Decreased Tiam1-mediated Rac1 activation is responsible for impaired directional persistence of chondrocyte migration in microtia.

The human auricle has a complex structure, and microtia is a congenital malformation characterized by decreased size and loss of elaborate structure in the affected ear with a high incidence. Our previous studies suggest that inadequate cell migration is the primary cytological basis for the pathogenesis of microtia, however, the underlying mechanism is unclear. Here, we further demonstrate that microtia chondrocytes show a decreased directional persistence during cell migration. Directional persistence can define a leading edge associated with oriented movement, and any mistakes would affect cell function and tissue morphology. By the screening of motility-related genes and subsequent confirmations, active Rac1 (Rac1-GTP) is identified to be critical for the impaired directional persistence of microtia chondrocytes migration. Moreover, Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) are detected, and overexpression of Tiam1 significantly upregulates the level of Rac1-GTP and improves directional migration in microtia chondrocytes. Consistently, decreased expression patterns of Tiam1 and active Rac1 are found in microtia mouse models, Bmp5se/J and Prkralear-3J/GrsrJ. Collectively, our results provide new insights into microtia development and therapeutic strategies of tissue engineering for microtia patients.

Demographic, Functional, and Risk Factors Characterization in Ecuadorian Mestizo Patients With Microtia: A Retrospective Study.

OBJECTIVE: This paper compares demographic, morphological, functional, and risk factors between isolated and familial forms of microtia in Ecuadorian mestizo patients. METHODS: The authors did an epidemiological, and retrospective study with 112 patients divided into isolated microtia (n = 91) and familial microtia (n = 21). Patients with syndromic microtia were not included. RESULTS: In isolated microtia, the mean age was 11.80 ± 16.9, and the most prevalent age group was from 5 to 9 years, with 45.0%; males were 58.2%, and 91.2% of patients were born in a city above 2500 meters about sea level. In familial microtia, the mean age was 15.57 ± 17.2. There were no statistically significant differences between the analyzed variables. In isolated microtia, 41.8% of patients had bilateral involvement, 40.7% had grade 1 microtia in the right ear (RE), and grade 1 in the left ear was 47.3%; external auditory canal atresia of RE was present in 62.6%, and in left ear in 31.6%. External auditory canal atresia sidedness was mostly unilateral in both groups. Most patients did not have tags or pits (78% and 81% in RE and 85.7% and 71.4%). Most patients had moderate hearing loss in both ears. CONCLUSION: The authors found an association between both microtia forms with external auditory canal atresia in RE; only 20% of patients had unilateral auricular tags or pits in both groups. The authors also found a high incidence (18.75%) of familial microtia, which suggests a distinct pathological genetic component than the more prevalent isolated cases. The authors found a high association of microtia cases from the Ecuadorian highlands above 2500 meters about sea level (over 90%). The presence of "social" intake of alcohol during pregnancy showed over twice the chance of having a child born with microtia.

Multi-omics analysis of a case of congenital microtia reveals aldob and oxidative stress associated with microtia etiology.

BACKGROUND: Microtia is reported to be one of the most common congenital craniofacial malformations. Due to the complex etiology and the ethical barrier of embryonic study, the precise mechanisms of microtia remain unclear. Here we report a rare case of microtia with costal chondrodysplasia based on bioinformatics analysis and further verifications on other sporadic microtia patients. RESULTS: One hundred fourteen deleterious insert and deletion (InDel) and 646 deleterious SNPs were screened out by WES, candidate genes were ranked in descending order according to their relative impact with microtia. Label-free proteomic analysis showed that proteins significantly different between the groups were related with oxidative stress and energy metabolism. By real-time PCR and immunohistochemistry, we further verified the candidate genes between other sporadic microtia and normal ear chondrocytes, which showed threonine aspartase, cadherin-13, aldolase B and adiponectin were significantly upregulated in mRNA levels but were significantly lower in protein levels. ROS detection and mitochondrial membrane potential (∆ Ψ m) detection proved that oxidative stress exists in microtia chondrocytes. CONCLUSIONS: Our results not only spot new candidate genes by WES and label-free proteomics, but also speculate for the first time that metabolism and oxidative stress may disturb cartilage development and this might become therapeutic targets and potential biomarkers with clinical usefulness in the future.

