Prenatal Ultrasound and Genetic Diagnosis of EFTUD2 Haploinsufficiency in Two Fetuses: A Case Series

Mandibulo-Facial Dysostosis with Microcephaly (MFDM) is a rare disease with a broad spectrum of symptoms, characterized by zygomatic and mandibular hypoplasia, microcephaly, and ear abnormalities. Here, we aimed at describing the external ear phenotype of MFDM patients, and train an Artificial Intelligence (AI)-based model to differentiate MFDM ears from non-syndromic control ears (binary classification), and from ears of the main differential diagnoses of this condition (multi-class classification): Treacher Collins (TC), Nager (NAFD) and CHARGE syndromes. The training set contained 1,592 ear photographs, corresponding to 550 patients. We extracted 48 patients completely independent of the training set, with only one photograph per ear per patient. After a CNN-(Convolutional Neural Network) based ear detection, the images were automatically landmarked. Generalized Procrustes Analysis was then performed, along with a dimension reduction using PCA (Principal Component Analysis). The principal components were used as inputs in an eXtreme Gradient Boosting (XGBoost) model, optimized using a 5-fold cross-validation. Finally, the model was tested on an independent validation set. We trained the model on 1,592 ear photographs, corresponding to 1,296 control ears, 105 MFDM, 33 NAFD, 70 TC and 88 CHARGE syndrome ears. The model detected MFDM with an accuracy of 0.969 [0.838-0.999] (p < 0.001) and an AUC (Area Under the Curve) of 0.975 within controls (binary classification). Balanced accuracies were 0.811 [0.648-0.920] (p = 0.002) in a first multiclass design (MFDM vs. controls and differential diagnoses) and 0.813 [0.544-0.960] (p = 0.003) in a second multiclass design (MFDM vs. differential diagnoses). This is the first AI-based syndrome detection model in dysmorphology based on the external ear, opening promising clinical applications both for local care and referral, and for expert centers.

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare autosomal dominant disorder caused by pathogenic variants in the EFTUD2 gene, presenting with craniofacial anomalies, microcephaly, and systemic abnormalities. Despite several reported cases, the genetic and molecular mechanisms underlying MFDM remain inadequately understood. This case study identifies and analyzes a previously unreported EFTUD2 splice variant (NM_004247.4:c.492+1del), investigates its clinical phenotype, and analyzes genotype-phenotype correlations to improve early recognition and diagnosis of MFDM. The patient had facial abnormalities (micrognathia, high-arched palate, microtia, and preauricular tags), a small head-to-body ratio, sensorineural hearing loss, delayed speech development, and cognitive impairment. Exome sequencing identified a splice variant, NM_004247.4:c.492+1del, in the EFTUD2 gene, which was classified as pathogenic according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). AlphaFold 2 predictions indicated that this variant had a significant impact on the protein structure. RNA-sequencing (RNA-seq) further demonstrated that the NM_004247.4:c.492+1del variant led to a pronounced splicing abnormality, causing exon 6 skipping during the transcription process of the EFTUD2 gene. This study reported a novel EFTUD2 splice variant in a child with MFDM, expanding its variant spectrum. Utilizing RNA-seq, we demonstrated the variant's pathogenicity and contributed to the understanding of MDFM's genotype-phenotype relationship, enriching the disease's variant spectrum with molecular insights.

Whole exome sequencing (WES) has been widely used in the pediatric setting to increase diagnostic yield, provide treatment options, and to estimate reoccurrence risks. However, there is limited knowledge regarding the utility of this technology in adults with neurodevelopmental disabilities. We present a 23-year-old male diagnosed with autism spectrum disorder, intellectual disability, cleft palate, micrognathia, microcephaly, bifid uvula, conductive hearing impairment, and hypotonia. The patient had several dysmorphic features including epicanthal folds, bulbous nasal tip, bilateral lop ear deformities, and tapered digits. His previous genetic workup includes negative Fragile X, microarray, FISH, and sub-telomeric testing. He has had several reconstructive surgeries and a cleft palate repair. Despite suspecting a genetic syndrome, WES testing was never completed due to its novelty and cost. Recently, WES testing found that he harbors a pathogenic variant in the EFTUD2 gene (c.1705 C>T) which is associated with Mandibulofacial Dysostosis With Microcephaly (MFDM). This variant was found to be de novo, providing the family with a refined reoccurrence risk. Testing adults can provide insight into the treatment options for young families and a better understanding of the course of the condition. Thus, this case adds to the literature on the management and spectrum of symptoms as well as highlights the importance of re-evaluating adult patients. Further research should be conducted to determine the utility and cost-effectiveness for WES testing in this population.

