Atypical mandibulofacial dysostosis with microcephaly diagnosed through the identification of a novel pathogenic mutation in EFTUD2.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536) is a rare monogenic disease that is caused by a mutation in the elongation factor Tu GTP binding domain containing 2 gene (EFTUD2, OMIM* 603892). It is characterized by mandibulofacial dysplasia, microcephaly, malformed ears, cleft palate, growth and intellectual disability. MFDM can be easily misdiagnosed due to its phenotypic overlap with other craniofacial dysostosis syndromes. The clinical presentation of MFDM is highly variable among patients. METHODS: A patient with craniofacial anomalies was enrolled and evaluated by a multidisciplinary team. To make a definitive diagnosis, whole-exome sequencing was performed, followed by validation by Sanger sequencing. RESULTS: The patient presented with extensive facial bone dysostosis, upward slanting palpebral fissures, outer and middle ear malformation, a previously unreported orbit anomaly, and spina bifida occulta. A novel, pathogenic insertion mutation (c.215_216insT: p.Tyr73Valfs*4) in EFTUD2 was identified as the likely cause of the disease. CONCLUSIONS: We diagnosed this atypical case of MFDM by the detection of a novel pathogenetic mutation in EFTUD2. We also observed previously unreported features. These findings enrich both the genotypic and phenotypic spectrum of MFDM.

Structural basis of human U5 snRNP late biogenesis and recycling.

Pre-mRNA splicing by the spliceosome requires the biogenesis and recycling of its small nuclear ribonucleoprotein (snRNP) complexes, which are consumed in each round of splicing. The human U5 snRNP is the ~1 MDa 'heart' of the spliceosome and is recycled through an unknown mechanism involving major architectural rearrangements and the dedicated chaperones CD2BP2 and TSSC4. Late steps in U5 snRNP biogenesis similarly involve these chaperones. Here we report cryo-electron microscopy structures of four human U5 snRNP-CD2BP2-TSSC4 complexes, revealing how a series of molecular events primes the U5 snRNP to generate the ~2 MDa U4/U6.U5 tri-snRNP, the largest building block of the spliceosome.

The feedback loop of EFTUD2/c-MYC impedes chemotherapeutic efficacy by enhancing EFTUD2 transcription and stabilizing c-MYC protein in colorectal cancer.

BACKGROUND: Chemoresistance presents a significant obstacle in the treatment of colorectal cancer (CRC), yet the molecular basis underlying CRC chemoresistance remains poorly understood, impeding the development of new therapeutic interventions. Elongation factor Tu GTP binding domain containing 2 (EFTUD2) has emerged as a potential oncogenic factor implicated in various cancer types, where it fosters tumor growth and survival. However, its specific role in modulating the sensitivity of CRC cells to chemotherapy is still unclear. METHODS: Public dataset analysis and in-house sample validation were conducted to assess the expression of EFTUD2 in 5-fluorouracil (5-FU) chemotherapy-resistant CRC cells and the potential of EFTUD2 as a prognostic indicator for CRC. Experiments both in vitro, including MTT assay, EdU cell proliferation assay, TUNEL assay, and clone formation assay and in vivo, using cell-derived xenograft models, were performed to elucidate the function of EFTUD2 in sensitivity of CRC cells to 5-FU treatment. The molecular mechanism on the reciprocal regulation between EFTUD2 and the oncogenic transcription factor c-MYC was investigated through molecular docking, ubiquitination assay, chromatin immunoprecipitation (ChIP), dual luciferase reporter assay, and co-immunoprecipitation (Co-IP). RESULTS: We found that EFTUD2 expression was positively correlated with 5-FU resistance, higher pathological grade, and poor prognosis in CRC patients. We also demonstrated both in vitro and in vivo that knockdown of EFTUD2 sensitized CRC cells to 5-FU treatment, whereas overexpression of EFTUD2 impaired such sensitivity. Mechanistically, we uncovered that EFTUD2 physically interacted with and stabilized c-MYC protein by preventing its ubiquitin-mediated proteasomal degradation. Intriguingly, we found that c-MYC directly bound to the promoter region of EFTUD2 gene, activating its transcription. Leveraging rescue experiments, we further confirmed that the effect of EFTUD2 on 5-FU resistance was dependent on c-MYC stabilization. CONCLUSION: Our findings revealed a positive feedback loop involving an EFTUD2/c-MYC axis that hampers the efficacy of 5-FU chemotherapy in CRC cells by increasing EFTUD2 transcription and stabilizing c-MYC oncoprotein. This study highlights the potential of EFTUD2 as a promising therapeutic target to surmount chemotherapy resistance in CRC patients.

