Wai-Man Chan

Homozygous HOXA1 mutations disrupt human brainstem, inner ear, cardiovascular and cognitive development

We identified homozygous truncating mutations in HOXA1 in three genetically isolated human populations. The resulting phenotype includes horizontal gaze abnormalities, deafness, facial weakness, hypoventilation, vascular malformations of the internal carotid arteries and cardiac outflow tract, mental retardation and autism spectrum disorder. This is the first report to our knowledge of viable homozygous truncating mutations in any human HOX gene and of a mendelian disorder resulting from mutations in a human HOX gene critical for development of the central nervous system.

Structural Grading of Foveal Hypoplasia Using Spectral-Domain Optical Coherence Tomography: A Predictor of Visual Acuity?

PURPOSE To characterize and grade the spectrum of foveal hypoplasia based on different stages of arrested development of the fovea. Grading was performed using morphologic findings obtained by ultra high-resolution spectral-domain optical coherence tomography. Best-corrected visual acuity (BCVA) was calculated for different grades. DESIGN Observational case series. PARTICIPANTS AND CONTROLS Sixty-nine patients with foveal hypoplasia (albinism, n = 34; PAX6 mutations, n = 10; isolated cases, n = 14; achromatopsia, n = 11) and 65 control subjects were examined. METHODS A 7×7-mm retinal area was sampled using a 3-dimensional scanning protocol (743×75, A scans×B scans) with ultra high-resolution spectral-domain optical coherence tomography (SOCT Copernicus HR; 3-μm axial resolution). Gross morphologic abnormalities were documented. B-scans at the fovea were segmented using a longitudinal reflectivity profile. Logarithm of the minimum angle of resolution BCVA was obtained. MAIN OUTCOME MEASURES Grading was based on presence or absence of foveal pit and widening of the outer nuclear layer (ONL) and outer segment (OS) at the fovea. Quantitative measurements were obtained for comparing atypical foveal hypoplasia in achromatopsia. Best-corrected visual acuity was compared with the grade of foveal hypoplasia. RESULTS Four grades of foveal hypoplasia were distinguished: grade 1, shallow foveal pit, presence of ONL widening, presence of OS lengthening; grade 2, grade 1 but absence of foveal pit; grade 3, grade 2 but absence of OS lengthening; grade 4, grade 3 but absence of ONL widening. There was significant difference in visual acuity (VA) associated with each grade (P<0.0001). Grade 1 was associated with the best VA (median VA, 0.2), whereas grades 2, 3, and 4 were associated with progressively poorer VA with a median VA of 0.44, 0.60, and 0.78, respectively. The atypical features seen with foveal hypoplasia associated with achromatopsia were characterized by decreased retinal and ONL thickness and deeper foveal depth. CONCLUSIONS A structural grading system for foveal hypoplasia was developed based on the stage at which foveal development was arrested, which helps to provide a prognostic indicator for VA and is applicable in a range of disorders associated with foveal hypoplasia. Atypical foveal hypoplasia in achromatopsia shows different characteristics. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Human CHN1 Mutations Hyperactivate α2-Chimaerin and Cause Duane's Retraction Syndrome

Duanes retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

Human CHN1 Mutations Hyperactivate α2-Chimaerin and Cause Duanes Retraction Syndrome

Duanes retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation

Microtubules are essential components of axon guidance machinery. Among β-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αβ-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that β-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

Horizontal gaze palsy with progressive scoliosis can result from compound heterozygous mutations in ROBO3

Background: Horizontal gaze palsy with progressive scoliosis (HGPPS) is an autosomal recessive disorder characterised by congenital absence of horizontal gaze, progressive scoliosis, and failure of the corticospinal and somatosensory axon tracts to decussate in the medulla. We previously reported that HGPPS patients from consanguineous pedigrees harbour homozygous mutations in the axon guidance molecule ROBO3. Methods: We now report two sporadic HGPPS children of non-consanguineous parents who harbour compound heterozygous mutations in ROBO3. The mother of one of the children also had scoliosis DNA was extracted from a blood sample from each participant using a standard protocol, and the coding exons of ROBO3 were amplified and sequenced as previously described. Results: Each patient harboured two unique heterozygous mutations in ROBO3, having inherited one mutation from each parent. Conclusions: HGPPS can result from compound heterozygous mutations. More comprehensive examinations of parents and siblings of HGPPS patients are required to determine if the incidence of scoliosis in individuals harbouring heterozygous ROBO3 mutations is greater than in the general population.

Three novel mutations in KIF21A highlight the importance of the third coiled-coil stalk domain in the etiology of CFEOM1

