr) missense variant could modify the interaction between connexins and the connexon assembly, affecting the homotypic GJD3 gap junction between cells. Our studies in mice revealed that the mouse ortholog Gjd3 - encoding Gjd3 or mouse connexin 30.2 (Cx30.2) - was expressed in the organ of Corti and vestibular organs, particularly in the tectorial membrane, the base of inner and outer hair cells and the nerve fibers. The present results describe a novel association between GJD3 and familial FMD, providing evidence that FMD is related to changes in the inner ear channels; in addition, it supports a new role of tectorial membrane proteins in FMD.

Menieres Disease (MD) is a chronic inner ear disorder defined by recurring episodes of vertigo, fluctuating sensorineural hearing loss, tinnitus, and/or fullness in the ear. Its prevalence varies by region and ethnicity, with scarce epidemiological data in Brazilian population. Although most MD cases are sporadic, Familial MD (FMD) is observed in 5% to 20% of European cases. By exome sequencing, we have found a rare missense variant in the OTOG gene in a Brazilian MD individual with probable European ancestry (chr11:17599671C>T), which was previously reported in a Spanish cohort. Two additional rare missense heterozygous OTOG variants were found in the same proband. Splice Site analysis showed that chr11:17599671C>T may lead to substantial changes generating exonic cis regulatory elements, and protein modelling revealed structural changes in the presence of chr11:17599671C>T, chr11:17576581G>C and chr11:17594108C>T, predicted to highly destabilize protein structure. These findings indicate that missense variants may have an additive effect leading to an unstable Otogelin and support OTOG gene as a key player in the MD pathophysiology.

PurposeInvestigate the genetic components of sensorineural hearing loss (SNHL) by performing genome-wide meta-analyses using the data from FinnGen and Estonian Biobank. MethodsWe studied genome-wide associations of SNHL in FinnGen and the Estonian Biobank in the general population and in sex- and age-of-onset stratified subgroups. The study-specific GWASs were combined through inverse variance-weighted genome-wide meta-analyses, encompassing a total of 531,059 individuals (Ncases=35,960). Age-stratified meta-analyses included 28,198 individuals diagnosed at the age of 55 years or after and 7,762 individuals diagnosed before the age of 55 years, with 495,099 controls. Sex-stratified meta-analyses included 313,501 females (Ncases=17,761) and 217,558 males (Ncases=18,199). ResultsIn the meta-analysis focusing on the general population, 22 SNHL-associated loci ({+/-}1 Mb window) were observed, seven of which were previously unreported. In the sex-stratified analysis, two previously unreported SNHL loci were observed in the female subgroup and one locus in the male subgroup. Additionally, in the age-stratified analysis, three previously unreported SNHL loci were observed in the subgroup of those that were diagnosed at the age of 55 years or after. In those diagnosed before the age of 55 years, one previously unreported locus was observed. Overall, 32 loci were associated with SNHL at p<5x10-8 in at least one of the study groups. Of these, 14 loci have not been previously reported in association with SNHL. We also estimated if there were significant differences in the effect sizes of the lead variants at each locus between the analytical subgroups and observed differences for 14 variants. ConclusionsPreviously unreported SNHL risk loci and differences in effect sizes found in this study provide additional insight into the genetic underpinnings of SNHL. Our results validate the role of mechano-transduction and genetic components affecting the structure of the inner ear in the background of SNHL. Our study contributes to our understanding of the genetic causes of SNHL and may open the door for further research into innovative therapies and preventative measures.

Cleft lip and/or palate (CL/P) occur in approximately 1 in 700 live births in the United States. High hereditary rates (50-80%) of CL/P indicate a strong genetic cause. The concept of strong genetic causes has been well-demonstrated in previous studies such as GWAS studies that identified IRF6 for Van der Woode syndrome. Since the risk for genetic factors is strongly associated with CL/P, we hypothesized that RNA sequencing (RNA-seq) from CL/P patients may reveal enriched genes. Differential expression analysis examined changes in gene expression in CL/P patients compared to healthy controls, and gene-enrichment in relevant pathways. To explore the relationship between variants driving the observed changes in gene expression, we performed variant analysis and reported all CL/P-specific single nucleotide polymorphisms (SNPs). Our findings demonstrate that the normally upregulated MUC7 gene is significantly downregulated in CL/P patients. Using our list of prioritized differentially expressed genes (DEGs), we observed significantly enriched pathways for biological processes related to cornification, skin and epidermis development, and keratinocyte and epidermal cell differentiation. By performing variant analyses, a single nucleotide polymorphism (SNP) in MUC7, and 47 SNPs in 20 additional genes (CLCA4, ETNK2, ERLNC1, HAL, HOPX, IVL, KLK11, LIPK, LY6D, MUC21, NCCRP1, NEBL, PHYH, SERPINB11, SERPINB4, SORD, SPINK5, SULT2B1, TMEM154, TMPRSS11A) were revealed. To our knowledge, this is the first report on the potential role of MUC7 in contributing to CL/P. Together, these findings provide further insight into the genetic causes of CL/P.

