A Novel De Novo KIF21A Variant in a Patient With Congenital Fibrosis of the Extraocular Muscles With a Syndromic CFEOM Phenotype

Clinical Correspondence Section Editors: Robert Avery, DO Karl C. Golnik, MD A Novel De Novo KIF21A Variant in a Patient With Congenital Fibrosis of the Extraocular Muscles With a Syndromic CFEOM Phenotype Luca Soliani, MD, Carlotta Spagnoli, MD, Grazia G. Salerno, MD, Miika Mehine, PhD, Susanna Rizzi, MD, Daniele Frattini, MD, Juha Koskenvuo, MD, PhD, Carlo Fusco, MD Downloaded from http://journals.lww.com/jneuro-ophthalmology by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC4/OAVpDDa8KKGKV0Ymy+78= on 05/04/2022 C ongenital fibrosis of the extraocular muscles (CFEOM) is a group of disorders in which a severe restriction of eye movements and ptosis are present because of abnormalities of extraocular muscles innervation caused by abnormal oculomotor and trochlear nerve development (1). The classification of CFEOM is evolving from one based on phenotypic manifestations to a genetic classification based on molecular analysis. CFEOM (CFEOM-1) presents autosomal dominant inheritance and is characterized by bilateral blepharoptosis and ophthalmoplegia with the eyes fixed in an infraducted (downward) primary position. Neuroimaging typically shows hypoplasia of the oculomotor muscles and nerve. CFEOM-1 results mostly from heterozygous mutations in the kinesin family member 21 (KIF21A) gene (gene ID: 300158; OIM 608283) encoding a kinesin motor protein (2). CFEOM-3 is a different subtype of the disorder, often inherited as an autosomal dominant trait, in some cases with incomplete penetrance, resulting in unilateral or bilateral ptosis and/or restrictions in ocular motility. The majority of described families with CFEOM3 carry heterozygous pathogenic variants in the TUBB3 gene (gene ID: 22152; OMIM 602661) encoding for the neuron-specific beta-tubulin isotype III (3). A small number of the individuals with CFEOM3, with specific mutations in TUBB3 gene, also manifest facial paralysis, intellectual impairments, and social disability, as well as structural cerebral malformations (i.e., dysgenesis of the corpus callosum, basal ganglia, or corticospinal tracts), abnormal white matter organization, and peripheral polyneuropathy (1,5,6). Department of Pediatrics (LS, CS, GGS, SR, DF, CF), Child Neurology Unit, Presidio Ospedaliero Provinciale Santa Maria Nuova, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy; Blueprint Genetics (MM, JK), Helsinki, Finland; and Pediatric Neurophysiology Laboratory (CF), Presidio Ospedaliero Provinciale Santa Maria Nuova, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy. Two authors are employed by Blueprint Genetics (M.M. and J.K.). The remaining authors report no conflicts of interest. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www. jneuro-ophthalmology.com). Address correspondence to Luca Soliani, MD, Department of Pediatrics, Child Neurology Unit, Presidio Ospedaliero Provinciale Santa Maria Nuova, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy; E-mail: [e-mail] Soliani et al: J Neuro-Ophthalmol 2021; 41: e85-e88 A subset of patients with CFEOM3 phenotype harbor a KIF21A pathogenic variant (4). Here we present a patient now aged 13 years. He was born at 38 weeks of gestation through spontaneous vaginal delivery, after normal pregnancy from healthy nonconsanguineous Caucasian parents, with negative family history. He presented to medical attention shortly after birth for bilateral blepharoptosis, exotropia, peripheral facial nerve palsy (right .. left), and severe limitation in eye movements (Fig. 1). Auditory function, as assessed by auditory brainstem response and audiometric testing (in the neonatal period), showed normal results. At 1 year of age, nerve conduction studies (NCS) and electromyography (EMG) showed normal results. At 11 years of age, clinical examination showed a cranial circumference of 53 cm (50th centile), peroneal muscle atrophy, bilateral cavus feet (Fig. 1), and lower-limb areflexia, with progressive deterioration in his motor skills. NCS and EMG were then repeated revealing axonal sensory-motor peripheral neuropathy with axonal denervation (see Supplemental Digital Content, Table E2, http://links.lww.com/WNO/A388 and Table E3, http://links.lww.com/WNO/A389). Currently, at 13 years of age, he walks with lower-limb ankle-foot ortheses. His neuropsychiatric history is significant for developmental delay and moderate intellectual disability [total IQ with Leiter at 6 years of age: 54; WISC IV performed at 11 years: Verbal Comprehension Index: 48, Perceptual Reasoning Index: 41]. He has a special needs teacher at school and is described as having normal social and communication skills. Serial brain MRI