SART3 associates with a post-splicing complex.

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.

MDF is a conserved splicing factor and modulates cell division and stress response in Arabidopsis.

The coordination of cell division with stress response is essential for maintaining genome stability in plant meristems. Proteins involved in pre-mRNA splicing are important for these processes in animal and human cells. Based on its homology to the splicing factor SART1, which is implicated in the control of cell division and genome stability in human cells, we analyzed if MDF has similar functions in plants. We found that MDF associates with U4/U6.U5 tri-snRNP proteins and is essential for correct splicing of 2,037 transcripts. Loss of MDF function leads to cell division defects and cell death in meristems and was associated with up-regulation of stress-induced genes and down-regulation of mitotic regulators. In addition, the mdf-1 mutant is hypersensitive to DNA damage treatment supporting its role in coordinating stress response with cell division. Our analysis of a dephosphomutant of MDF suggested how its protein activity might be controlled. Our work uncovers the conserved function of a plant splicing factor and provides novel insight into the interplay of pre-mRNA processing and genome stability in plants.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

A functional loop between YTH domain family protein YTHDF3 mediated m6A modification and phosphofructokinase PFKL in glycolysis of hepatocellular carcinoma.

BACKGROUND & AIMS: N6-methyladenosine (m6A) modification plays a critical role in progression of hepatocellular carcinoma (HCC), and aerobic glycolysis is a hallmark of cancer including HCC. However, the role of YTHDF3, one member of the core readers of the m6A pathway, in aerobic glycolysis and progression of HCC is still unclear. METHODS: Expression levels of YTHDF3 in carcinoma and surrounding tissues of HCC patients were evaluated by immunohistochemistry. Loss and gain-of-function experiments in vitro and in vivo were used to assess the effects of YTHDF3 on HCC cell proliferation, migration and invasion. The role of YTHDF3 in hepatocarcinogenesis was observed in a chemically induced HCC model with Ythdf3-/- mice. Untargeted metabolomics and glucose metabolism phenotype assays were performed to evaluate relationship between YTHDF3 and glucose metabolism. The effect of YTHDF3 on PFKL was assessed by methylated RNA immunoprecipitation assays (MeRIP). Co-immunoprecipitation and immunofluorescence assays were performed to investigate the connection between YTHDF3 and PFKL. RESULTS: We found YTHDF3 expression was greatly upregulated in carcinoma tissues and it was correlated with poor prognosis of HCC patients. Gain-of-function and loss-of-function assays demonstrated YTHDF3 promoted proliferation, migration and invasion of HCC cells in vitro, and YTHDF3 knockdown inhibited xenograft tumor growth and lung metastasis of HCC cells in vivo. YTHDF3 knockout significantly suppressed hepatocarcinogenesis in chemically induced mice model. Mechanistically, YTHDF3 promoted aerobic glycolysis by promoting phosphofructokinase PFKL expression at both mRNA and protein levels. MeRIP assays showed YTHDF3 suppressed PFKL mRNA degradation via m6A modification. Surprisingly, PFKL positively regulated YTHDF3 protein expression, not as a glycolysis rate-limited enzyme, and PFKL knockdown effectively rescued the effects of YTHDF3 overexpression on proliferation, migration and invasion ability of Sk-Hep-1 and HepG2 cells. Notably, co-immunoprecipitation assays demonstrated PFKL interacted with YTHDF3 via EFTUD2, a core subunit of spliceosome involved in pre-mRNA splicing process, and ubiquitination assays showed PFKL could positively regulate YTHDF3 protein expression via inhibiting ubiquitination of YTHDF3 protein by EFTUD2. CONCLUSIONS: our study uncovers the key role of YTHDF3 in HCC, characterizes a positive functional loop between YTHDF3 and phosphofructokinase PFKL in glucose metabolism of HCC, and suggests the connection between pre-mRNA splicing process and m6A modification.

A forward genetic screen in C. elegans identifies conserved residues of spliceosomal proteins PRP8 and SNRNP200/BRR2 with a role in maintaining 5' splice site identity.