Biallelic variants in ABCA4 cause Stargardt disease (STGD1), the most frequent heritable macular disease. Determination of the pathogenicity of variants in ABCA4 proves to be difficult due to 1) the high number of benign and pathogenic variants in the gene; 2) the presence of complex alleles; 3) the extensive variable expressivity of this disease and 4) reduced penetrance of hypomorphic variants. Therefore, the classification of many variants in ABCA4 is currently of uncertain significance. Here we complemented the ABCA4 Leiden Open Variation Database (LOVD) with data from [~]11,000 probands with ABCA4-associated inherited retinal diseases from literature up to the end of 2020. We carefully adapted the ACMG/AMP classifications to ABCA4 and assigned these classifications to all 2,247 unique variants from the ABCA4 LOVD to increase the knowledge of pathogenicity. In total, 1,247 variants were categorized with a Likely Pathogenic or Pathogenic classification, whereas 194 variants were categorized with a Likely Benign or Benign classification. This uniform and improved structured reclassification, incorporating the largest dataset of ABCA4-associated retinopathy cases so far, will improve both the diagnosis as well as genetic counselling for individuals with ABCA4-associated retinopathy.

1.Alkaptonuria or black urine disease is a rare autosomal recessive disorder caused by dysfunctional homogentisate 1,2-dioxygenase (HGD) gene (3q13.33) leading to accumulation of homogentisic acid in the body. This inborn error in metabolism of phenylalanine and tyrosine causes accumulation of homogentisic acid leading to ochronosis, pigmentation in the sclera, ear cartilage, mitral valve calcification and osteoarthropathy. Advances in sequencing technologies have helped us to map genetic variants associated with alkaptonuria in diverse populations and regions. Currently, no centralized resource of all the reported actionable variants with uniformity in annotation exists for the HGD gene. We have compiled HGD exonic variants from various data sources and systematically annotated their pathogenicity according to American College of Medical Genetics and the Association of Molecular Pathologists (ACMG/AMP) variant classification framework. A total of 1686 exonic variants were catalogued and manually curated, creating one of the most comprehensive Alkaptonuria variant databases (AKUHub) which is publicly available.

Intellectual disability (ID) is a neurodevelopmental disorder affecting up to 1-3% of people worldwide. Genetic factors, including rare de novo or rare homozygous mutations, explain many cases of autosomal dominant or recessive forms of ID. ID is clinically and genetically heterogeneous, with hundreds of genes associated with it. In this study, we performed high-depth whole-genome sequencing of twenty individuals from five consanguineous families from Pakistan, with nine individuals affected by mild or severe ID. We identified one splice and five missense rare variants (at allele frequencies below 0.001%) in a homozygous state in the affected individuals with supporting and moderate evidence of pathogenicity based on guidance from the American College of Medical Genetics and Genomics. These six variants mapped to different genes (SRD5A3, RDH11, RTF2, PCDHA2, ADAMTS17, and TRPC3), and only SRD5A3 had previously been known to cause ID. The p.Tyr169Cys mutation inside SRD5A3 was predicted to be deleterious and affect protein structure by multiple in silico tools. In addition, we found one missense mutation, p.Pro1505Ser, inside UNC13B with conflicting evidence of pathogenic and benign effects. Further functional studies are required to confirm the pathogenicity of these variants and understand their role in ID. Our findings provide additional needed information for interpreting rare variants in the genetic testing of ID.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder that causes sudden death in young adults. Rare genetic variants in the SCN5A gene, encoding the Nav1.5 sodium channel, and common non-coding variants at this locus, are robustly associated with the condition. BrS is particularly prevalent in Southeast Asia but the underlying ancestry-specific factors remain largely unknown. Here, we performed genome sequencing of BrS probands from Thailand and population-matched controls and identified a rare non-coding variant in an SCN5A intronic enhancer that is highly enriched in BrS cases (3.9% in cases, odds ratio 20.2-45.2) and predicted to disrupt a Mef2 transcription factor binding site. Heterozygous introduction of the enhancer variant in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) caused significantly reduced SCN5A expression from the variant-containing allele and a 30% reduction in Nav1.5-mediated sodium-current density compared to isogenic controls. This is the first example of a validated rare non-coding variant at the SCN5A locus and partly explains the increased prevalence of BrS in this geographic region.

