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

Abstract

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.

This study reported an untypical patient with mandibulofacial dysostosis with microcephaly (MFDM), who exhibited previously unreported phenotypes, including orbit anomalies and spina bifida occulta. A novel pathogenic insertion mutation of EFTUD2 was identified by whole‐exome sequencing as the etiology for this MFDM case, highlighting the importance of genetic diagnosis in patients with craniofacial dysostosis.

1. INTRODUCTION

Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536), or mandibulofacial dysostosis, Guion‐Almeida type (MFDGA), is a rare craniofacial syndrome. Guion‐Almeida et al. first described it as a new form of mandibulofacial dysostosis that is characterized by a combination of mandibulofacial dysostosis, microcephaly, malformed ears with skin tags, cleft palate and growth and intellectual disability (Guion‐Almeida et al., 2006). The most common and striking phenotypes of MFDM are craniofacial anomalies, which include micrognathia, due to mandibular hypoplasia, malar hypoplasia, microcephaly, abnormality of the pinna, choanal atresia and cleft palate. Patients with MFDM also have a high possibility of one or more extracranial phenotypes, such as tracheoesophageal fistula (TEF)/esophageal atresia (EA), congenital heart diseases, thumb anomalies and seizures (Abell et al., 2021; Yu et al., 2018). Development delay, manifesting as intellectual disability, speech impediments, motor development delays, and short stature, is a significant feature of MFDM (Yu et al., 2018). The syndrome is highly penetrant and exhibits extreme clinical variability. Clinical findings in affected individuals vary from subclinical features, which make it difficult to establish an unequivocal diagnosis, to lethal anomalies in multiple systems (McDermott et al., 2017). Although MFDM is considered a rare disease, with a prevalence of less than 1/1,000,000, some scholars believe that the prevalence is underestimated due to lack of awareness or misdiagnosis as other mandibulofacial dysostosis syndromes (Silva et al., 2019).

Mutations in the Elongation Factor Tu GTP Binding Domain Containing 2 (EFTUD2) gene were proven to be responsible for MFDM in 2012 (Lines et al., 2012). To date, in accordance with the Human Gene Mutation Database (https://www.hgmd.cf.ac.uk/), more than 160 pathogenic mutations have been found in affected individuals. These include missense mutations, nonsense mutations, insertions, small deletions/duplications, splice site mutations and large gene deletions. The mutations are found in the exonic (67.7%) or intronic (31.6%) regions, with no mutation hotspot found within EFTUD2 (Ulhaq et al., 2024). Most of the mutations are predicted to result in haploinsufficiency of EFTUD2. It has been reported that 75% of patients are sporadic cases that result from de novo mutations, whereas 19% are familial cases with an autosomal dominant inheritance pattern. The remaining 6% of cases are due to germline mosaicism (Huang et al., 2016). A clear phenotype–genotype correlation has yet to be established (Yu et al., 2018). Rarely, individuals with a large deletion that involves EFTUD2 may present with not only craniofacial anomalies but other additional features or severe intellectual disability (Gandomi et al., 2015; Zarate et al., 2015). Currently, there are only approximately 150 reported cases of MFDM that have been confirmed to carry EFTUD2 mutations and most of these are individuals of European descent. There are still limited numbers of patients with an Asian background (Ulhaq et al., 2024). The phenotypic and genotypic spectrum of MFDM needs to be expanded to fully understand the clinical characteristics and pathogenesis.

In this study, we described a patient who presented with mandibulofacial dysostosis with an ambiguous diagnosis. Utilizing whole‐exome sequencing (WES) analysis, we identified a novel, pathogenic, heterozygous, insertion mutation in EFTUD2 and made a definitive MFDM diagnosis. This highlighted the importance of genetic diagnosis. The patient presented with several previously unreported features, which included orbit anomaly and spina bifida occulta. This expanded the pool of known phenotypes associated with MFDM.

2. MATERIALS AND METHODS

2.1. Ethics statement

This study was observational. We enrolled an adult male patient who had been referred to our hospital with the chief complaints of auricle deformation and micrognathia. All study protocols were approved by the ethical committee of the Eye & ENT Hospital of Fudan University (2020069). Informed consent was obtained from the patient before enrollment.

