Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.

A recurrent missense variant in the E3 ubiquitin ligase substrate recognition subunit FEM1B causes a rare syndromic neurodevelopmental disorder.

PURPOSE: Fem1 homolog B (FEM1B) acts as a substrate recognition subunit for ubiquitin ligase complexes belonging to the CULLIN 2-based E3 family. Several biological functions have been proposed for FEM1B, including a structurally resolved function as a sensor for redox cell status by controlling mitochondrial activity, but its implication in human disease remains elusive. METHODS: To understand the involvement of FEM1B in human disease, we made use of Matchmaker exchange platforms to identify individuals with de novo variants in FEM1B and performed their clinical evaluation. We performed functional validation using primary neuronal cultures and in utero electroporation assays, as well as experiments on patient's cells. RESULTS: Five individuals with a recurrent de novo missense variant in FEM1B were identified: NM_015322.5:c.377G>A NP_056137.1:p.(Arg126Gln) (FEM1BR126Q). Affected individuals shared a severe neurodevelopmental disorder with behavioral phenotypes and a variable set of malformations, including brain anomalies, clubfeet, skeletal abnormalities, and facial dysmorphism. Overexpression of the FEM1BR126Q variant but not FEM1B wild-type protein, during mouse brain development, resulted in delayed neuronal migration of the target cells. In addition, the individuals' cells exhibited signs of oxidative stress and induction of type I interferon signaling. CONCLUSION: Overall, our data indicate that p.(Arg126Gln) induces aberrant FEM1B activation, resulting in a gain-of-function mechanism associated with a severe syndromic developmental disorder in humans.

Expanding the genetics and phenotypes of ocular congenital cranial dysinnervation disorders.

PURPOSE: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). METHODS: We coupled phenotyping with exome or genome sequencing of 467 probands (550 affected and 1108 total individuals) with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. RESULTS: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. CONCLUSION: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

IFN-γ and androgens disrupt mitochondrial function in murine myocytes.

The effect of cytokines on non-traditional immunological targets under conditions of chronic inflammation is an ongoing subject of study. Fatigue is a symptom often associated with autoimmune diseases. Chronic inflammatory response and activated cell-mediated immunity are associated with cardiovascular myopathies which can be driven by muscle weakness and fatigue. Thus, we hypothesize that immune dysfunction-driven changes in myocyte mitochondria may play a critical role in fatigue-related pathogenesis. We show that persistent low-level expression of IFN-γ in designated IFN-γ AU-Rich Element deletion mice (ARE mice) under androgen exposure resulted in mitochondrial and metabolic deficiencies in myocytes from male or castrated ARE mice. Most notably, echocardiography unveiled that low ejection fraction in the left ventricle post-stress correlated with mitochondrial deficiencies, explaining how heart function decreases under stress. We report that inefficiencies and structural changes in mitochondria, with changes to expression of mitochondrial genes, are linked to male-biased fatigue and acute cardiomyopathy under stress. Our work highlights how male androgen hormone backgrounds and active autoimmunity reduce mitochondrial function and the ability to cope with stress and how pharmacological blockade of stress signal protects heart function. These studies provide new insight into the diverse actions of IFN-γ in fatigue, energy metabolism, and autoimmunity. © 2023 The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

TWIST1, a gene associated with Saethre-Chotzen syndrome, regulates extraocular muscle organization in mouse.

Heterozygous loss of function mutations in TWIST1 cause Saethre-Chotzen syndrome, which is characterized by craniosynostosis, facial asymmetry, ptosis, strabismus, and distinctive ear appearance. Individuals with syndromic craniosynostosis have high rates of strabismus and ptosis, but the underlying pathology is unknown. Some individuals with syndromic craniosynostosis have been noted to have absence of individual extraocular muscles or abnormal insertions of the extraocular muscles on the globe. Using conditional knock-out alleles for Twist1 in cranial mesenchyme, we test the hypothesis that Twist1 is required for extraocular muscle organization and position, attachment to the globe, and/or innervation by the cranial nerves. We examined the extraocular muscles in conditional Twist1 knock-out animals using Twist2-cre and Pdgfrb-cre drivers. Both are expressed in cranial mesoderm and neural crest. Conditional inactivation of Twist1 using these drivers leads to disorganized extraocular muscles that cannot be reliably identified as specific muscles. Tendons do not form normally at the insertion and origin of these dysplastic muscles. Knock-out of Twist1 expression in tendon precursors, using scleraxis-cre, however, does not alter EOM organization. Furthermore, developing motor neurons, which do not express Twist1, display abnormal axonal trajectories in the orbit in the presence of dysplastic extraocular muscles. Strabismus in individuals with TWIST1 mutations may therefore be caused by abnormalities in extraocular muscle development and secondary abnormalities in innervation and tendon formation.