The spliceosome undergoes extensive rearrangements as it assembles onto precursor messenger RNAs. In the earliest assembly step, U1snRNA identifies the 5' splice site. However, U1snRNA leaves the spliceosome relatively early in assembly, and 5' splice site identity is subsequently maintained through interactions with U6snRNA, protein factor PRP8, and other components during the rearrangements that build the catalytic site. Using a forward genetic screen in Caenorhabditis elegans, we have identified suppressors of a locomotion defect caused by a 5'ss mutation. Here we report three new suppressor alleles from this screen, two in PRP8 and one in SNRNP200/BRR2. mRNASeq studies of these suppressor strains indicate that they also affect specific native alternative 5'ss, especially for suppressor PRP8 D1549N. A strong suppressor at the unstructured N-terminus of SNRNP200, N18K, indicates a novel role for this region. By examining distinct changes in the splicing of native genes, examining double mutants between suppressors, comparing these new suppressors to previously identified splicing suppressors from yeast, and mapping conserved suppressor residues onto cryoEM structural models of assembling human spliceosomes, we conclude that there are multiple interactions at multiple stages in spliceosome assembly responsible for maintaining the initial 5'ss identified by U1snRNA for entry into the catalytic core.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion.

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc-/-) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc-/- embryos with pifithrin-α, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-α-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc-/-; Trp53ncc-/-) mutant embryos. Eftud2ncc-/- embryos that were treated with pifithrin-α or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc-/-; Trp53ncc-/- embryos compared to Eftud2ncc-/- mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc-/-; Trp53ncc-/- embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc-/- embryos.

A de novo start-loss in EFTUD2 associated with mandibulofacial dysostosis with microcephaly: case report.

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare genetic disorder inherited in an autosomal dominant pattern. Major characteristics include developmental delay, craniofacial malformations such as malar and mandibular hypoplasia, and ear anomalies. Here, we report a 4.5-yr-old female patient with symptoms fitting MFDM. Using whole-genome sequencing, we identified a de novo start-codon loss (c.3G > T) in the EFTUD2 We examined EFTUD2 expression in the patient by RNA sequencing and observed a notable functional consequence of the variant on gene expression in the patient. We identified a novel variant for the development of MFDM in humans. To the best of our knowledge, this is the first report of a start-codon loss in EFTUD2 associated with MFDM.

Clinical and molecular delineation of mandibulofacial dysostosis with microcephaly in six Korean patients: When to consider EFTUD2 analysis?

Mandibulofacial dysostosis with microcephaly (MFDM, OMIM#610536) is an extremely rare genetic syndrome characterised by microcephaly, external ear deformity, hearing loss, and distinct facial appearance, including zygomatic hypoplasia and micrognathia. Occasionally, various malformations in other internal organs, including oesophageal atresia or tracheoesophageal fistula, may lead to life-threatening situations. Haploinsufficiency of EFTUD2 is responsible for MFDM. Here, we present the phenotypic and genetic characteristics of six Korean children who were diagnosed with MFDM by molecular genetic testing. All but one patient had occipitofrontal circumferences below the -2.0 standard deviation score. Micrognathia was identified in all patients. A cleft palate (66.7%) and other facial dysmorphisms, including facial asymmetry (50%) and malar hypoplasia (50%), were also frequently observed. Hearing loss was observed in all patients along with one or more internal and external ear deformities, including ossicular anomalies, auditory canal stenosis, and microtia. Two patients (33.3%) had undergone surgery for tracheoesophageal fistula type C. Most patients were initially misdiagnosed as other better-known syndromes with overlapping characteristics, such as Treacher Collins or CHARGE syndrome. The first three patients were diagnosed using exome sequencing. However, after increased awareness of MFDM in the first three patients, MFDM was considered one of the initial differential diagnoses and could be diagnosed by target gene analysis in the remaining three cases. Thus, we recommend targeted EFTUD2 analysis as the initial workup for the rapid diagnosis of MFDM in patients with facial dysostosis, microcephaly, and otologic problems.