Genetic variants in EFTUD2 were proven to influence variable phenotypic expressivity in mandibulofacial dysostosis Guion-Almeida type (MFDGA) or mandibulofacial dysostosis with microcephaly (MFDM). Yet, the association between the severity of clinical findings with variants within the EFTUD2 gene has not been established. Thus, we aim to elucidate a possible genotype-phenotype correlation in MFDM. Forty articles comprising 156 patients were evaluated. The genotype-phenotype correlation was analyzed using a chi-square or Fisher's exact test. The proportion of patients with MFDM was higher in Caucasian relative to Asian populations. Although, in general, there was no apparent genotype-phenotype correlation in patients with MFDM, Asians tended to have more severe clinical manifestations than Caucasians. In addition, cardiac abnormality presented in patients with intronic variants located in canonical splice sites was a predisposing factor in affecting MFDM severity. Altogether, this article provides the pathogenic variants observed in EFTUD2 and possible genotype-phenotype relationships in this disease.

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.

Objective:To explore the clinical diagnosis, otological treatment and molecular etiology in a rare syndromic hearing loss case characterized by mandibulofacial dysostosis with microcephaly（MFDM）. Methods: The proband underwent detailed history collection, systematic physical examination and phenotypic analysis, as well as audiological examination, chest X-ray, temporal bone CT and brain MRI and other imaging examinations. The blood DNA of the proband and his parents was extracted and tested by the whole exom sequencing. The EFTUD2-related-MFDM literatures published by the end of 2020 were searched and sifted in PubMed and CNKI databases,the clinical characteristics of MFDM were summarized. Results:In this study, the patient presented with hypoplasia of auricle, micrognathia, microcephaly, developmental retardation, severe sensorineural hearing loss in both ears, and developmental malformation of middle and inner ear. Genetic analysis revealed a de novo deletion c.623_624delAT in EFTUD2 gene. According to the clinical features and genetic test results, the patient was diagnosed as MFDM. In order to solve the problem of hearing loss, the patient was further performed bilateral cochlear implantation, and part of the electrodes responded well during and after operation. Conclusion:This is the first domestic reported case of MFDM caused by EFTUD2 gene mutation. The key problem of cochlear implantation for this kind of patient is to avoid damaging the malformed facial nerve during the operation.The effect of speech rehabilitation after cochlear implant operation is related to many factors such as intelligence development of the patients.

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is due to haploinsufficiency of spliceosomal GTPase EFTUD2. Features include microcephaly, craniofacial dysmorphology, developmental disability, and other anomalies. We surveyed parents of individuals with MFDM to expand knowledge about health, development, and parental concerns. Participants included attendees of the inaugural MFDM family conference in June 2019 and members of the MFDM online group. To explore MFDM variable expressivity, we offered targeted Sanger sequencing for untested parents. Forty-seven parents participated in the survey. 59% of individuals with MFDM were male, with mean age 6.4 years (range 8 months to 49 years). Similar to the literature (n = 123), common features include microcephaly, cleft palate, choanal stenosis, tracheoesophageal fistula, heart problems, and seizures. New information includes airway intervention details, age-based developmental outcomes, rate of vision refractive errors, and lower incidences of prematurity and IUGR. Family concerns focused on development, communication, and increased support. Targeted Sanger sequencing for families of seven individuals demonstrated de novo variants, for a total of 91.9% de novo EFTUD2 variants (n = 34/37). This study reports the largest single cohort of individuals with MFDM, expands phenotypic spectrum and inheritance patterns, improves understanding of developmental outcomes and care needs, and identifies development as the biggest concern for parents.

Achondroplasia and mandibulofacial dysostosis with microcephaly (MFDM) are rare monogenic, dominant disorders, caused by gain-of-function fibroblast growth factor receptor 3 (FGFR3) gene variants and loss-of-function elongation factor Tu GTP binding domain-containing 2 (EFTUD2) gene variants, respectively. The coexistence of two distinct Mendelian disorders in a single individual is uncommon and challenges the traditional paradigm of a single genetic disorder explaining a patient's symptoms, opening new avenues for diagnosis and management. We present a case of a female patient initially diagnosed with achondroplasia due to a maternally inherited pathogenic FGFR3 variant. She was referred to our genetic department due to her unusually small head circumference and short stature, which were both significantly below the expected range for achondroplasia. Additional features included distinctive facial characteristics, significant speech delay, conductive hearing loss, and epilepsy. Given the complexity of her phenotype, she was recruited to the DDD (Deciphering Developmental Disorders) study and the 100,000 Genomes project for further investigation. Subsequent identification of a complex EFTUD2 intragenic rearrangement confirmed an additional diagnosis of mandibulofacial dysostosis with microcephaly (MFDM). This report presents the first case of a dual molecular diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly in the same patient. This case underscores the complexity of genetic diagnoses and the potential for coexistence of multiple genetic syndromes in a single patient. This case expands our understanding of the molecular basis of dual Mendelian disorders and highlights the importance of considering the possibility of dual molecular diagnoses in patients with phenotypic features that are not fully accounted for by their primary diagnosis.

