AbstractBackgroundMandibulofacial 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.MethodsWe 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.ResultsWe 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).ConclusionOur 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.