BMP signaling maintains auricular chondrocyte identity and prevents microtia development by inhibiting protein kinase A.

Elastic cartilage constitutes a major component of the external ear, which functions to guide sound to the middle and inner ears. Defects in auricle development cause congenital microtia, which affects hearing and appearance in patients. Mutations in several genes have been implicated in microtia development, yet, the pathogenesis of this disorder remains incompletely understood. Here, we show that Prrx1 genetically marks auricular chondrocytes in adult mice. Interestingly, BMP-Smad1/5/9 signaling in chondrocytes is increasingly activated from the proximal to distal segments of the ear, which is associated with a decrease in chondrocyte regenerative activity. Ablation of Bmpr1a in auricular chondrocytes led to chondrocyte atrophy and microtia development at the distal part. Transcriptome analysis revealed that Bmpr1a deficiency caused a switch from the chondrogenic program to the osteogenic program, accompanied by enhanced protein kinase A activation, likely through increased expression of Adcy5/8. Inhibition of PKA blocked chondrocyte-to-osteoblast transformation and microtia development. Moreover, analysis of single-cell RNA-seq of human microtia samples uncovered enriched gene expression in the PKA pathway and chondrocyte-to-osteoblast transformation process. These findings suggest that auricle cartilage is actively maintained by BMP signaling, which maintains chondrocyte identity by suppressing osteogenic differentiation.

Co-culture of RhoA-overexpressed microtia chondrocytes and adipose-derived stem cells in the construction of tissue-engineered ear-shaped cartilage.

Microtia is a congenital auricle dysplasia with a high incidence and tissue engineering technology provides a promising strategy to reconstruct auricles. We previously described that the engineered cartilage constructed from microtia chondrocytes exhibited inferior levels of biochemical and biomechanical properties, which was proposed to be resulted of the decreased migration ability of microtia chondrocytes. In the current study, we found that Rho GTPase members were deficient in microtia chondrocytes. By overexpressing RhoA, Rac1, and CDC42, respectively, we further demonstrated that RhoA took great responsibility for the decreased migration ability of microtia chondrocytes. Moreover, we constructed PGA/PLA scaffold-based cartilages to verify the chondrogenic ability of RhoA overexpressed microtia chondrocytes, and the results showed that overexpressing RhoA was of limited help in improving the quality of microtia chondrocyte engineered cartilage. However, coculture of adipose-derived stem cells (ADSCs) significantly improved the biochemical and biomechanical properties of engineered cartilage. Especially, coculture of RhoA overexpressed microtia chondrocytes and ADSCs produced an excellent effect on the wet weight, cartilage-specific extracellular matrix, and biomechanical property of engineered cartilage. Furthermore, we presented that coculture of RhoA overexpressed microtia chondrocytes and ADSCs combined with human ear-shaped PGA/PLA scaffold and titanium alloy stent fabricated by CAD/CAM and 3D printing technology effectively constructed and maintained auricle structure in vivo. Collectively, our results provide evidence for the essential role of RhoA in microtia chondrocytes and a developed strategy for the construction of patient-specific tissue-engineered auricular cartilage.

Genotype-phenotype associations in microtia: a systematic review.