Whole-Slide Imaging, Mutual Information Registration for Multiplex Immunohistochemistry and Immunofluorescence.

Multiplex immunohistochemistry/immunofluorescence (mIHC/mIF) is a developing technology that facilitates the evaluation of multiple, simultaneous protein expressions at single-cell resolution while preserving tissue architecture. These approaches have shown great potential for biomarker discovery, yet many challenges remain. Importantly, streamlined cross-registration of multiplex immunofluorescence images with additional imaging modalities and immunohistochemistry (IHC) can help increase the plex and/or improve the quality of the data generated by potentiating downstream processes such as cell segmentation. To address this problem, a fully automated process was designed to perform a hierarchical, parallelizable, and deformable registration of multiplexed digital whole-slide images (WSIs). We generalized the calculation of mutual information as a registration criterion to an arbitrary number of dimensions, making it well suited for multiplexed imaging. We also used the self-information of a given IF channel as a criterion to select the optimal channels to use for registration. Additionally, as precise labeling of cellular membranes in situ is essential for robust cell segmentation, a pan-membrane immunohistochemical staining method was developed for incorporation into mIF panels or for use as an IHC followed by cross-registration. In this study, we demonstrate this process by registering whole-slide 6-plex/7-color mIF images with whole-slide brightfield mIHC images, including a CD3 and a pan-membrane stain. Our algorithm, WSI, mutual information registration (WSIMIR), performed highly accurate registration allowing the retrospective generation of an 8-plex/9-color, WSI, and outperformed 2 alternative automated methods for cross-registration by Jaccard index and Dice similarity coefficient (WSIMIR vs automated WARPY, P < .01 and P < .01, respectively, vs HALO + transformix, P = .083 and P = .049, respectively). Furthermore, the addition of a pan-membrane IHC stain cross-registered to an mIF panel facilitated improved automated cell segmentation across mIF WSIs, as measured by significantly increased correct detections, Jaccard index (0.78 vs 0.65), and Dice similarity coefficient (0.88 vs 0.79).

Recessive variants in COL25A1 gene as novel cause of arthrogryposis multiplex congenita with ocular congenital cranial dysinnervation disorder.

A proper interaction between muscle-derived collagen XXV and its motor neuron-derived receptors protein tyrosine phosphatases σ and δ (PTP σ/δ) is indispensable for intramuscular motor innervation. Despite this, thus far, pathogenic recessive variants in the COL25A1 gene had only been detected in a few patients with isolated ocular congenital cranial dysinnervation disorders. Here we describe five patients from three unrelated families with recessive missense and splice site COL25A1 variants presenting with a recognizable phenotype characterized by arthrogryposis multiplex congenita with or without an ocular congenital cranial dysinnervation disorder phenotype. The clinical features of the older patients remained stable over time, without central nervous system involvement. This study extends the phenotypic and genotypic spectrum of COL25A1 related conditions, and further adds to our knowledge of the complex process of intramuscular motor innervation. Our observations indicate a role for collagen XXV in regulating the appropriate innervation not only of extraocular muscles, but also of bulbar, axial, and limb muscles in the human.

Genotypic and Phenotypic Spectrum of Foveal Hypoplasia: A Multicenter Study.