As one of the most common structural birth defects, orofacial clefts (OFCs) have been studied for decades, and recent studies have demonstrated that there are genetic differences between the different phenotypic presentations of OFCs. However, the contribution of rare genetic variation genome-wide to different subtypes of OFCs has been understudied, with most studies focusing on common genetic variation or rare variation within targeted regions of the genome. Therefore, we used whole-genome sequencing data from the Gabriella Miller Kids First Pediatric Research Program to conduct a gene-based burden analysis to test for genetic modifiers of cleft lip (CL) vs cleft lip and palate (CLP). We found that there was a significantly increased burden of rare variants in SEC24D in CL cases compared to CLP cases (p=6.86x10-7). Of the 15 variants within SEC24D, 53.3% were synonymous, but overlapped a known craniofacial enhancer. We then tested whether these variants could alter predicted transcription factor binding sites (TFBS), and found that the rare alleles destroyed binding sites for 9 transcription factors (TFs), including Pax1 (p=0.0009), and created binding sites for 23 TFs, including Pax6 (p=6.12x10-5) and Pax9 (p= 0.0001), which are known to be involved in normal craniofacial development, suggesting a potential mechanism by which these synonymous variants could have a functional impact. Overall, this study demonstrates that rare genetic variation contributes to the phenotypic heterogeneity of OFCs and suggests that regulatory variation may also contribute and warrant further investigation in future studies of genetic variants controlling risk to OFC.

Age-related hearing loss is a widespread sensory impairment affecting a significant proportion of the elderly population, yet the genetic underpinnings of this condition remain incompletely understood. In this study, we investigate a non-synonymous variant (rs2242416) in the CRIP3 gene, which is expressed in auditory hair cells, in the context of hearing loss. Firstly, we find the variant shows strong and consistent association with hearing loss across multiple genome-wide association studies. Secondly, this variant, substitutes the nonpolar isoleucine for the polar threonine at an amino acid site that is otherwise highly conserved across placental mammals. Thirdly, by causing the amino acid change, the variant subtly alters the structure of the CRIP3 protein. Together, these three analyses provide phenotypic, evolutionary, and molecular evidence for the functionality of CRIP3 and its role in hearing loss. Moreover, the population genetics of the CRIP3 locus reveals an increased frequency of the derived threonine allele of rs2242416 in Eurasian populations following the out-of-Africa migration of humans more than 50,000 years ago. Nevertheless, the role of Darwinian selection in this increased frequency remains inconclusive. Overall, our results make a compelling argument to auditory researchers to make a CRIP3 mouse model to pinpoint the precise role of the protein in auditory function. Such a model will pave the way for therapeutic interventions targeting the CRIP3 protein to mitigate age-related hearing loss.

Common diseases exhibit substantial heritability, and GWAS of these diseases have revealed hundreds of thousands of high-frequency disease susceptibility variants throughout the genome. These studies offer the prospect of using genomic data to improve disease prediction and diagnosis, however, the relative performance of different predictive modeling approaches is not well-characterized. To investigate this systematically, we constructed a Monte Carlo simulation generating model genomes with large numbers of SNPs, with a proportion of SNPs carrying risk alleles that are parameterized by the strength of their effects and by different modes of inheritance - additive, dominant, recessive, and combinations thereof. After generating genotypes for cases and controls, several machine learning classifiers (logistic regression, naive Bayes, random forests, and neural networks, with and without feature selection) were applied to predict disease phenotype from genotypes. Each classifiers rates of false positives and false negatives were evaluated and compared using AUC. We found that random forest models were the most accurate predictors of disease phenotype over the range of inheritance parameters, followed by logistic regression and naive Bayes, while the feedforward multilayer neural network-based predictive model had lower AUC. Furthermore, with the small fraction of null sites in our model, there was almost no difference in the performance of classifiers with or without LASSO-based feature selection. We also investigate the association of AUC with the difference in polygenic risk score (PRS) between disease and control samples by comparing AUC in the simulations to the values predicted from the PRS distributions based on odds-risk and liability models.

