A Novel De Novo TUBB3 Variant Causing Developmental Delay, Epilepsy and Mild Ophthalmological Symptoms in a Chinese Child

Heterozygous missense mutations in TUBB3 have been implicated in various neurological disorders encompassing either isolated congenital fibrosis of the extraocular muscles type 3 (CFEOM3) or complex cortical dysplasia with other brain malformations 1 (CDCBM1). The description of seizures in patients with TUBB3 mutations is rare. Here, we reported a patient who had febrile seizures before and focal seizure this time, which was diagnosed as epilepsy in combination with an abnormal EEG. MRI showed hypoplastic corpus callosum. Mutation analysis showed a novel de novo heterozygous variant of the TUBB3 gene (NM_006086), c.763G > A (p.V255I). The patient had global developmental delay, photophobia and elliptic pupils, but lacking extraocular muscle involvement and malformations of cortical development, which might be a less severe phenotype of TUBB3 mutations. This is the first report of elliptic pupils in a patient with TUBB3 mutations and expands the spectrum of TUBB3 phenotypes. It indicates that the phenotypic range of TUBB3 mutations might exist on more of a continuum than as a discrete entity, with severity ranging from mild to severe. Further studies are needed to elucidate the complete spectrum of TUBB3-related phenotypes.


Introduction
Tubulins represent the major constituents of microtubules; they are dimeric proteins consisting of two closely related α and β subunits encoded by tubulin genes (i.e., TUBA1A, TUBB2B, TUBB3, TUBB4A, TUBB2A, TUBB, TUB8A) (Wu et al. 2014). Mutations in the α-and β-tubulin genes, related to the functional area of the protein involved, lead to a complex and wide spectrum of cerebral malformations defined as "tubulinopathies" (Mutch et al. 2016;Tischfield and Engle 2010;Romaniello et al. 2019). In addition, because neurons are particularly susceptible to microtubule defects, the deregulation of the microtubule cytoskeleton caused by mutations in tubulin genes is considered to be a possible pathogenesis of neurodegenerative disorders (Sferra et al. 2020). Beta-III tubulin (TUBB3, MIM 602,661) is the most dynamic β-tubulin isoform expressed in neurons, and is highly expressed in the central nervous system (Panda et al. 1994). Mutations altering TUBB3, which is expressed in post-mitotic neurons but not glia, segregate into two distinct phenotypic groups: congenital fibrosis of the extraocular muscles (CFEOM), a congenital disorder in which maldevelopment of cranial nerve axons leads to ptosis (drooping eye lids) and restricted eye movements Demer et al. 2010;Dentici et al. 2020), and a spectrum of cortical dysplasias collectively termed malformations of cortical development (MCD), such as neuronal migration defects, complex cortical dysgenesis and gyral simplification, cerebellar vermis dysplasia, thinning or agenesis of corpus callosum, and oculomotor and olfactory nerve hypoplasia (Poirier et al. 2010). However, the relationship between TUBB3 and epileptic seizures had not been thoroughly investigated. Here, we reported a patient who presented with epilepsy and global developmental delay, and genetic analysis showed a novel de novo TUBB3 variant.

Ethics Statement
This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. The boy's parents had given written informed consent to publish these cases details.

Methods
Patients and data collection. The patient was admitted to our hospital in February 2021. Clinical records were reviewed to obtain information including sex, history of febrile seizures, age at seizure onset, seizure types, electroclinical features, and treatment. The psychomotor development was assessed according to clinical judgment. Biochemical studies, blood and urinary metabolic screenings, video electroencephalography (VEEG) and magnetic resonance imaging (MRI) were performed. VEEG monitoring. VEEG monitoring was performed using a Nihon Kohden digital video-EEG-1100 K instrument for about 4 h, covering both awake and sleep states. EEG electrodes were positioned over the scalp according to the international 10-20 system, with the corresponding polyelectromyography to record the activities of muscles including the deltoid, quadriceps femoris and so on. EEG and PEMG activities were recorded with band passes of 0.3-70 Hz and 5.3-120 Hz. The EEG traces were evaluated by a qualified neurophysiologist.
MRI examination. The patient underwent 3.0 T MRI of sequences, including T1-weighted, T2-weighted, FLAIR, and DWI sequencing in the axial, sagittal, and coronal planes.
Whole exome sequencing. Genetic analysis was performed by whole exome sequencing. Exome capture was performed using the xGen Exome Research Panel v1.0, which consists of 429,826 individually synthesized and quality-controlled xGen Lockdown Probes. The captured libraries were sequenced using an Illumina NovaSeq 6000 sequencer at 100 × coverage. The reads were mapped to the Ensembl GRCh37/hg19 by a Burrows-Wheeler aligner (BWA 0.7.11) with default parameters. Duplicate reads were marked with Picard Tools, version 2.9.0. The Genome Analysis Tool Kit (GATK 3.5) was used for local realignment and base quality score recalibration. Variants were called jointly in all samples using GATK's HaplotypeCaller. Variant Effect Predictor (VEP 93) was used to annotate variants with predicted effects on protein-coding genes. The transcript database used was RefSeq transcripts (Li et al. 2019). The pathogenicity of the identified missense substitution was predicted based on the lack of such mutations in the control samples and on the conservation of substituted nucleotides and amino acids. We also predicted the functional alteration of the novel missense mutations using the polymorphism phenotyping-2 tool (PolyPhen-2, http:// genet ics. bwh. harva rd. edu/ pph2/) and MutationTaster (http:// www. muta-tiont aster. org/).