2.2. Clinical examination

The physical examination and systemic review were performed by a multidisciplinary team, which included craniofacial surgery specialists, otolaryngologists, audiologists, radiologists, and medical geneticists. A computed tomography (CT) scan was also performed.

2.3. Mutational study

A blood sample was collected from the patient, and DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The exome library was captured and sequenced using an Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, USA), following standard protocols. Raw sequencing data were aligned to the human reference genome sequence, GRCh37 (hg19). After the detection and annotation of variants, we removed the variants that were synonymous, in segment duplication, or with minor allele frequency >0.01. Public databases, in addition to clinical and experimental data collected from the literature, were utilized to filter and prioritize variants. The DNASTAR Lasergene software (DNASTAR, Inc., Madison, WA, USA) was used to predict the change in amino acid sequence after the mutation point. The fragment that encompassed the candidate MFDM mutation was amplified with a polymerase chain reaction and Sanger sequencing was used for validation.

3. RESULTS

3.1. Clinical evaluation

The patient was born at term to non‐affected parents and the pregnancy was uneventful. He had no siblings and there was no family history of craniofacial abnormalities. The patient did not have feeding difficulties or dysphagia, growth restriction, or short stature. He was in good general health, with no reports of intellectual disability. His occipitofrontal circumference (OFC) was 55.2 cm, which was within the normal range. The most prominent feature was micrognathia, which was the result of mandible hypoplasia. He also had upslanted palpebral fissures; low‐set, cup‐like ears and stenosis of the external auditory canals (Figure 1). His limbs and thumbs were normal. The audiologist found his speech recognition normal and his oral language articulate. The patient refused a standard audiometry test to obtain a precise hearing threshold.

FIGURE 1.

Pedigree and manifestation of the affected patient (a) Pedigree of the affected patient. Males are represented as squares, females are represented as circles and the filled symbol is the affected proband. (b) and (c) show the anterior and lateral craniofacial features, respectively. The patient had upslanted palpebral fissures; cup‐like, low‐set ears and severe micrognathia.

3.2. Imaging examination

The CT scan confirmed asymmetric and extensive hypoplasia of the craniofacial bones, which was more severe on the left side (Figure 2). The significant hypoplasia of the mandible resulted in the deformity and dislocation of the temporomandibular joint. The zygomatic arch showed aplasia (Figure 2a). In addition, the CT scan indicated abnormal cervical vertebral bodies, which showed improper segmentation and spina bifida occulta of the vertebrae (Figure 2a,b). Soft tissue hypoplasia was also observed, including a small parotid gland and hypoplasia of the masseter (Figure 2c). No malformation was found in the inner ear and vestibule. However, the bilateral middle ears showed multiple anomalies, which may lead to conductive hearing loss. The right middle ear cavity was enlarged and filled with soft tissue of low density, suggesting previous middle ear inflammation or cholesteatoma (Figure 2d,f). The left middle ear cavity was smaller than normal, with the malformed ossicle chain hanging within (Figure 2e). Left external auditory canal stenosis (<4 mm) was confirmed. The CT scan also showed other unexpected anomalies, such as the curve‐shaped Ramus of the mandible (Figure 2d) and hypoplasia of the inferior‐lateral bony wall of the orbit (Figure 2f).

FIGURE 2.

Computed Tomography revealed bony and soft tissue anomalies in the patient with MFDM. (a) The reconstructed image shows the severe micrognathia, aplasia of the zygomatic arch (star); and improper segmentation of vertebral bodies (arrow); (b) Spina bifida occulta of the vertebrae (arrow). (c) Hypoplasia of the parotid gland (star) and the masseter (arrow) on the left side, in comparison to the right side. (d) and (f) Enlargement of the right middle ear cavity, without ossicle chain (triangle); (d) also shows the shortening and malformation of the ramus of the mandible (arrow). (e) Small middle ear cavity on the left side, with an abnormal ossicle chain (arrow). (f) Partial absence of the bony wall of the orbit (arrow).