A novel de novo missense mutation in EFTUD2 identified by whole-exome sequencing in mandibulofacial dysostosis with microcephaly.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is a rare multiple malformation syndrome characterized by malar and mandibular hypoplasia and congenital- or postnatal-onset microcephaly induced by haploinsufficiency of (elongation factor Tu GTP-binding domain-containing 2) EFTUD2. METHODS: We report the case of a 16-month-old boy with MFDM symptoms, including malar and mandibular hypoplasia, microcephaly, micrognathia, midline cleft palate, microtia, auditory canal atresia, severe sensorineural hearing loss, and developmental delay. Whole-exome sequencing (WES) analysis of the patient's family was performed to identify the genetic etiology responsible for this phenotype. RESULTS: We identified a novel de novo missense mutation (c.671G>T, p.Gly224Val) in the EFTUD2. According to the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines, the c.671G>T mutation was classified as likely pathogenic (PS2, PM1, PM2, and PP3). Based on our findings, prenatal diagnosis was performed on the second baby of the proband's parents to exclude the mutation and it was confirmed that the baby did not have the MFDM phenotype after 14 months of follow-up. Furthermore, the zebrafish model confirmed that the EFTUD2 c.671G>T mutation caused a loss of gene function in EFTUD2, and the pathogenicity of the EFTUD2 c.671G>T mutation was classified as pathogenic (PS2, PS3, PM1, and PM2). CONCLUSION: Our results indicate that WES is a useful tool for identifying potentially pathogenic mutations, particularly in rare disorders, and is advantageous for genetic counseling and subsequent prenatal diagnosis. Moreover, the importance of functional assays cannot be underestimated, which could further confirm the pathogenicity of the genetic variants.

The role of splicing factor PRPF8 in breast cancer.

BACKGROUND: Alternative splicing is a mechanism to produce different proteins with diverse functions from one gene. Many splicing factors play an important role in cancer progression. PRPF8 is a core protein component of the spliceosome complex, U4/U6-U5 tri-snRNP. OBJECTIVE: However, PRPF8 involved in mRNA alternative splicing are rarely included in the prognosis. METHODS: We found that PRPF8 was expressed in all examined cancer types. Further analyses found that PRPF8 expression was significantly different between the breast cancer and paracancerous tissues. RESULTS: Survival analyses showed that PRPF8-high patients had a poor prognosis, and the expression of PRPF8 is associated with distant metastasis-free survival (DMFS) and post progression survival (PPS). Gene Set Enrichment Analysis (GSEA) has revealed that PRPF8 expression is correlated with TGF-ß, JAK-STAT, and cell cycle control pathways. Consistent with these results, upon PRPF8 silencing, the growth of MCF-7 cells was reduced, the ability of cell clone formation was weakened, and p⁢21 expression was increased. CONCLUSIONS: These results have revealed that PRPF8 is a significant factor for splicing in breast cancer progression.

Expanding the genotypic spectrum of TXNL4A variants in Burn-McKeown syndrome.

The developmental disorder Burn-McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre-messenger RNA splicing factor TXNL4A. Most patients have a loss-of-function variant in trans with a 34-base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs *21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258-3C>G) and a type 1 Δ34 promoter deletion. We show the c.258-3C>G variant and a previously reported c.258-2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56 bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22 bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22 bp repeated motif could be relevant to BMKS aetiology. Finally, our data emphasises the need to analyse the non-coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation.

Phenotypic and Molecular Heterogeneity in Mandibulofacial Dysostoses: A Case Series From India.

OBJECTIVE: Facial dysostosis is a group of rare craniofacial congenital disabilities requiring multidisciplinary long-term care. This report presents the phenotypic and genotypic information from South India. DESIGN: The study is a case series. SETTING: This was an international collaborative study involving a tertiary craniofacial clinic and medical genetics unit. PATIENTS, PARTICIPANTS: The participants were 9 families with 17 affected individuals of facial dysostosis. INTERVENTION: Exome analysis focused on known genes associated with acrofacial and mandibulofacial syndromes. MAIN OUTCOME MEASURE: The outcome measure was to report phenotyptic and genetic heterogeneity in affected individuals. RESULTS: A Tessier cleft was seen in 7 (41%), lower eyelid coloboma in 12 (65%), ear anomalies in 10 (59%), uniolateral or bilateral aural atresia in 4 (24%), and deafness in 6 (35%). The facial gestalt of Treacher Collins syndrome (TCS) showed extensive phenotypic variations. Pathogenic variants in TCOF1 (Treacher Collins syndrome) were seen in six families, POLR1A (acrofacial dysostosis, Cincinnati type) and EFTUD2 (mandibulofacial dysostosis with microcephaly) in one each. One family (11.1%) had no detectable variation. Five out of six probands with Treacher Collins syndrome had other affected family members (83.3%), including a non-penetrant mother, identified after sequencing. CONCLUSION: Our report illustrates the molecular heterogeneity of mandibulofacial dysostosis in India.