BACKGROUND: Microtia is a congenital ear malformation that can occur as isolated microtia or as part of a syndrome. The etiology is currently poorly understood, although there is strong evidence that genetics has a role in the occurrence of microtia. This systematic review aimed to determine the genes involved and the abnormalities in microtia patients' head and neck regions. METHODS: We used seven search engines to search all known literature on the genetic and phenotypic variables associated with the development or outcome of microtia. The identified publications were screened and selected based on inclusion and exclusion criteria and assessed for methodological quality using the Joanna Briggs Institute (JBI) critical appraisal tools. We found 40 papers in this systematic review with phenotypic data in microtia involving 1459 patients and 30 articles containing genetic data involved in microtia. RESULT: The most common accompanying phenotype of all microtia patients was external ear canal atresia, while the most common head and neck abnormalities were the auricular, mental, and oral regions. The most common syndrome found was craniofacial microsomia syndrome. In the syndromic microtia group, the most common genes were TCOF1 (43.75%), SIX2 (4.69%), and HSPA9 (4.69%), while in the non-syndromic microtia group, the most frequently found gene was GSC exon 2 (25%), FANCB (16.67%), HOXA2 (8.33%), GSC exon 3 (8.33%), MARS1 (8.33%), and CDT1 (8.33%). CONCLUSIONS: Our systematic review shows some genes involved in the microtia development, including TCOF1, SIX2, HSPA9, GSC exon 2, FANCB, HOXA2, GSC exon 3, MARS1, and CDT1 genes. We also reveal a genotype-phenotype association in microtia. In addition, further studies with more complete and comprehensive data are needed, including patients with complete data on syndromes, phenotypes, and genotypes.

A 76-base pair duplication within the enhancer region of the HMX1 gene causes sheep microtia.

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Profiling of Long Non-Coding RNAs in Auricular Cartilage of Patients with Isolated Microtia.

Introduction: Microtia is the second most common maxillofacial birth defect worldwide. However, the involvement of long non-coding RNAs (lncRNAs) in isolated microtia is not well understood. This study aimed at identifying lncRNAs that regulate the expression of genes associated with isolated microtia. Methods: We used our microarray data to analyze the expression pattern of lncRNA in the auricular cartilage tissues from 10 patients diagnosed with isolated microtia, alongside 15 control subjects. Five lncRNAs were chosen for validation using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: We identified 4651 differentially expressed lncRNAs in the auricular cartilage from patients with isolated microtia. By Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway (GO/KEGG) analysis, we identified 27 differentially expressed genes enriched in pathways associated with microtia. In addition, we predicted 9 differentially expressed genes as potential cis-acting targets of 12 differentially expressed lncRNAs. Our findings by qRT-PCR demonstrate significantly elevated expression levels of ZFAS1 and DAB1-AS1, whereas ADIRF-AS1, HOTAIRM1, and EPB41L4A-AS1 exhibited significantly reduced expression levels in the auricular cartilage tissues of patients with isolated microtia. Conclusions: Our study sheds light on the potential involvement of lncRNAs in microtia and provides a basis for further investigation into their functional roles and underlying mechanisms.

The circular RNA expression profile of human auricle cartilage and the role of circCOL1A2 in isolated microtia.

Microtia is one of the most common craniofacial birth defects worldwide, and its primary clinical manifestation is auricle deformity. Epigenetic factors are known to contribute to the etiology of microtia, yet the involvement of circular RNAs (circRNAs) in human auricle development and their association with microtia remains poorly understood. In this study, we aimed to analyze differentially expressed circRNAs and explore their functional implications in isolated microtia. By employing circRNA microarray analysis and bioinformatics approaches, we identified 340 differentially expressed circRNAs in auricle cartilage of patients with isolated microtia, comprising 152 upregulated and 188 downregulated circRNAs. A circRNA-mRNA co-expression network was constructed, followed by gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Subsequently, we selected four significantly upregulated circRNAs from the co-expression network based on their association with cartilage development and validated their expressions in 30 isolated microtia and 30 control clinical auricle cartilage samples. Among these circRNAs, circCOL1A2, the most significantly upregulated circRNA, was selected as a representative circRNA for investigating its role in isolated microtia. Overexpression of circCOL1A2 significantly inhibited chondrocyte proliferation and chondrogenic differentiation of human mesenchymal stem cells. Additionally, circCOL1A2 upregulated Dermatan Sulfate Epimerase Like (DSEL) expression by sponging miR-637 through the competing endogenous RNA (ceRNA) mechanism. Notably, the downregulation of DSEL attenuated the inhibitory effect of circCOL1A2 overexpression on cell proliferation and chondrogenic differentiation. Collectively, these findings highlight the involvement of circCOL1A2 in the pathogenesis of isolated microtia and emphasize the potential significance of dysregulated circRNAs in disease development.