3.3. Genetic study

Since the initial examination did not reveal specific symptoms or features conclusive for a diagnosis, we attempted to make a definitive diagnosis using WES analysis. We identified that the EFTUD2 (NM_004247.4) gene contained a heterozygous insertion (c.215_216insT: p.Tyr73Valfs*4) in exon 3. This mutation was subsequently confirmed by Sanger sequencing (Figure 3a). The insertion was predicted, to result in a premature termination codon in exon 3 (Figure 3b) and a truncated protein of only 75 amino acids (Figure 3c).

FIGURE 3.

Mutation validation, location and predicted change in the protein (a) Using WES, a heterozygous, insertion mutation (c.215_216insT) was identified in EFTUD2. The result was confirmed by Sanger sequencing. The arrow indicates the position of the insertion. (b) Intron‐exon structure of EFTUD2. The downward arrow indicates the location of the insertion, which results in a premature termination codon in exon 3. The boxes indicate the exons of EFTUD2. (c) Schematic domain architecture of the EFTUD2 protein, shown in the correct proportions. The arrow indicates the location of the truncation point (amino acid 76), which results from the premature termination codon, to give a shortened protein of 75 amino acids. The frameshift mutation causes a loss of 897 amino acids, which includes the GTP binding domain and domains II–V. The colored boxes indicate the domains of EFTUD2 and the numbers indicate the position of each domain in the full length of the EFTUD2 protein.

3.4. Treatment

This patient required a comprehensive multidisciplinary therapeutic approach to correct the abnormalities and to improve his facial esthetics. He underwent mandibular distraction osteogenesis and was satisfied with the result. Precise audiometry and regular visits for stenosis of the external auditory canal were also advised. Given his good general health, the patient declined further examinations, such as an esophagogram or ultrasonography, to rule out extracranial anomalies. We also discussed the results of the genetic analysis with the patient, detailing the pathogenic mutation in EFTUD2 and discussing potential strategies for preventing the transmission of the mutation to offspring.

4. DISCUSSION

This study was based on an adult patient with craniofacial dysostosis, and outer and middle ear malformation. WES was used to resolve the ambiguous diagnosis provided by other examinations. We identified a novel pathogenic insertion mutation in EFTUD2, which led to the diagnosis of MFDM.

The EFTUD2 gene is located on chromosome 17q21.31 and contains 28 exons. It encodes a highly conserved 972 amino acid spliceosomal GTPase, which has a core component of U5 small nuclear ribonucleoproteins and plays an important role in the splicing process (Beauchamp et al., 2020). The EFTUD2 protein (also called U5‐116kD) contains six domains that include a N‐terminal acidic domain, a GTP‐binding domain essential for the hydrolysis of GTP, and additional conserved domains, II–V, that are shared amongst EF‐2 ribosomal translocase proteins. While the N‐terminal acidic domain is not present in other members of this protein family, the remainder of EFTUD2 is very similar to the ribosomal translation elongation factor, EF‐2 (Beauchamp et al., 2020; Lines et al., 2012). In our case, the insertion mutation was predicted to yield a truncated EFTUD2 protein, with the loss of the GTP‐binding domain and an incomplete N‐terminal acidic domain. In animal models, zebrafish with a heterozygous mutation in the N‐acidic terminal domain exhibited a shortened jawbone and deformity of Meckel's cartilage, which mimics the craniofacial anomalies of human MFDM (Wu et al., 2019). Homozygous conditional deletion of the exon 2 of Eftud2 in neural crest cells in mice causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients (Beauchamp et al., 2021). Hence, the insertion was identified as a pathogenic loss‐of‐function mutation in the patient with MFDM. Regretfully, the non‐affected parents of this patient were not involved in the molecular study. They would have served as the best controls to further validate the relationship between the mutation and MFDM.

Identification of MFDM remains a diagnostic dilemma. A number of heterogeneous monogenic diseases present with craniofacial phenotypes similar to those observed in MFDM patients. These diseases include Treacher Collins syndrome (OMIM# 154500), Nager syndrome (OMIM# 154400), oculoauriculovertebral spectrum, CHARGE syndrome (OMIM# 214800), postaxial acrofacial dysostosis (or Miller syndrome, OMIM# 263750), microsomia and many other congenital diseases that affect the craniofacial region. The overlapping phenotypes include craniofacial hypoplasia, outer and middle ear anomalies associated with hearing loss, choanal atresia, and cleft palate. The similarities with other diseases cause a major challenge in making a definitive diagnosis using clinical features, particularly when the manifestations are atypical or mild.