Case Report
Our patient is a 21-month-old boy, the first child (the third pregnancy) of non-consanguineous healthy parents with unremarkable family history. Abortion was performed on the 50th day of the first gestation due to absence of fetal heart rate. The second pregnancy aborted at 18 6/7 weeks due to cervical insufficiency. Pregnancy and perinatal of this child were uneventful, and birth was at term with a spontaneous vaginal delivery. Birth weight was 4.3 kg. Developmental milestones were delayed. Crawl was achieved at 11 months, independent standing at 16 months, and unsupported walking with a waddling gait at 19 months. Currently, the 21-month-old boy can walk independently with a waddling gait for about 3 m and can speak only several reduplicated words such as "baba" and "mama." The auxological parameters were within normal range, with weight of 12.5 kg (50th-75th centile), height of 86 cm (50th-75th centile) and occipital frontal circumference (OFC) of 46 cm (5th-10th centile). Photophobia and elliptic pupils were found by the parents at an age of several months without special diagnosis and treatment. He had three febrile seizures since the age of 1 year, with a decreasing fever peak (39.9, 38.5 and 37.8 °C, respectively), and two seizures occurred during the third febrile episode. This time, the child was admitted to our hospital for an afebrile convulsion during an awake state, which manifested as blinking, eyes slowly turning horizontally, clenched fists, straightened and lifted right upper limb, left upper limb at the side of the body, limbs stiff and shaking, with loss of consciousness, lasting for about 10 min. The results of neurological physical examination, including cranial nerve examination, meningeal irritation sign and pathologic reflex, were unremarkable. Blood examinations revealed normal blood cell count and C-reactive protein level, and normal levels of serum ammonia, glucose, sodium and calcium (Table S1). The EEG revealed multifocal spikes and waves and slow wave discharges, predominantly in bilateral anterior regions (Fig. 1). Brain MRI disclosed a significantly hypoplastic corpus callosum (Fig. 2). Chromosome karyotype, plasma amino acid and organic acid analyses were normal.

Genetic Analysis
Mutation analysis showed a novel de novo heterozygous variant of TUBB3 (NM_006086), c.763G > A (p.V255I) in exon 4 (Fig. 3). Based on the Human Gene Mutation Database (HGMD), the variant has not been reported before. The missense variant c.763G > A in TUBB3 affects evolutionarily conserved residues. It is predicted by PolyPhen-2 to be "probably damaging" on protein function, with a probabilistic score of 0.986, and predicted by MutationTaster to be "disease causing" with a probabilistic score of 0.999.