[Genetic analysis of a child with Meier-Gorlin syndrome due to a variant of ORC6 gene].

OBJECTIVE: To analyze the genetic characteristics of a child with Meier-Gorlin syndrome (MGS) due to a homozygous variant of the ORC6 gene. METHODS: A child who was admitted to the Children's Hospital Affiliated to Soochow University on March 25, 2019 due to growth retardation was selected as the study subject. Clinical data of the child was collected. Whole exome sequencing was carried out for the child. Candidate variant was validated by Sanger sequencing and bioinformatic analysis. RESULTS: The child, a 8-year-and-3-month-old male, has featured short stature, small ears, bilateral cryptorchidism and patellar dysplasia. His parents were of first cousins. The child was found to harbor a homozygous c.712A>T (p.K238*) missense variant of the ORC6 gene, which may lead to premature termination of protein translation. Sanger sequencing confirmed that both of his parents were heterozygous carriers. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was classified as pathogenic (PVS1_Moderate+PM2_Supporting+PM3+PP3+PP4). CONCLUSION: The homozygous c.712A>T (p.K238*) variant probably underlay the MGS in this child.

Pathogenic Genes for Congenital Microtia: A Bioinformatics Analysis.

OBJECTIVE: The purpose of this study is to accurately find the pathogenic genes of congenital microtia, so as to lay a theoretical foundation for genetic screening, diagnosis, and gene therapy of congenital microtia in the further stage. METHODS: In this study, the authors used public data from the Mouse Genome Informatics database. The authors used the String database ( https://string-db.org/ ) to construct the Protein-Protein Interaction network. Then Gene Ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed for the pathogenic genes. RESULTS: The authors searched the Mouse Genome Informatics database and found 84 pathogenic genes of congenital microtia. The Protein-Protein Interaction network for pathogenic genes was constructed, which contained 81 nodes and 148 lines with MCM5, CDT1, POLA1, CDC45, CDC6, EFTUD2, ORC1, ORC4, ORC6, and TCOF1 . The authors conducted a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis on pathogenic genes, and the results showed that pathogenic genes were involved in O-mannan biosynthesis, cell cycle, RNA polymerase, and other signaling pathways. CONCLUSIONS: The authors' results indicated that the occurrence of congenital microtia is attributed to a variety of genes. Furthermore, the interactions of pathogenic genes were further elucidated by using a bioinformatics approach. This study will help to reveal the pathogenesis of congenital microtia and lay the foundation for accurate diagnosis and treatment of congenital microtia in the future.

DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation.

Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.

Inheritance analysis and family history for microtia: A hospital-based study in China.

OBJECTIVES: Microtia is a congenital anomaly of the outer ear. Although genetic and environmental factors could play a role, no consensus has been established on the pathogenesis and cause of this condition. In this study, we surveyed the frequency and pattern of family history in patients with microtia in a Chinese specialty clinic population. METHODS: We evaluated data from 672 patients (mean age = 9.2, male-to-female ratio = 2.6:1) with microtia admitted to the Department of Auricular Reconstruction at the Plastic Surgery Hospital of Peking Union Medical College from December 2014 to February 2016. Family history of congenital ear anomalies across three generations was recorded. Pearson chi-square test or Fisher exact test was used to test the associations between the characteristics of microtia and hereditary features. RESULTS: A family history of auricle anomalies was identified in 202 patients (30.1%), of whom, 95 families showed vertical transmission, 14 families skipped a generation, and 120 families showed family aggregations. The incidence of family history varied with grades of microtia (P = 0.001). Patients with preauricular tags or pits (38.3%) had a higher familial incidence of microtia than those with simple microtia (24.1%) (P < 0.001). CONCLUSION: Patients with a lower grade of microtia demonstrated a higher incidence of family history. Patients with microtia had significantly more relatives with preauricular tags or pits. Microtia and preauricular tags or pits are different manifestations of the same defect, and their significant concurrency among relatives suggests that a considerable proportion of microtia is inherited and could recur with varying degrees of severity in other family members.