PURPOSE: To characterize the genotypic and phenotypic spectrum of foveal hypoplasia (FH). DESIGN: Multicenter, observational study. PARTICIPANTS: A total of 907 patients with a confirmed molecular diagnosis of albinism, PAX6, SLC38A8, FRMD7, AHR, or achromatopsia from 12 centers in 9 countries (n = 523) or extracted from publicly available datasets from previously reported literature (n = 384). METHODS: Individuals with a confirmed molecular diagnosis and availability of foveal OCT scans were identified from 12 centers or from the literature between January 2011 and March 2021. A genetic diagnosis was confirmed by sequence analysis. Grading of FH was derived from OCT scans. MAIN OUTCOME MEASURES: Grade of FH, presence or absence of photoreceptor specialization (PRS+ vs. PRS-), molecular diagnosis, and visual acuity (VA). RESULTS: The most common genetic etiology for typical FH in our cohort was albinism (67.5%), followed by PAX6 (21.8%), SLC38A8 (6.8%), and FRMD7 (3.5%) variants. AHR variants were rare (0.4%). Atypical FH was seen in 67.4% of achromatopsia cases. Atypical FH in achromatopsia had significantly worse VA than typical FH (P < 0.0001). There was a significant difference in the spectrum of FH grades based on the molecular diagnosis (chi-square = 60.4, P < 0.0001). All SLC38A8 cases were PRS- (P = 0.003), whereas all FRMD7 cases were PRS+ (P < 0.0001). Analysis of albinism subtypes revealed a significant difference in the grade of FH (chi-square = 31.4, P < 0.0001) and VA (P = 0.0003) between oculocutaneous albinism (OCA) compared with ocular albinism (OA) and Hermansky-Pudlak syndrome (HPS). Ocular albinism and HPS demonstrated higher grades of FH and worse VA than OCA. There was a significant difference (P < 0.0001) in VA between FRMD7 variants compared with other diagnoses associated with FH. CONCLUSIONS: We characterized the phenotypic and genotypic spectrum of FH. Atypical FH is associated with a worse prognosis than all other forms of FH. In typical FH, our data suggest that arrested retinal development occurs earlier in SLC38A8, OA, HPS, and AHR variants and later in FRMD7 variants. The defined time period of foveal developmental arrest for OCA and PAX6 variants seems to demonstrate more variability. Our findings provide mechanistic insight into disorders associated with FH and have significant prognostic and diagnostic value.

Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans.

Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.

TUBB3 Arg262His causes a recognizable syndrome including CFEOM3, facial palsy, joint contractures, and early-onset peripheral neuropathy.

Microtubules are formed from heterodimers of alpha- and beta-tubulin, each of which has multiple isoforms encoded by separate genes. Pathogenic missense variants in multiple different tubulin isoforms cause brain malformations. Missense mutations in TUBB3, which encodes the neuron-specific beta-tubulin isotype, can cause congenital fibrosis of the extraocular muscles type 3 (CFEOM3) and/or malformations of cortical development, with distinct genotype-phenotype correlations. Here, we report fourteen individuals from thirteen unrelated families, each of whom harbors the identical NM_006086.4 (TUBB3):c.785G>A (p.Arg262His) variant resulting in a phenotype we refer to as the TUBB3 R262H syndrome. The affected individuals present at birth with ptosis, ophthalmoplegia, exotropia, facial weakness, facial dysmorphisms, and, in most cases, distal congenital joint contractures, and subsequently develop intellectual disabilities, gait disorders with proximal joint contractures, Kallmann syndrome (hypogonadotropic hypogonadism and anosmia), and a progressive peripheral neuropathy during the first decade of life. Subsets may also have vocal cord paralysis, auditory dysfunction, cyclic vomiting, and/or tachycardia at rest. All fourteen subjects share a recognizable set of brain malformations, including hypoplasia of the corpus callosum and anterior commissure, basal ganglia malformations, absent olfactory bulbs and sulci, and subtle cerebellar malformations. While similar, individuals with the TUBB3 R262H syndrome can be distinguished from individuals with the TUBB3 E410K syndrome by the presence of congenital and acquired joint contractures, an earlier onset peripheral neuropathy, impaired gait, and basal ganglia malformations.

Recessive MYF5 Mutations Cause External Ophthalmoplegia, Rib, and Vertebral Anomalies.

MYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myogenesis. Here, we report three consanguineous families with biallelic homozygous loss-of-function mutations in MYF5 who define a clinical disorder characterized by congenital ophthalmoplegia with scoliosis and vertebral and rib anomalies. The clinical phenotype overlaps strikingly with that reported in several Myf5 knockout mouse models. Affected members of two families share a haploidentical region that contains a homozygous 10 bp frameshift mutation in exon 1 of MYF5 (c.23_32delAGTTCTCACC [p.Gln8Leufs∗86]) predicted to undergo nonsense-mediated decay. Affected members of the third family harbor a homozygous missense change in exon 1 of MYF5 (c.283C>T [p.Arg95Cys]). Using in vitro assays, we show that this missense mutation acts as a loss-of-function allele by impairing MYF5 DNA binding and nuclear localization. We performed whole-genome sequencing in one affected individual with the frameshift mutation and did not identify additional rare variants in the haploidentical region that might account for differences in severity among the families. These data support the direct role of MYF5 in rib, spine, and extraocular muscle formation in humans.