scans disclosed multiple malformations, dominated by cerebellar vermis hypoplasia, small cerebellar hemispheres, arachnoid cyst, prominent retrocerebellar cerebrospinal fluid space, thin corpus callosum, reduced white matter, dysmorphic midbrain, small caudate bodies, and small extraocular muscles (superior, inferior, medial recti) (Fig. 2). Karyotype was normal 46, XY. Array CGH was not contributory, showing a microdeletion of 76 kb on 13q33.1 (101,212,939-101,289,072) x1, of paternal origin, which contains 2 protein coding genes, GGACT and TMTC4, that are not found to be morbid genes. Targeted Next e85 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence FIG. 1. A. Face at rest, showing primary position of the eyes with bilateral exotropia and bilateral blepharoptosis (previous palpebral ptosis surgery at 2 years of age and surgical correction of strabismus at 5 years of age). B–E. Range of eye movements. B. Bilateral upward gaze limitation with exotropia (right . left) C. Looking left, right eye failure in adduction. D. Looking right, left eye failure in adduction, right eye limitation in abduction (E) looking down, left eye failure in depression, right eye failure in depression, and exotropia with intorsion. F. Attempt to smile with eyes closed, peripheral right facial nerve palsy with lagophthalmos in right eye, disappearance of nasolabial fold and inability to lift right mouth angle, mild peripheral left facial nerve palsy with inability to completely close the left eye. G. Attempt to smile with eyes open. H. Feet of the patient with evident bilateral cavism and distal atrophy. Generation Sequencing panel for brain malformations and direct sequencing of TUBB3, TRPV4, DNM2, FOXL2, and COL6A1 also showed negative results. To exclude a congenital myasthenic syndrome, molecular analysis for RAPSN, CHRNA1, and CHRNE genes was performed with negative results. Sialotransferrins isoelectrofocusing was also negative. The patient’s peripheral blood sample was evaluated by high-quality clinical whole exome sequencing including detection for both sequence variants (with mean coverage FIG. 2. Brain MRI: (A–C) neonatal period. E–G. At 10 years of age. A–D. FLAIR sequence and T-2 SE weighted axial images: dismorphic aspect of the lateral ventricles, associated to reduced white matter, dysmorphic midbrain, small caudate bodies. B–E. Coronal T-2 weighted and FLAIR sequence images: mildly simplified gyral pattern, arachnoid cyst in left posterior cranial fossa with hypoplasic vermis and small hemispheric cerebellum (left . right). C–F. Sagittal T-1–weighted image: Hypoplasic cerebellum, abnormally thin corpus callosum (midpoint to posterior third). G. Axial orbital imaging showing small bilateral medial rectus. H. Orbital coronal magnetic resonance image shows small medial rectus, inferior and superior rectus, while lateral rectus shows normal muscular trophism (arrows). e86 Soliani et al: J Neuro-Ophthalmol 2021; 41: e85-e88 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence of 165x and 99.5% of the target nucleotides had .20x read depth) and CNVs (sensitivity of 92% at one exon level) (7) (https://blueprintgenetics.com/tests/whole-exomesequencing/whole-exome-family/). This enabled us to identify the novel missense variant c.2015 T.C (p.Leu672Pro) on the KIF21A gene (NM_017641.3, chr12:39734764 A.G (GRCh37). Segregation analysis with direct Sanger sequencing in both parents was negative, confirming its de novo origin. We did not identify any rare (,1%) sequence variants in exonic or intronic (20bp from exonic regions) regions in tubulin genes (TUBA1A, TUBB, TUBB2B, TUBB3, TUBB4A, and TUBG1) nor other variants than the reported KIF21A de novo alteration. The detected variant has not been reported in the literature or in the disease-related variation databases ClinVar and HGMD. There are no individuals with the same variant in the Genome Aggregation Database (gnomAD, n . 120,000 exomes and .15,000 genomes). It is predicted as deleterious by in silico tools SIFT, PolyPhen, and Mutation Taster. According to the American College of Medical Genetics and Genomics classification, this variant fulfills the following points: PM2 (absent in gnomAD control population), PM6 (assumed de novo, but without confirmation of paternity and maternity), PP2 (missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease), PP3 (multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.), and PP4 (patient’s phenotype or family history is highly specific for a disease with a single genetic etiology), thus classified as likely pathogenic (8). Our patient has a complex clinical phenotype, highly reminiscent of CFEOM-3 caused by TUBB3 mutations, characterized by CFEOM, brain malformations, intellectual disability, and peripheral