Effective research and clinical application in audiology and hearing loss (HL) often require the integration of diverse data. However, the absence of a dedicated database impeded understanding and insight extraction in HL. To address this, the Genetic Deafness Commons (GDC) was developed by consolidating extensive genetic and genomic data from 51 public databases and the Chinese Deafness Genetics Consortium, encompassing 5,983,613 variants across 201 HL genes. This comprehensive dataset detailed the genetic landscape of HL, identifying six novel mutational hotspots within DNA binding domains of transcription factor genes, which were eligible for evidence-based variant pathogenicity classification. Comparative phenotypic analyses highlighted considerable disparities between human and mouse models, with only 130 human HL genes exhibiting hearing abnormality in mice. Moreover, gene expression analyses in the cochleae of mice and rhesus macaques demonstrated a notable correlation (R2 = 0.76). Utilizing gene expression, function, pathway, and phenotype data, a SMOTE-Random Forest model identified 18 candidate HL genes, including TBX2 and ERCC2, newly confirmed as HL genes. The GDC, as a comprehensive and unified repository, significantly advances audiology research and clinical practice by enhancing data accessibility and usability, thereby facilitating deeper insights into hearing disorders.

Identification of host genetic factors that predispose individuals to severe COVID-19 is important, not only for understanding the disease and guiding the development of treatments, but also for risk prediction when combined to form a polygenic risk score (PRS). Using population controls, Pairo-Castineira et al. identified 12 SNPs (a panel of 8 SNPs and a panel of 6 SNPs, with two SNPs in both panels) associated with severe COVID-19. Using controls with asymptomatic or mild COVID-19, we were able to replicate the association with severe COVID-19 for only three of their SNPs and found marginal evidence for an association for one other. When combined as an 8-SNP PRS and a 6-SNP PRS, we found no evidence of association with severe COVID-19. The difference in our results and the results of Pairo-Castineira et al. might be the choice of controls: population controls vs controls with asymptomatic or mild COVID-19.

BackgroundOculoauriculovertebral Spectrum (OAVS) encompasses abnormalities on derivatives from the first and second pharyngeal arches including macrostomia, hemifacial microsomia, micrognathia, preauricular tags, ocular and vertebral anomalies. We present genetic findings on a three-generation family affected with macrostomia, preauricular tags and uni- or bilateral ptosis following an autosomal dominant pattern. MethodsWe generated whole genome sequencing data for the proband, affected parent and unaffected paternal grandparent followed by Sanger sequencing on 23 family members for the top 10 candidate genes: KCND2, PDGFRA, CASP9, NCOA3, WNT10A, SIX1, MTF1, KDR/VEGFR2, LRRK1, and TRIM2 We performed parent and sibling-based transmission disequilibrium tests and burden analysis via a penalized linear mixed model, for segregation and mutation burden respectively. Next, via bioinformatic tools we predicted protein function, mutation pathogenicity and pathway enrichment to investigate the biological relevance of mutations identified. ResultsRare missense mutations in SIX1, KDR/VEGFR2, and PDGFRA showed the best segregation with the OAV phenotypes in this family. When considering any of the 3 OAVS phenotypes as an outcome, SIX1 had the strongest associations in parent-TDTs and sib-TDTs (p=0.025, p=0.052) (unadjusted p-values). Burden analysis identified SIX1 (RC=0.87) and PDGFRA (RC=0.98) strongly associated with OAVS severity. Using phenotype-specific outcomes, sib-TDTs identified SIX1 with uni- or bilateral ptosis (p=0.049) and ear tags (p=0.01), and PDGFRA and KDR/VEGFR2 with ear tags (both p<0.01). ConclusionSIX1, PDGFRA, and KDR/VEGFR2 are strongly associated to OAVS phenotypes. SIX1 has been previously associated with OAVS ear malformations and is co-expressed with EYA1 during ear development. Efforts to strengthen the genotype-phenotype co-relation underlying the OAVS are key to discover etiology, family counseling and prevention.