Until now, there have not been any well‐established diagnostic standards for MFDM. However, based on phenotypic characteristics, some scholars have proposed that MFDM should be considered as the first‐line clinical diagnosis for patients with facial dysostosis, microcephaly, and otologic problems (Ryu et al., 2022). However, these criteria are still too broad to exclude some phenotypically similar syndromes (Table 1). By comparing various mandibulofacial dysostosis syndromes, we aimed to identify the most characteristic manifestations to help in diagnosing MFDM (Table 1) (Abell et al., 2021; Beleza‐Meireles et al., 2015; Lehalle et al., 2014; Marszalek‐Kruk et al., 2021; Thomas et al., 2022; Yu et al., 2018). In the reported MFDM cases validated with EFTUD2 mutations, the most frequent phenotypes were micrognathia of variable severity, external ear anomalies and malar hypoplasia. The most specific manifestation of MFDM is microcephaly, which is more common in MFDM cases (88%) than in Treacher Collins syndrome (3%) or other craniofacial syndromes. Congenital anomalies of the esophagus, such as TEF and EA, are more commonly seen in patients with MFDM than in other mandibulofacial dysostosis syndromes (Table 1). Notably, eye defects were found in 28% of the MFDM cases and consisted of optometric anomalies, epicanthic folds, lacrimal duct stenosis and epibulbar dermoid (Lehalle et al., 2014). Manifestations that affect appearance of the eyes, such as microphthalmia, coloboma or retinal dystrophy, are rarely reported in individuals with EFTUD2 mutations. A novel deletion in EFTUD2 was identified in a patient with syndromic microphthalmia, anophthalmia and coloboma, though the ocular phenotype was considered coincidental (Deml et al., 2015). In summary, typical MFDM patients may present with a striking mandibulofacial dysostosis phenotype and ear malformation. Most also have a small OFC and show symptoms of TEF/EA, with normal eye size and shape.

TABLE 1.

Comparison of the prevalence of phenotypes in different mandibulofacial dysostosis syndromes.

	MFDM a	Treacher Collins syndrome b	Oculoauriculovertebral spectrum c	Nager Syndrome d	CHARGE syndrome e
Prevalence	Unknown	1/50,000	3.8/100,000	Unknown	1/8,500
Causative gene	EFTUD2	TCOF1 (63%–93%) POLR1D (6%) POLR1B (1.3%) POLR1C (1.2%)	NR	SF3B4	CHD7
Craniofacial feature
Microcephaly	88%	3%	4%–8%	43%	27%
Micrognathia	98%	78%–91%	NR	100%	43%
Outer and/or middle ear malformation	Up to 97%	Up to 77%	25%–100%	86%	External ear 79% Middle ear 50%
Hearing loss	83%	83%–92%	50%–85%	57%	87%
Facial asymmetry	56%	52%	49%–90%	29%	NR
Eye defect	28% f	54%–69% g	4%–25%	5/7	83% h
Downslanting palpebral fissures	NR	89%–100%	NR	86%	NR
Cleft palate	47%	21%–33%	11%–12%	86%	25%
Extracranial feature
Limb or thumb anomalies	31	1.5%	3%–21%	Thumb 86% feet/toes 71%	NR
TEF/EA or Gastrointestinal anomalies	27%–33%	NR	2%–12%	NR	17%
Heart defect	31%	11%	4%–33%	29%	75%
Developmental delay/Intellectual disability	100%	1.7%–10%	9%–18%	14%	84%

Abbreviations: EA, esophageal atresia; NR, not reported; TEF, tracheoesophageal fistula.

^a Obtained from Huang et al. (2016) and Lehalle et al. (2014).

^b Summarized by Marszalek‐Kruk et al. (2021).

^c Obtained from Beleza‐Meireles et al. (2015).

^d Obtained from Czeschik et al. (2013).

^e Reviewed by Thomas et al. (2022).

^f Symptoms including astigmatism, myopia, hypermetropia, strabismus, epicanthic folds, lacrimal duct stenosis and epibulbar dermoid.

^g Eyelid coloboma.

^h Iris or retina coloboma.