Genetic Screening of Targeted Region on the Chromosome 22q11.2 in Patients with Microtia and Congenital Heart Defect.

Microtia is a congenital malformation characterized by a small, abnormally shaped auricle (pinna) ranging in severity. Congenital heart defect (CHD) is one of the comorbid anomalies with microtia. However, the genetic basis of the co-existence of microtia and CHD remains unclear. Copy number variations (CNVs) of 22q11.2 contribute significantly to microtia and CHD, respectively, thus suggesting a possible shared genetic cause embedded in this genomic region. In this study, 19 sporadic patients with microtia and CHD, as well as a nuclear family, were enrolled for genetic screening of single nucleotide variations (SNVs) and CNVs in 22q11.2 by target capture sequencing. We detected a total of 105 potential deleterious variations, which were enriched in ear- or heart-development-related genes, including TBX1 and DGCR8. The gene burden analysis also suggested that these genes carry more deleterious mutations in the patients, as well as several other genes associated with cardiac development, such as CLTCL1. Additionally, a microduplication harboring SUSD2 was validated in an independent cohort. This study provides new insights into the underlying mechanisms for the comorbidity of microtia and CHD focusing on chromosome 22q11.2, and suggests that a combination of genetic variations, including SNVs and CNVs, may play a crucial role instead of single gene mutation.

The expanding genetic and clinical landscape associated with Meier-Gorlin syndrome.

High-throughput sequencing has become a standard first-tier approach for both diagnostics and research-based genetic testing. Consequently, this hypothesis-free testing manner has revealed the true breadth of clinical features for many established genetic disorders, including Meier-Gorlin syndrome (MGORS). Previously known as ear-patella short stature syndrome, MGORS is characterized by growth delay, microtia, and patella hypo/aplasia, as well as genital abnormalities, and breast agenesis in females. Following the initial identification of genetic causes in 2011, a total of 13 genes have been identified to date associated with MGORS. In this review, we summarise the genetic and clinical findings of each gene associated with MGORS and highlight molecular insights that have been made through studying patient variants. We note interesting observations arising across this group of genes as the number of patients has increased, such as the unusually high number of synonymous variants affecting splicing in CDC45 and a subgroup of genes that also cause craniosynostosis. We focus on the complicated molecular genetics for DONSON, where we examine potential genotype-phenotype patterns using the first 3D structural model of DONSON. The canonical role of all proteins associated with MGORS are involved in different stages of DNA replication and in addition to summarising how patient variants impact on this process, we discuss the potential contribution of non-canonical roles of these proteins to the pathophysiology of MGORS.

Identification of a homozygous frameshift mutation in the FGF3 gene in a consanguineous Iranian family: First report of labyrinthine aplasia, microtia, and microdontia syndrome in Iran and literature review.