Association of Elongation Factor Tu GTP-binding Domain-containing 2 Gene (EFTUD2) Polymorphism with the Risk of Hepatitis B Virus Infection.

BACKGROUND: The elongation factor Tu GTP-binding domain-containing 2 gene (EFTUD2) participates in antiviral immune responses. However, the association between genetic polymorphisms of EFTUD2 and hepatitis B virus (HBV) infection susceptibility has not been well-studied. We analyzed the relationship between single nucleotide polymorphisms (SNPs) of EFTUD2 and HBV infection susceptibility and clarified the potential function. METHODS: In total, 448 control subjects and 379 patients with chronic HBV infection from Zhangjiagang First People's Hospital (Jiangsu, China) were enrolled. Sequenom iPLEX assay was used to detect genotypes of four SNPs (rs1071682, rs2277617, rs2289674, and rs3809756). Dual-luciferase reporter vectors with wild-type A and mutant-type C alleles of EFTUD2 rs3809756 were transfected into HepG2 cells to explore effects on transcription activity. RESULTS: Only rs3809756 was significantly associated with HBV infection susceptibility (P < .05). The risk of HBV infection was higher in individuals carrying the rs3809756-CC genotype than in those carrying the rs3809756-AA genotype (odds ratio [OR] = 1.945, 95% confidence interval [CI] = 1.129-3.351, P = .017). Subgroup analysis based on the dominant model revealed that rs3809756-AC and rs3809756-CC carriers had a significantly higher risk of HBV infection than rs3809756-AA carriers among patients who were male (OR = 1.732, 95% CI = 1.218-2.464, P = .002), were aged ≥47 years (OR = 1.502, 95% CI = 1.050-2.148, P = .026), or without liver cirrhosis (OR = 1.407, 95% CI = 1.077-1.838, P = .012). In the dual-luciferase reporter assay, the relative luciferase activity of rs3809756-C was significantly lower than that of rs3809756-A (P < .05). CONCLUSION: EFTUD2 rs3809756A>C was associated with HBV infection susceptibility and might be involved in the downregulation of promoter activity.

Investigation of Genetic Causes in a Developmental Disorder: Oculoauriculovertebral Spectrum.

OBJECTIVE: Oculoauriculovertebral spectrum (OAVS) is a genetically and clinically heterogeneous disorder that occurs due to a developmental field defect of the first and second pharyngeal arches. Even though recent whole exome sequencing studies (WES) have led to identification of several genes associated with this spectrum in a subset of individuals, complete pathogenesis of OAVS remains unsolved. In this study, molecular genetic etiology of OAVS was systematically investigated. DESIGN/SETTING/PATIENTS: A cohort of 23 Turkish patients with OAVS, referred to Hacettepe University Hospital, Department of Pediatric Genetics from 2008 to 2018, was included in this study. Minimal diagnostic criteria for OAVS were considered as unilateral microtia or hemifacial microsomia with preauricular skin tag. The cohort was clinically reevaluated for craniofacial and extracranial findings. Molecular etiology was investigated using candidate gene sequencing following copy number variant (CNV) analysis. WES was also performed for 2 of the selected patients. RESULTS: Patients in the study cohort presented similar demographic and phenotypic characteristics to previously described patients in the literature except for a higher frequency of bilaterality, cardiac findings, and intellectual disability/developmental delay. CNV analysis revealed a possible genetic etiology for 3 patients (13%). Additional WES in 1 of the 2 patients uncovered a novel heterozygous nonsense variant in Elongation factor Tu GTP-binding domain-containing 2 (EFTUD2) causing mandibulofacial dysostosis with microcephaly (MFDM), which clinically overlaps with OAVS. CONCLUSION: Detailed clinical evaluation for any patient with OAVS is recommended due to a high rate of accompanying systemic findings. We further expand the existing genetic heterogeneity of OAVS by identifying several CNVs and a phenotypically overlapping disorder, MFDM.