Discussion
Tubulin proteins play a key role in several cellular processes crucial for proper cortical development during neuronal proliferation, migration, differentiation, cortical laminar organization and axonal guidance of the radial glia (axon outgrowth and maintenance) (Romaniello et al. 2019(Romaniello et al. , 2015. TUBB3 expression is greatest during periods of axon guidance and maturation; levels decrease in the adult central nervous system (CNS) but remain high in the peripheral nervous system (PNS) Jiang and Oblinger 1992). Heterozygous missense mutations in TUBB3 have been implicated in various neurological disorders encompassing either isolated CFEOM3 (MIM 600,638) or complex cortical dysplasia with other brain malformations 1 (CDCBM1) (MIM 614,039). CFEOM encompasses several different inherited strabismus syndromes characterized by congenital restrictive ophthalmoplegia affecting the extraocular muscles innervated by the oculomotor and/or trochlear nerves. If all affected members of a family have classic CFEOM with bilateral involvement and inability to raise the eyes above midline, the phenotype is classified as CFEOM1 (MIM 135,700). CFEOM2 (MIM 602,078) shows autosomal recessive inheritance. CFEOM3 is characterized by autosomal dominant inheritance of a more variable phenotype than classic CFEOM1. Individuals with CFEOM3 may not have bilateral involvement, may be able to raise the eyes above midline, or may not have blepharoptosis (Yamada et al. 2004;Heidary et al. 2008). CDCBM is a disorder of aberrant neuronal migration and disturbed axonal guidance. Affected individuals have mild to severe mental retardation, strabismus, axial hypotonia, and spasticity. Brain imaging shows variable MCD, including polymicrogyria, gyral disorganization, and fusion of the basal ganglia, as well as a thin corpus callosum, hypoplastic brainstem, and dysplastic cerebellar vermis. Extraocular muscles are not involved (Poirier et al. 2010).
Although the clinical phenotype related to TUBB3 has been widely reported, the description of seizures in patients with TUBB3 mutations is rare. It might because that TUBB3 was involved primarily in axon guidance mechanisms and scantly associated with MCDs and, consequently, with low levels of epileptogenicity Romaniello et al. 2015). After reviewing the literature published previously, we found only six cases that had seizures during the courses (Table 1) (Poirier et al. 2010;Chew et al. 2013;Oegema et al. 2015;Costain et al. 2019;Ziats et al. 2020). It seemed that both the phenotype CFEOM and CDCBM could present epileptic seizures. Poirier et al. (Poirier et al. 2010) reported two cases carrying TUBB3 mutations that had seizures, consisting of refractory and multifocal epilepsy in one case, and occasional prolonged febrile seizures in another patient. Chew et al. (Chew et al. 2013) and Oegema et al. (Oegema et al. 2015) each reported one case with complex partial seizures. Costain et al. (Costain et al. 2019) reported one patient with generalized tonic-clonic seizures and tonic spasms. Ziats et al. (Ziats et al. 2020) found a child with epilepsy carrying a TUBB3 mutation among 523 patients by clinical exome sequencing. All six patients had intellectual disability and/or motor delay, as well as corpus callosum abnormalities. Ophthalmological symptoms (three of four cases) and MCD in cranial MRI (three of four cases) were also observed. Similarly, our patient here had febrile seizures before and focal seizure this time, which was diagnosed as epilepsy in combination with an abnormal EEG. He had global developmental delay, and the MRI showed a hypoplastic corpus callosum. Recently, (Xu et al. 2017) reported that TUBB3 expression was increased in patients with temporal lobe epilepsy and experimental models of epileptic seizures, which supported that TUBB3 played an important role in regulating epileptic seizures via GABA-A receptor-mediated synaptic transmission and probably explained the rare epileptic cases that described TUBB3-mutated patients.
Of note, our patient here had photophobia and elliptic pupils, but lack of additional ophthalmological impairment indicated an ocular motility disorder, CFEOM3. Although intermittent or permanent nonparalytic strabismus had been reported previously (Poirier et al. 2010), this was the first report of elliptic pupils in a patient with TUBB3 mutations. We speculated that Fig. 1 The EEG results of the patient. Multifocal spike and waves and slow wave discharges, predominantly in the right frontal pole, frontal, and anterior temporal regions (a) and left frontal and anterior temporal regions (b) it might be due to fine oculomotor nerve defects resulting in damage to the intraocular muscle, considering that TUBB3 mutations could affect extraocular muscles innervated by the oculomotor and/or trochlear nerves. However, elucidation of the exact mechanism requires further research.
It has been suggested that TUBB3 mutations cause CFEOM or CDCBM. The former is not associated with cortical malformations, and the latter is not associated with CFEOM or other forms of ophthalmoplegia, suggesting that each set of mutations alters the microtubule functions in a specific fashion (Poirier et al. 2010;Oegema et al. 2015;Bahi-Buisson et al. 2014). The genotype/phenotype correlations were assumed not only by the primary sequence of the protein, but also by the tertiary sequence and the three-dimensional arrangement of the   (Poirier et al. 2010). In disagreement with this clinical stratification, our patient with a novel c.763G > A (p.V255I) of TUBB3 manifested as epilepsy with global neurodevelopmental delay, a mild ocular phenotype and hypoplastic corpus callosum, with no evidence of MCD and extraocular muscle involvement, which did not seem to meet the diagnostic criteria of either CFEOM or CDCBM. Besides, two TUBB3 mutations (G71R and G98S) had been found in four patients with CFEOM, intellectual disability, and MCD (Whitman et al. 2016). Thus, tubulinopathies caused by TUBB3 might be associates with a wider range of clinical phenotypes such as intraocular muscle involvement or merge the two distinct phenotypes (CFEOM and CDCBM) in the same patient, suggesting a common microtubule dysfunction shared by both phenotypes (Whitman et al. 2016). Consequently, our findings expand the spectrum of TUBB3 phenotypes to global developmental delay, epilepsy, photophobia and elliptic pupils, and a hypoplastic corpus callosum, without extraocular muscle involvement or MCD, which might be a less severe phenotype of TUBB3 mutations. This indicates that the phenotypic range of TUBB3 mutations might be more continuous than discrete, with severity ranging from mild to severe. However, it is worth mentioning that the possibility that some clinical manifestations had not presented completely could not be excluded, considering the patient here was only 21 months old at the last follow-up. Further studies are needed to elucidate the complete spectrum of TUBB3-related phenotypes.