A novel variant of NEMF in a family with congenital fibrosis of the extraocular muscles: a case report and literature review

doi:10.21203/rs.3.rs-4913975/v1

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Case Report

A novel variant of NEMF in a family with congenital fibrosis of the extraocular muscles: a case report and literature review

https://doi.org/10.21203/rs.3.rs-4913975/v1

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Background

Congenital fibrosis of the extraocular muscles (CFEOM) is one of the congenital cranial dysinnervation disorders (CCDDs) characterized by hypoplastic oculomotor nerves (CN3), congenital ptosis and non-progressive restrictive ophthalmoplegia. The NEMF gene encodes a protein that resolves stalled ribosomes during translation. Variants in NEMF have been associated with neurodevelopmental and neuromuscular disorders, including intellectual disability, developmental delay, central nervous system impairment and peripheral neuropathy.

Case presentation:

We describe a family with CFEOM harboring a novel variant of NEMF gene. CFEOM3 was diagnosed in two siblings, a 9 year old girl and a 6 year old boy. Whole-exome sequencing (WES) revealed that the siblings carried a novel heterozygous NEMF variant c.1972A > C, p. (Lys658Gln). This variant was predicted to lead to the loss of hydrogen bonds, potentially affecting the spatial structure and stability of the NEMF protein.

Conclusions

Our findings support that the NEMF variant may be the cause of CFEOM in our case. This report expands the phenotypic spectrum of NEMF-associated diseases and provides a new candidate gene for CFEOM.

Congenital fibrosis of the extraocular muscles

NEMF

heterozygous

phenotypic spectrum

NEMF plays a critical role in the ribosome-associated quality control (RQC) pathway[1], which is essential for maintaining proteostasis and preventing neurodegeneration[2, 3]. NEMF is highly expressed in the human brain. Variants in NEMF(OMIM#608378) can cause a range of neurodevelopmental disorders, such as intellectual disability (ID), developmental delay, central nervous system impairment and peripheral neuropathy[4–6]. Most reported cases are autosomal recessive inheritance. Patients with central nervous system impairment due to NEMF variants may exhibit abnormal eye movement phenotypes such as strabismus[6], but no association with congenital fibrosis of the extraocular muscles (CFEOM; HP:0001491) has been reported.

CFEOM, as a class of congenital cranial dysinnervation disorders (CCDDs), is characterized by congenital ptosis and non-progressive restrictive ophthalmoplegia[7]. Hypoplasia of oculomotor nerves (CN3) is the most common MRI finding of CFEOM. CFEOM is mainly divided into three subtypes, CFEOM1, CFEOM2 and CFEOM3. CFEOM1 and CFEOM3 are autosomal dominant disorders, whereas CFEOM2 is an autosomal recessive disorder[8]. CFEOM1 presents with bilateral symmetrical ptosis and the eyes fixed in downgaze. CFEOM3 is a more variable eye phenotype that can include asymmetrical ptosis and oculomotility restrictions. CFEOM2 is typically characterized by bilateral restrictive ophthalmoplegia, pupil abnormalities, and ptosis. At present, several pathogenic genes are known to be related to CFEOM, including KIF21A, TUBB3, PHOX2A, TUBB2B and TUBA1A.

Here, we report a novel NEMF missense variant associated with CFEOM in a family. This abnormal eye movement, which has not been previously reported, expands the phenotype spectrum of NEMF-related diseases.

The patients are children of non-consanguineous Chinese parents (Fig. 1A), born at term with normal birth parameters. Both siblings reached the developmental milestones within the normal age range. No significant abnormalities were observed on their cranial MRI.

The elder sister was a 9-year-old girl who presented to our ophthalmology clinic with abnormal eye movement since birth. She had already undergone right lateral rectus recession and medial rectus resection surgery at 4 years of age. On examination, she had right exotropia of 40^△ on modified Krimsky test and right ptosis. Ocular motility examination showed that she had − 3 limitation of abduction in the right eye. Her right eye moved into abduction on attempted adduction (Fig. 1C). MRI of the brain and orbits revealed the hypoplasia of right CN3 (Fig. 1E: e1) and bilateral abducens nerves (CN6) (Fig. 1E: e2). In addition, her right medial rectus muscle (MR) appeared thinner (Fig. 1E: e3).