sensory-motor neuropathy. In our case, autistic behavior and cyclic vomiting were not present, in contrast with some previous descriptions (5). TUBB3 gene direct sequencing was negative. In CFEOM-3, several TUBB3 disease-causing variants have been described, associated with different phenotypes (3,5,6). A TUBB3 Glu410Lys variant is associated with a clinical picture similar to that of our patient, characterized by developmental delay, facial weakness, sensorimotor polyneuropathy, and some of his central nervous system (CNS) features (i.e., corpus callosum dysmorphisms), but no cerebellar abnormalities. Another variant, Gly98Ser in TUBB3, on the contrary, showed cerebellar vermis hypoplasia but not polyneuropathy (3,5,6). KIF21A pathogenic variants have been demonstrated as causative of an abnormal kinesin activity by attenuating the physiologically preferred autoinhibited state, resulting in enhanced association between KIF21A and microtubules. Experimental data suggest high vulnerability of the oculomotor nerve to perturbations of the axon cytoskeleton (9). Soliani et al: J Neuro-Ophthalmol 2021; 41: e85-e88 Most of the so-far described disease-causing variants in KIF21A gene are missense (as in our case) or single amino acid substitutions, and affect both the stalk and the motor regions of the protein, interfering with its function. It has been shown that gain-of-function variants in KIF21A, especially those affecting one of the coiled coil domains, cause CFEOM1. So far, 80% of the unique recurrent, segregating, and de novo KIF21A variants locate in coiled coil domains, more specifically in the CC-domain2 (931–1,019) according to Uniprot/NM_017641.310 definition. Of note, UniProt defines the coiled-coil region differently from previous KIF21A reports: CC-domain-1 corresponds to residues 365–575, CC-domain-2 to 931– 1,019, and CC-domain-3 to residues 1,053–1,083. As in 20% of previously described cases, our patient’s variant c.2015 T.C (p.Leu672Pro) locates outside the coiledcoil domains. Thus, it seems likely variants locating also in other regions of the protein contribute to the phenotype, possibly through gain-of-function mechanism. A comparison with previously reported pathogenic variants is available as supplementary material (see Supplementary Digital Content, Table E1, http://links.lww.com/WNO/A387). KIF21A pathogenic variants (typically causative of a CFEOM-1 phenotype) (4) are uncommonly associated with CNS malformations (1,11). In detail, cerebellar abnormalities have been seldom reported: Di Fabio and coworkers described cerebellar atrophy in 2 members of a family with KIF21A-positive CFEOM-1 (11). Furthermore, peripheral sensory-motor neuropathy has not been previously reported with KIF21A variants, while it has been associated with CFEOM3 secondary to TUBB3 mutations and linked to motor protein trafficking defects. However, in a murine model, variants in TUBB3 gene alter microtubule stability and decrease its interaction with KIF21A (3). We hypothesize that the presented variant in KIF21A, like variants in TUBB3, by altering the normal function of kinesin 21A, might cause an abnormal binding between kinesin and microtubules, leading to our patient’s phenotype, which was so far associated exclusively to TUBB3 mutations. This might be the explanation for the alteration in axon guidance both in central and peripheral nervous system, leading to congenital malformations in CNS and progressive sensory-motor polyneuropathy. In conclusion, our observation suggests a wider phenotypic spectrum associated with disease-causing variants in KIF21A, including a clinical phenotype so far only associated with TUBB3 variants, highlighting the clinical overlap between different CFEOM subtypes. STATEMENT OF AUTHORSHIP Category 1: a. Conception and design: L. Soliani, C. Spagnoli, and C. Fusco; b. Acquisition of data: L. Soliani, C. Spagnoli, C. Fusco, J. Koskenvuo, and M. Mehine; c. Analysis and interpretation of data: L. Soliani, C. Spagnoli, C. Fusco, J. Koskenvuo, and M. Mehine. e87 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Clinical Correspondence Category 2: a. Drafting the manuscript: L. Soliani and C. Spagnoli; b. Revising it for intellectual content: L. Soliani, C. Spagnoli, C. Fusco, J. Koskenvuo, M. Mehine, G. G. Salerno, S. Rizzi, and D. Frattini. Category 3: a. Final approval of the completed manuscript: L. Soliani, C. Spagnoli, C. Fusco, J. Koskenvuo, M. Mehine, G. G. Salerno, S. Rizzi, and D. Frattini. REFERENCES 1. Whitman M, Hunter DG, Engle EC. Congenital fibrosis of the extraocular muscles. In: AdamMP, ArdingerHH, PagonRA, WallaceSE, BeanLJH, StephensK, AmemiyaA, eds, Seattle, WA: GeneReviews University of Washington:1993–2019. 2. 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