We could not make the initial diagnosis of MFDM because the patient had a normal OFC, and showed no intellectual disability or unique symptoms associated with TEF/EA. In some MFDM cases, the OFC is below the normal range but can reach normal range by adulthood. This underlies the need to retrospectively assess the patient's childhood condition when collecting medical histories (Huang et al., 2016). Moreover, dispite the initial misdiagnosis, we found some rare features. The CT scan displayed hypoplasia of the bony wall of the orbit, which may cause diplopia in some patients. The spina bifida occulta, which is the failure of fusion of neural tube, is a new form of the spine anomaly in MFDM. While this anatomic variant does not seem to cause symptoms, it can rarely develop into meningocele or myelomeningocele that needs repair via surgery (Manenti et al., 2017). To our knowledge, these features have not been reported.

Identification of a harmful mutation in EFTUD2 is the gold standard for diagnosis of MFDM. Some scholars choose Array Comparative Genomic Hybridization (Array‐CGH) as the first‐tier diagnostic test (Gandomi et al., 2015), while others use targeted EFTUD2 analysis for patients who have been clinically diagnosed with MFDM (Yu et al., 2018). As the rapidly developing WES technology has been proven effective for the diagnosis of genetic disorders, we propose that WES should be the first‐line method to identify causative mutations in monogenic diseases such as MFDM. When a patient meets the diagnostic criteria of MFDM, WES should be advised for the affected individual.

This study emphasizes the need for genetic testing. The correct diagnosis is necessary to allow clinicians to give the right prognosis and the proper treatment for affected individuals. Furthermore, genetic tests can offer valuable genetic information to the affected families during genetic consultations, prenatal screening, and preimplantation diagnosis. These efforts will decrease the risk of birth defects.

5. CONCLUSION

We recruited a Chinese patient who had multiple anomalies that included craniofacial dysostosis, ear malformation, and a previously unreported form of cervical spine defect. We used WES analysis to detect a novel, pathogenic, insertion mutation c.215_216insT: p.Tyr73Valfs*4 in EFTUD2, which was subsequently confirmed by Sanger sequencing. The identified mutation was predicted to be pathogenic, introduces a premature termination codon, which results in a truncated EFTUD2 protein lacking key domains to function normally. Hence, this loss‐of‐function mutation was considered to be the disease‐causing mutation in our patient. The result expands the genotypic and phenotypic knowledge of MFDM and also reminds clinicians that MFDM may manifest with atypical features that need special attention.

AUTHOR CONTRIBUTIONS

All co‐authors have reviewed and approved of the manuscript prior to submission. Ying Chen performed the molecular study and wrote the original draft. Run Yang performed the molecular study and data curation. Xin Chen performed clinical data curation and help to wrote the original draft. Naier Lin, Chenlong Li, Yaoyao Fu, Aijuan He participated in multidisciplinary clinical evaluation. Yimin Wang performed bioinformatic analysis. Tianyu Zhang designed this work, and reviewed the manuscript. Jing Ma designed this work, and reviewed the manuscript.

FUNDING INFORMATION

This study was financially supported by the Science and Technology Commission of Shanghai Municipality (No. 21DZ2200700), the National Natural Science Foundation of China (No. 82271889, No. 82371173), Science and Technology Innovation Plan Of Shanghai Science and Technology Commission (No. 23ZR1409400), and the National Key Research and Development Program of China (No. 2021YFC2701000).

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest.

ETHICS STATEMENT

The institutional review board approved the present study (No. 2020069).

PATIENT CONSENT STATEMENT

The patient in this study has signed the informed consent of the Eye & ENT Hospital of Fudan University to collect clinical data and biological samples for scientific study and academic publication. Informed consent was obtained from the patient before enrollment.

Chen, Y. , Yang, R. , Chen, X. , Lin, N. , Li, C. , Fu, Y. , He, A. , Wang, Y. , Zhang, T. , & Ma, J. (2024). Atypical mandibulofacial dysostosis with microcephaly diagnosed through the identification of a novel pathogenic mutation in EFTUD2 . Molecular Genetics & Genomic Medicine, 12, e2426. 10.1002/mgg3.2426

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the finding of this study are available within the article. Raw genomic data can be provided by the corresponding author, upon reasonable request, such as for academic research purposes.