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance.

To date, mutations in 15 actin- or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts.

Characterization of the tumor immune microenvironment in human papillomavirus-positive and -negative head and neck squamous cell carcinomas.

Approximately 15% of advanced head and neck squamous cell carcinomas (HNSCC) respond to anti-PD-(L)1 monotherapies. Tumor PD-L1 expression and human papillomavirus (HPV) status have been proposed as biomarkers to identify patients likely to benefit from these treatments. We aimed to understand the potential immune effects of HPV in HNSCC and to characterize additional potentially targetable immune-regulatory pathways in primary, treatment-naïve tumors. CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, IDO-1, and GITR cell densities were determined in 27 HNSCC specimens. IHC for PD-L1 assessed percentage of positive tumor cells and immune cells separately or as a combined positive score (CPS), and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). HPV testing with p16 IHC was confirmed by HPV genotyping. When compared to HPV(-) tumors (n = 14), HPV+ tumors (n = 13) contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (P < 0.02), and there was a trend towards increased density of FoxP3 + cells. PD-L1 expression patterns did not vary by tumor viral status, suggesting possible heterogeneous mechanisms driving constitutive vs adaptive PD-L1 expression patterns in HNSCC. IDO-1 expression was abundant (> 500 IDO-1+ cells/mm2 in 17/27 specimens) and was found on tumor cells as well as immune cells in 12/27 (44%) cases (range 5-80% tumor cells+). Notably, the studied markers varied on a per-patient basis and were not always related to the degree of T cell infiltration. These findings may inform therapeutic co-targeting strategies and raise consideration for a personalized treatment approach.

Differentiating Moebius syndrome and other congenital facial weakness disorders with electrodiagnostic studies.

INTRODUCTION: Congenital facial weakness (CFW) can result from facial nerve paresis with or without other cranial nerve and systemic involvement, or generalized neuropathic and myopathic disorders. Moebius syndrome is one type of CFW. In this study we explored the utility of electrodiagnostic studies (EDx) in the evaluation of individuals with CFW. METHODS: Forty-three subjects enrolled prospectively into a dedicated clinical protocol and had EDx evaluations, including blink reflex and facial and peripheral nerve conduction studies, with optional needle electromyography. RESULTS: MBS and hereditary congenital facial paresis (HCFP) subjects had low-amplitude cranial nerve 7 responses without other neuropathic or myopathic findings. Carriers of specific pathogenic variants in TUBB3 had, in addition, a generalized sensorimotor axonal polyneuropathy with demyelinating features. Myopathic findings were detected in individuals with Carey-Fineman-Ziter syndrome, myotonic dystrophy, other undefined myopathies, or CFW with arthrogryposis, ophthalmoplegia, and other system involvement. DISCUSSION: EDx in CFW subjects can assist in characterizing the underlying pathogenesis, as well as guide diagnosis and genetic counseling.

Optic Nerve Head and Retinal Abnormalities Associated with Congenital Fibrosis of the Extraocular Muscles.

Congenital fibrosis of the extraocular muscles (CFEOM) is a congenital cranial dysinnervation disorder caused by developmental abnormalities affecting cranial nerves/nuclei innervating the extraocular muscles. Autosomal dominant CFEOM arises from heterozygous missense mutations of KIF21A or TUBB3. Although spatiotemporal expression studies have shown KIF21A and TUBB3 expression in developing retinal ganglion cells, it is unclear whether dysinnervation extends beyond the oculomotor system. We aimed to investigate whether dysinnervation extends to the visual system by performing high-resolution optical coherence tomography (OCT) scans characterizing retinal ganglion cells within the optic nerve head and retina. Sixteen patients with CFEOM were screened for mutations in KIF21A, TUBB3, and TUBB2B. Six patients had apparent optic nerve hypoplasia. OCT showed neuro-retinal rim loss. Disc diameter, rim width, rim area, and peripapillary nerve fiber layer thickness were significantly reduced in CFEOM patients compared to controls (p < 0.005). Situs inversus of retinal vessels was seen in five patients. Our study provides evidence of structural optic nerve and retinal changes in CFEOM. We show for the first time that there are widespread retinal changes beyond the retinal ganglion cells in patients with CFEOM. This study shows that the phenotype in CFEOM extends beyond the motor nerves.