eP246: Novel EFTUD2 variant adds to understanding of phenotypic spectrum of mandibulofacial dysostosis with microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a sporadic malformation syndrome with severe craniofacial abnormalities, microcephaly, developmental delay, and dysmorphic features. Most cases of clinically diagnosed MFDM remain genetically unexplained, and to the best of our knowledge a total of 35 patients, 31 different mutations, 4 deletions, and 6 reports have been published. Our proband was born at 36 weeks gestation with microcephaly, microcrania, cleft palate, severe retrognathia, oral and pharyngeal dysphagia, bilateral proximal radioulnar synostosis, 11 thoracic ribs, abnormal magnetic resonance imaging (MRI) findings including simplified gyral pattern and mild dilatation of the posterior bodies of the lateral ventricles secondary to thinning of the white matter, high-pitched cry due to unilateral vocal cord paralysis, and dysmorphic features. Array comparative genomic hybridization (aCGH) + single nucleotide polymorphism (SNP) analysis identified a likely de novo pathogenic deletion on chromosome 17q21.31, encompassing the EFTUD2 gene. Our case represents the fifth reported proband to have MFDM caused by small deletions involving EFTUD2. All known mutations involving EFTUD2 result in genetic haploinsufficiency, consistent with our proband's case as well. Her phenotypic features both overlap and expand on the clinical features of previously reported cases, and her genetic diagnosis also supports the use of aCGH as a first-tier testing option for this disorder.

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare condition that causes abnormalities of the head and face. Other major extracranial malformations may also be found. The authors present a case...

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome with characteristic craniofacial and external ear abnormalities and microcephaly. It is inherited in an autosomal dominant mode of inheritance with high penetrance and variable expressivity. In this report, we present a molecularly confirmed case of MFDM ( EFTUD2 [NM_004247.4]: c.670G&gt;A, p.Gly224Arg) in a 2-year female child along with a comprehensive review of literature.

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal requirement for Eftud2 during craniofacial development. We generated a mouse with a conditional mutation of exon 2 of Eftud2 (Eftud2 flox) using the CRISPR/Cas9 editing system. We used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Embryos carrying one mutated allele of Eftud2 were normal. However, when both alleles were mutated, all embryos displayed hypoplasia of the midbrain and pharyngeal arches at E9.5. By E11.5, most of these embryos showed an open neural tube and all live embryos exhibited exencephaly at E14.5. At this stage, only 3 out of 12 embryos were still alive. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel’s cartilage, and abnormal inner ear development. Crosses with the Rosa26RlacZ mice reporter line revealed a reduced number of neural crest cells in the pharyngeal region at E9.5 and E10.5, but not at E9.0. We next performed RNA sequencing analysis from the head of Eftud2 neural crest cells mutant embryos at E9.0, before any phenotype was observed. The P53 pathway was the most significantly upregulated in the mutant embryos, compared to their wild‐type littermates. Overexpression of P53 target genes identified in the RNAseq (Ccng1, Phlad3 and Trp53inp1) was validated by qRT‐PCR. Additionally, 373 genes showed increased skipped exons in the mutant embryos. Among them, only 2 genes were also overexpressed, including Mdm2, a major regulator of P53. We found that mutant embryos had significantly increased levels of the Mdm2 transcript lacking exon 3, which was previously reported to produce a truncated MDM2 protein, allowing stabilization of P53. Finally, we evaluated the contribution of P53 in craniofacial malformation of Eftud2 mutants by treating pregnant females with daily i.p. injection of pifithrinα, a P53 inhibitor, from E6.5 to E8.5. 37% of pifithrinα‐treated mutant embryos had normal midbrain development at E9.5. Also, the area perimeter of the first pharyngeal arch of pifithrinα‐treated mutant embryos was significantly higher than the vehicle‐treated ones. Altogether, these data suggest that Eftud2 specifically regulates Mdm2 splicing and leads to neural crest cell death due to overactivation of the P53 pathway. We propose that cranial neural crest cell death is responsible for craniofacial malformations in MFDM patients.Support or Funding InformationCanadian Institutes of Health Research

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.