The younger brother was a six-year-old boy who was brought to our clinic with similar complaints as his older sister. At the time of presentation, he had undergone bilateral lateral rectus recession and medial rectus resection for exotropia and frontalis aponeurosis flap suspension for right ptosis. On examination, he was orthophoric in primary gaze with right ptosis and nystagmus. He presented with a compensatory left head turn. Ocular motility examination showed restricted horizontal movement and limitation in downgaze in his left eye. Additionally, he had upshoot of the right eye on attempted adduction (Fig. 1D). MRI revealed the hypoplasia of bilateral CN3 (Fig. 1F: f1), the absence of bilateral CN6 (Fig. 1F: f2) and hypoplastic MR bilaterally (Fig. 1F: f3-f4).

Whole exome sequencing (WES) revealed that two siblings carried a novel heterozygous variant of NEMF (NM_004713: c.1972A > C, p. (Lys658Gln) (chr14: 50269389T>G, hg19)) and inherited from their unaffected father (Fig. 1B). The heterozygous variant was validated by Sanger sequencing. According to the American College of Medical Genetics and Genomics (ACMG) genetic variant classification criteria and guidelines, the NEMF gene missense variant p. (Lys658Gln) was defined as a pathogenic variant (PVS1 + 2PM + 3PP). The variant resulted in a lysine to glutamine change at amino acid position 658 (Lys658Gln), which was not present in the Genome Aggregation Database (gnomAD), 1000 Genomes project, the Exome Aggregation Consortium (ExAC), or the NHLBI Exome Sequencing Project (ESP6500si). The conservation analysis based on the phyloP demonstrated that p. (Lys658Gln) substitution resulted in highly conserved amino acid changes, which was also analyzed in several common animals through the UGENE software (Fig. 2B). A variety of bioinformatics prediction softwares, such as SIFT (deleterious), MutationTaster (disease causing), PolyPhen-2 (probably damaging) and LRT (deleterious), predicted the p. (Lys658Gln) substitution to be harmful. Protein-function prediction suggested that p. (Lys658Gln) substitution leads to a reduction in hydrogen bonds, affecting the stability of protein spatial structure (Fig. 2C). We use the 3D protein structure of human NEMF predicted by AlphaFold (AF-O60524-F1) to conduct the computational-stability analysis. Multiple online servers indicated that the variant resulted in a decrease in the stability of the protein structure in terms of a significant decline in Gibbs free energy (Table 1).

Table 1

The stability analysis of *NEMF* K658Q by different servers.
Mutation		I-Mutant 2.0	mCSM	SDM	DUET	ENCoM	DynaMut	MAESTRO
NEMF- K685Q	ΔΔG (kcal/mol)	-0.910	-0.996	-1.010	-0.940	-0.102	-0.333	0.058
NEMF- K685Q	Prediction	Destabilizing	Destabilizing	Destabilizing	Destabilizing	Destabilizing	Destabilizing	Destabilizing

We report a family with CFEOM harboring a novel NEMF variant. Our patients present with unilateral ptosis and ophthalmoplegia of varying severity. MRI findings of hypoplastic CN3 and extraocular muscles in two siblings were consistent with CFEOM. CFEOM is a subset of CCDDs and belongs to the neurodevelopmental disorders. At present, the known pathogenic genes related to CFEOM include KIF21A, TUBB3, PHOX2A, TUBB2B and TUBA1A. Among them, KIF21A and TUBB3 genes are the most common pathogenic genes, and variants of them have been reported to affect neurodevelopment and axon guidance[9, 10]. Heterozygous missense variants in KIF21A and TUBB3 can cause CFEOM, while some variants also appear to exhibit incomplete penetrance.

The NEMF gene has also been linked to neurodevelopmental diseases. The NEMF (Nuclear Export Mediator Factor) gene, located on chromosome 14q22, encodes a component of the RQC complex[11]. RQC ensures the quality of translation products by monitoring and eliminating incomplete nascent polypeptides produced from interrupted translation. RQC is a key molecular pathway that prevents neurodegeneration[6, 12]. In a previous study, Anazi et al. reported NEMF as a new candidate gene for intellectual disability (ID)[4]. They identified homozygous truncating variants in two families with ID and developmental delay. Brain MRI of these patients showed no significant abnormalities. Next, Martin et al. described NEMF variants in seven families associated with juvenile neuromuscular disease, which presented with ID and motor neuron disease phenotypes of varying severities[5]. They proposed that NEMF was a novel gene associated with neurodegeneration and neuromuscular disease in mice and humans. In addition, Ahmed et al. found that NEMF may affect neuronal projection during early brain development, and that variants in NEMF can lead to central nervous system impairment and peripheral neuropathy[6].