Altered White Matter Organization in the TUBB3 E410K Syndrome.

Seven unrelated individuals (four pediatric, three adults) with the TUBB3 E410K syndrome, harboring identical de novo heterozygous TUBB3 c.1228 G>A mutations, underwent neuropsychological testing and neuroimaging. Despite the absence of cortical malformations, they have intellectual and social disabilities. To search for potential etiologies for these deficits, we compared their brain's structural and white matter organization to 22 controls using structural and diffusion magnetic resonance imaging. Diffusion images were processed to calculate fractional anisotropy (FA) and perform tract reconstructions. Cortical parcellation-based network analysis and gyral topology-based FA analyses were performed. Major interhemispheric, projection and intrahemispheric tracts were manually segmented. Subjects had decreased corpus callosum volume and decreased network efficiency. While only pediatric subjects had diffuse decreases in FA predominantly affecting mid- and long-range tracts, only adult subjects had white matter volume loss associated with decreased cortical surface area. All subjects showed aberrant corticospinal tract trajectory and bilateral absence of the dorsal language network long segment. Furthermore, pediatric subjects had more tracts with decreased FA compared with controls than did adult subjects. These findings define a TUBB3 E410K neuroimaging endophenotype and lead to the hypothesis that the age-related changes are due to microscopic intrahemispheric misguided axons that are pruned during maturation.

Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita.

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC.

Ocular congenital cranial dysinnervation disorders (CCDDs): insights into axon growth and guidance.

Unraveling the genetics of the paralytic strabismus syndromes known as congenital cranial dysinnervation disorders (CCDDs) is both informing physicians and their patients and broadening our understanding of development of the ocular motor system. Genetic mutations underlying ocular CCDDs alter either motor neuron specification or motor nerve development, and highlight the importance of modulations of cell signaling, cytoskeletal transport, and microtubule dynamics for axon growth and guidance. Here we review recent advances in our understanding of two CCDDs, congenital fibrosis of the extraocular muscles (CFEOM) and Duane retraction syndrome (DRS), and discuss what they have taught us about mechanisms of axon guidance and selective vulnerability. CFEOM presents with congenital ptosis and restricted eye movements, and can be caused by heterozygous missense mutations in the kinesin motor protein KIF21A or in the ß-tubulin isotypes TUBB3 or TUBB2B. CFEOM-causing mutations in these genes alter protein function and result in axon growth and guidance defects. DRS presents with inability to abduct one or both eyes. It can be caused by decreased function of several transcription factors critical for abducens motor neuron identity, including MAFB, or by heterozygous missense mutations in CHN1, which encodes α2-chimaerin, a Rac-GAP GTPase that affects cytoskeletal dynamics. Examination of the orbital innervation in mice lacking Mafb has established that the stereotypical misinnervation of the lateral rectus by fibers of the oculomotor nerve in DRS is secondary to absence of the abducens nerve. Studies of a CHN1 mouse model have begun to elucidate mechanisms of selective vulnerability in the nervous system.

Loss of MAFB Function in Humans and Mice Causes Duane Syndrome, Aberrant Extraocular Muscle Innervation, and Inner-Ear Defects.

Duane retraction syndrome (DRS) is a congenital eye-movement disorder defined by limited outward gaze and retraction of the eye on attempted inward gaze. Here, we report on three heterozygous loss-of-function MAFB mutations causing DRS and a dominant-negative MAFB mutation causing DRS and deafness. Using genotype-phenotype correlations in humans and Mafb-knockout mice, we propose a threshold model for variable loss of MAFB function. Postmortem studies of DRS have reported abducens nerve hypoplasia and aberrant innervation of the lateral rectus muscle by the oculomotor nerve. Our studies in mice now confirm this human DRS pathology. Moreover, we demonstrate that selectively disrupting abducens nerve development is sufficient to cause secondary innervation of the lateral rectus muscle by aberrant oculomotor nerve branches, which form at developmental decision regions close to target extraocular muscles. Thus, we present evidence that the primary cause of DRS is failure of the abducens nerve to fully innervate the lateral rectus muscle in early development.

The Liberfarb syndrome, a multisystem disorder affecting eye, ear, bone, and brain development, is caused by a founder pathogenic variant in thePISD gene.

PURPOSE: We observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers. METHODS: Patients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing. RESULTS: Affected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion (NM_014338.3:c.904-12_904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts. CONCLUSION: We have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.