BackgroundCongenital fibrosis of the extraocular muscles types 1 and 3 (CFEOM1/CFEOM3) are autosomal dominant strabismus disorders that appear to result from maldevelopment of ocular nuclei and nerves. We previously reported that most individuals with CFEOM1 and rare individuals with CFEOM3 harbor heterozygous mutations in KIF21A. KIF21A encodes a kinesin motor involved in anterograde axonal transport, and the familial and de novo mutations reported to date predictably alter one of only a few KIF21A amino acids – three within the third coiled-coil region of the stalk and one in the distal motor domain, suggesting they result in altered KIF21A function. To further define the spectrum of KIF21A mutations in CFEOM we have now identified all CFEOM probands newly enrolled in our study and determined if they harbor mutations in KIF21A.ResultsSixteen CFEOM1 and 29 CFEOM3 probands were studied. Three previously unreported de novo KIF21A mutations were identified in three CFEOM1 probands, all located in the same coiled-coil region of the stalk that contains all but one of the previously reported mutations. Eight additional CFEOM1 probands harbored three of the mutations previously reported in KIF21A; seven had one of the two most common mutations, while one harbored the mutation in the distal motor domain. No mutation was detected in 5 CFEOM1 or any CFEOM3 probands.ConclusionAnalysis of sixteen CFEOM1 probands revealed three novel KIF21A mutations and confirmed three reported mutations, bringing the total number of reported KIF21A mutations in CFEOM1 to 11 mutations among 70 mutation positive probands. All three new mutations alter amino acids in heptad repeats within the third coiled-coil region of the KIF21A stalk, further highlighting the importance of alterations in this domain in the etiology of CFEOM1.

Congenital fibrosis of the vertically acting extraocular muscles maps to the FEOM3 locus

Abstract. The diagnosis of congenital fibrosis of the extraocular muscles (CFEOM) encompasses several different inherited strabismus syndromes characterized by congenital restrictive ophthalmoplegia affecting extraocular muscles innervated by the oculomotor and/or trochlear nerves. The OMIM database (http://www.ncbi.nlm.nih.gov/Omim/) currently contains four familial CFEOM phenotypes: CFEOM1–3, which map to the FEOM1–3 loci (MIM 135600, 602078, 604361), respectively, and congenital fibrosis of the vertically acting extraocular muscles (MIM 600638), reported in a single family without a corresponding genotype. We have had the opportunity to study the reported family with this fourth phenotype and now demonstrate that their phenotype can be reclassified as CFEOM3 and that it maps to FEOM3, flanked by D16S498 to 16qter, with a maximum lod score of 6.0.

A novel PHOX2A/ARIX mutation in an Iranian family with congenital fibrosis of extraocular muscles type 2 (CFEOM2).

PURPOSE To describe the clinical features of two affected members of an Iranian family with autosomal recessive congenital fibrosis of the extraocular muscles (CFEOM2) and to report their novel mutation in the PHOX2A/ARIX gene. DESIGN Experimental study. METHODS SETTING Institutional practice. patient population:Six members of an Iranian family with CFEOM underwent complete ocular examinations including assessment of ocular motility, visual acuity, slit-lamp biomicroscopy, tonometry, and ophthalmoscopy. EXPERIMENTAL PROCEDURE Mutation analysis of the PHOX2A gene was performed using polymerase chain reaction amplification of the coding exons and direct sequencing of polymerase chain reaction products. MAIN OUTCOME MEASURE Presence or absence of mutation in PHOX2A gene in two siblings with exotropia and recessive CFEOM. Exotropia and ptosis were corrected surgically in one of the two siblings. RESULTS The two affected siblings had bilateral ptosis and exotropia and severe limitation of all extraocular movements. One patient underwent strabismus surgery and ptosis repair. PHOX2A mutation analysis revealed a novel nonsense mutation in exon 2 (439C-->T). Both parents and the unaffected siblings were heterozygous,and the two affected siblings were homozygous for this mutation. CONCLUSIONS The 439C-->T mutation in this family changes a glutamine to a stop codon (Q90X) at the beginning of the PHOX2A homeodomain region. This is the fourth CFEOM2 mutation in PHOX2A and the first nonsense mutation to be identified. It confirms PHOX2A as the autosomal recessive CFEOM2 disease gene and provides evidence that the phenotypic differences between PHOX2A mutations in man and mouse do not result from hypomorphic PHOX2A alleles in humans.

Expansion of the CHN1 Strabismus Phenotype

PURPOSE Hyperactivating CHN1 mutations have been described in individuals with Duane retraction syndrome with or without vertical gaze abnormalities. This was a study of five family members with distinctive ocular dysmotility patterns that co-segregated with a novel hyperactivating CHN1 mutation. METHODS Participating members of a family segregating pleomorphic incomitant strabismus underwent ophthalmic examinations, and several underwent high-resolution magnetic resonance imaging (MRI) of the orbits and brain stem. Participant DNA was extracted and amplified for haplotype analysis encompassing the CHN1 region on chromosome 2q31.1, and mutation analysis of the CHN1 gene, which encodes the Rac-GAP signaling protein α2-chimaerin. In vitro functional studies of the co-inherited mutation were performed, including a Rac-GTP activation assay, quantification of α2-chimaerin translocation, and co-immunoprecipitation. RESULTS All five clinically affected family members exhibited monocular or binocular supraduction deficits, three in the absence of Duane retraction syndrome. MRI in four affected individuals demonstrated small or absent abducens nerves in all four, small oculomotor nerve in one, and small optic nerves in three. Superior oblique muscle volume was also decreased in three of the individuals, supporting trochlear nerve hypoplasia. Strabismus segregated with the CHN1 locus and affected individuals harbored a c.443A>T CHN1 mutation (p.Y148F). In vitro, this novel mutation behaved similarly to previously reported CHN1 mutations underlying familial Duane syndrome, hyperactivating α2-chimaerin by enhancing its dimerization and membrane association and lowering total intracellular Rac-GTP. CONCLUSIONS Analysis of the current pedigree expands the phenotypic spectrum of hyperactivating CHN1 mutations to include vertical strabismus and supraduction deficits in the absence of Duane retraction syndrome.