Eye movement phenotypes have also been reported in NEMF-related diseases. In a previous study, a total of six cases with NEMF-related eye movement phenotypes were reported[6], including three cases of strabismus, one case of impaired smooth-pursuit and saccade eye movements, one case of impaired smooth-pursuit and saccade eye movements with esotropia, and one case of Duane syndrome Type 1. The patients in our study were diagnosed with CFEOM based on a combination with clinical findings and MRI findings. Our patients exhibited an atypical eye movement phenotype, characterized by abduction or upshoot of the right eye when attempting adduction. This CFEOM phenotype associated with a NEMF variant has not yet been reported.

Most cases of human NEMF variants were reported to be associated with autosomal recessive inheritance. Martin et al. reported that a heterozygous variant in NEMF (c.1658T > C, p. (Ile553Thr)) caused later-onset peripheral neuropathy, including distal muscle atrophy and tremor[5]. Our patients carry a novel heterozygous variant in NEMF (c.1972A > C, p. (Lys658Gln)), which was inherited from their phenotypically unaffected father. Similarly, Senser et al. reported a member of a CFEOM family carrying a KIF21A variant who was clinically unaffected[13]. Additionally, Tischfield et al. discovered that a patient with CFEOM inherited the TUBB3 R62Q variant from his unaffected mother[10]. These observations suggest that the variants associated with CFEOM may exhibit incomplete penetrance.

Homozygous Nemf mice displayed a series of neurologic phenotypes such as growth reduction, neurogenic muscle atrophy, and reduced lifespan, while findings of denervation of postsynaptic terminals and progressive axon loss in mice also supported the association between NEMF and neurodevelopmental abnormalities[5]; Knocking down Nemf gene in cultured mouse primary cortical neurons resulted in shorter axon length and fewer synapses[6]. Different mutation sites may lead to varying degrees of impact on NEMF function and differences in severity of symptoms. Martin et al. constructed two mouse models, where the R86S mutation located in the N-terminal domain resulted in a shorter lifespan and more severe phenotypes compared to the R487G mutation located in the second coiled-coil domain[5]. The variant p. (Lys658Gln) identified in this study is located in the R domain[14] (Fig. 2A), and the substitution is predicted to reduce hydrogen bonds, affecting the structural stability of NEMF.

In conclusion, this study reports a novel missense variant in NEMF causing CFEOM in two siblings. Additional research is needed to elucidate the impact of the missense variant on NEMF function. This study expands the clinical phenotypic spectrum of NEMF-related disorders and provides a new candidate pathogenic gene for CFEOM.

CFEOM Congenital fibrosis of the extraocular muscles

CCDDs Congenital cranial dysinnervation disorders

CN3 Oculomotor nerves

RQC Ribosome-associated quality control

CN6 Abducens nerves

MR Medial rectus muscle

ID Intellectual disability

Ethics approval and consent to participate

This study was approved by the Institution Review Board of Beijing Tongren Hospital, Capital Medical University (TRECKY2018-035) and adhered to the ethical principles outlined in the Declaration of Helsinki.

Consent for publication

Written informed consent for publication of this case report was obtained from the parents.

Competing interests

The authors declare no competing interests.

Funding

This study was funded by the National Natural Science Foundation of China (No.82070999).

Author Contribution

CZ P designed the study, collected the data, reviewed the literature, analyzed the data, wrote the manuscript. RR Z analyzed the data, revised the manuscript. HY J analyzed the data, collected the data. QL C provided and analyzed the MRI results. D W collected the data. YH J designed the study, analyzed the data, supervised the study, revised the manuscript. All authors read and approved the final manuscript.

Acknowledgement

We are grateful to the patients and their family for their participation in the study.

Data availability

All data generated or analyzed in this study are presented in this article.

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No competing interests reported.

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A novel variant of NEMF in a family with congenital fibrosis of the extraocular muscles: a case report and literature review

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Abstract

Background

Case presentation:

Conclusions

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Background

Case presentation

Discussion and conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Funding

Author Contribution

Acknowledgement

Data availability

References

Additional Declarations

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