BACKGROUND: To date, over 400 syndromes with hearing impairment have been identified which altogether constitute almost 30% of hereditary hearing loss (HL) cases around the globe. Manifested as complete or partial labyrinthine aplasia (severe malformations of the inner ear structure), type I microtia (smaller outer ear with shortened auricles), and microdontia (small and widely spaced teeth), labyrinthine aplasia, microtia, and microdontia (LAMM) syndrome (OMIM 610706) is an extremely rare autosomal recessive condition caused by bi-allelic mutations in the FGF3 gene. METHODS: Using the whole-exome sequencing (WES) data of the proband, we analyzed a consanguineous Iranian family with three affected members presenting with congenital bilateral HL, type I microtia, and microdontia. RESULTS: We discovered the homozygous deletion c.45delC in the first exon of the FGF3 gene, overlapping a 38.72 Mb homozygosity region in chromosome 11. Further investigations using Sanger sequencing revealed that this variant co-segregated with the phenotype observed in the family. CONCLUSION: Here, we report the first identified case of LAMM syndrome in Iran, and by identifying a frameshift variant in the first exon of the FGF3 gene, our result will help better clarify the phenotype-genotype relation of LAMM syndrome.

Search for a genetic cause in children with unilateral isolated microtia and congenital aural atresia.

PURPOSE: Microtia describes a spectrum of auricular malformations ranging from mild dysplasia to anotia. A vast majority of microtia patients demonstrate congenital aural atresia (CAA). Isolated microtia has a right ear predominance (58-61%) and is more common in the male sex. Isolated microtia is a multifactorial condition involving genetic and environmental causes. The aim of this study is to describe the phenotype of children with unilateral isolated microtia and CAA, and to search for a common genetic cause trough DNA analysis. METHODS: Phenotyping included a complete clinical examination. Description on the degree of auricular malformation (Weerda classification-Weerda 1988), assessment for hemifacial microsomia and age-appropriate audiometric testing were documented. Computerized tomography of the temporal bone with 3-D rendering provided a histopathological classification (HEAR classification-Declau et al. 1999). Genetic testing was carried out by single nucleotide polymorphism (SNP) microarray. RESULTS: Complete data are available for 44 children (50% was younger than 33 days at presentation; 59.1% boys; 72.7% right ear). Type III microtia was present in 28 patients. Type 2b CAA existed in 32 patients. All patients had a normal hearing at the non-affected side. Genome wide deletion duplication analysis using microarray did not reveal any pathological copy number variant (CNV) that could explain the phenotype. CONCLUSIONS: Type III microtia (peanut-shell type) in combination with a type 2b CAA was the most common phenotype, present in 23 of 44 (52.3%) patients with isolated unilateral microtia. No abnormalities could be found by copy number variant (CNV) analysis. Whole exome sequencing in a larger sample with a similar phenotype may represent a future diagnostic approach.

Patterns of co-occurring birth defects in children with anotia and microtia.

Many infants with anotia or microtia (A/M) have co-occurring birth defects, although few receive syndromic diagnoses in the perinatal period. Evaluation of co-occurring birth defects in children with A/M could identify patterns indicative of undiagnosed/unrecognized syndromes. We obtained information on co-occurring birth defects among infants with A/M for delivery years 1999-2014 from the Texas Birth Defects Registry. We calculated observed-to-expected ratios (OER) to identify birth defect combinations that occurred more often than expected by chance. We excluded children diagnosed with genetic or chromosomal syndromes from analyses. Birth defects and syndromes/associations diagnosed ≤1 year of age were considered. We identified 1310 infants with non-syndromic A/M, of whom 38% (N = 492) were diagnosed with co-occurring major defects. Top combinations included: hydrocephalus, ventricular septal defect, and spinal anomalies (OER 58.4); microphthalmia and anomalies of the aorta (OER 55.4); and cleft lip with or without cleft palate and rib or sternum anomalies (OER 32.8). Some combinations observed in our study may represent undiagnosed/atypical presentations of known A/M associations or syndromes, or novel syndromes yet to be described in the literature. Careful evaluation of infants with multiple birth defects including A/M is warranted to identify individuals with potential genetic or chromosomal syndromes.

Damaging variants in FOXI3 cause microtia and craniofacial microsomia.

PURPOSE: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. METHODS: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. RESULTS: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. CONCLUSION: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.