CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.

PEBAT, an Intriguing Neurodegenerative Tubulinopathy Caused by a Novel Homozygous Variant in TBCD: A Case Series and Literature Review.

Progressive encephalopathy with brain atrophy and thin corpus callosum (PEBAT) is a severe and rare progressive neurodegenerative disease (OMIM 617913). This condition has been described in individuals with pathogenic variants affecting tubulin-specific chaperone protein D (TBCD), which is responsible for proper folding and assembly of tubulin subunits. Here we describe two unrelated infants from Central America presenting with worsening neuromuscular weakness, respiratory failure, polyneuropathy, and neuroimaging findings of severe cerebral volume loss with thin corpus callosum. These individuals harbored the same homozygous variant of uncertain significance in the TBCD gene on whole exome sequencing (WES). Predicted protein modeling of this variant confirmed disruption of the protein helix at the surface of TBCD. The goal of this report is to emphasize the importance of rapid WES, careful interpretation of uncertain variants, prognostication, and family counseling especially when faced with a neurodegenerative clinical course.

Femtosecond laser assisted superficial lamellar keratectomy as a successful treatment of corneal opacity in a patient with Thiel Behnke corneal dystrophy.

INTRODUCTION AND IMPORTANCE: Thiel Behnke corneal dystrophy (TBCD) and Reis Buckler corneal dystrophy (RBCD) are Bowman's layer dystrophies with overlapping clinical features causing diagnostic confusion. However, each entity has typical histopathological features. We describe in this case the successful use of Femtosecond laser (FSL) in the treatment of TBCD-related corneal opacity. CASE PRESENTATION: We present a 54-year-old male with bilateral superficial corneal opacities consistent with TBCD based on clinical appearance, anterior segment optical coherence tomography (AS-OCT), and In vivo confocal microscopy. Management options were discussed with the patient before proceeding with Femtosecond Laser Assisted Superficial Lamellar Keratectomy (FSLASLK). The histopathological findings of the excised left anterior lamellar corneal flap were typical of TBCD and the patient had a satisfactory outcome. CLINICAL DISCUSSION: TBCD typically affects Bowman's layer centrally with progressive opacities involving the deeper layer of the corneal stroma and the periphery with advancing age. Histopathology typically shows subepithelial fibrosis with interrupted basement membrane and totally replaced Bowman's layer by uneven fibrous tissue forming the characteristic saw tooth pattern. The treatment of such cases is challenging with variable success and recurrence rates. Our case was managed successfully using FSL. CONCLUSION: TBCD, even though a rare type of dystrophy, should be suspected based on the appearance of the corneal opacities clinically. It can be diagnosed by typical AS-OCT findings supported by histopathological confirmation and can be successfully treated by FSASLK.

Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy.

Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type.

Novel Compound Heterozygous Variants in TBCD Gene Associated with Infantile Neurodegenerative Encephalopathy.

Mutations in tubulin-specific chaperon D (TBCD), the gene encoding one of the co-chaperons required for the assembly and disassembly of the α/ß-tubulin heterodimers, have been reported to cause perturbed microtubule dynamics, resulting in debilitating early-onset progressive neurodegenerative disorder. Here, we identified two novel TBCD variants, c.1340C>T (p.Ala447Val), and c.817+2T>C, presented as compound heterozygotes in two affected siblings born to unaffected carrier parents. Clinical features included early-onset neurodegeneration, failure to thrive, respiratory failure, hypotonia, muscle weakness and atrophy and seizures. We established the genotype-phenotype relationship of these TBCD pathogenic variants and provided insight into the protein structural alteration that may contribute to this chaperone-associated tubulinopathy.

Tubulin Folding Cofactor D Deficiency: Missing the Diagnosis With Whole Exome Sequencing.

Two siblings with an early onset of a neurodegenerative disease were presented with muscular hypotonia, secondary microcephaly, and severe developmental delay. Seizures were refractory to treatment but could be controlled with a ketogenic diet. Over the course of 5 years, whole exome sequencing (WES) was performed twice in both children. The first time the diagnosis was missed. The next one revealed compound heterozygous mutations in the gene coding for the tubulin folding cofactor D. Technical improvements in WES mandated a new investigation after a few years in children where the diagnosis has not been found.

Hematological findings associated with tubulin-folding cofactors D-related encephalopathy: Expanding the phenotype.

The dysfunction of microtubules (α/ß-tubulin polymers) underlies a wide range of nervous system genetic abnormalities. Defects in TBCD, a tubulin-folding cofactor, cause diseases highlighted with early-onset encephalopathy with or without neurodegeneration, intellectual disability, seizures, microcephaly and tetraparaperesis. Utilizing various molecular methods, we describe nine patients from four unrelated families with two novel exon 18 variants in TBCD exhibiting the typical neurological phenotype of the disease. Interestingly, all the investigated patients had previously unreported hematological findings in the form of neutropenia and mild degree of anemia and thrombocytopenia. In addition to delineating the neurological phenotype in several patients with TBCD variants, our study stresses on the new association of neutropenia, in particular, with the disease.

Biallelic pathogenic variants in TBCD-related neurodevelopment disease with mild clinical features.

BACKGROUND: Microtubule dynamics is crucial for neuronal function and survival. The disrupted function of microtubule dynamics would lead to neurodegenerative and neurodevelopmental disorders. Tubulin-specific chaperone D (TBCD) is one of five tubulin co-chaperones acted in assembly and disassembly dynamics of microtubule. The biallelic pathogenic variants of TBCD gene were reported to be associated with severe degenerative encephalopathy accompanied with seizures previously. RESULTS: Compound heterozygous variants were identified in three patients from three families. The in silico prediction software and ACMG standards and guidelines proved the pathogenicity of the TBCD pathogenic variants. The clinical features of the three patients presented with mild neurodevelopmental manifestations including autism spectrum disorder (ASD) and occasional generalized tonic-clonic seizures (GTCSs) responding well to antiepileptic drugs. CONCLUSION: Our research expanded the clinical spectrum of TBCD-related neurodevelopmental disease which contributed to understanding the genotype-phenotype correlations of the disease.

Cortical atrophy and hypofibrinogenemia due to FGG and TBCD mutations in a single family: a case report.

BACKGROUND: Blended phenotypes or co-occurrence of independent phenotypically distinct conditions are extremely rare and are due to coincidence of multiple pathogenic mutations, especially due to consanguinity. Hereditary fibrinogen deficiencies result from mutations in the genes FGA, FGB, and FGG, encoding the three different polypeptide chains that comprise fibrinogen. Neurodevelopmental abnormalities have not been associated with fibrinogen deficiencies. In this study, we report an unusual patient with a combination of two independently inherited genetic conditions; fibrinogen deficiency and early onset cortical atrophy. CASE PRESENTATION: The study describes a male child from consanguineous family presented with hypofibrinogenemia, diffuse cortical atrophy, microcephaly, hypertonia and axonal motor neuropathy. Through a combination of homozygosity mapping and exome sequencing, we identified bi-allelic pathogenic mutations in two genes: a homozygous novel truncating mutation in FGG (c.554del; p.Lys185Argfs*14) and a homozygous missense mutation in TBCD (c.1423G > A;p.Ala475Thr). Loss of function mutations in FGG have been associated with fibrinogen deficiency, while the c.1423G > A mutation in TBCD causes a novel syndrome of neurodegeneration and early onset encephalopathy. CONCLUSIONS: Our study highlights the importance of homozygosity mapping and exome sequencing in molecular prenatal diagnosis, especially when multiple gene mutations are responsible for the phenotype.

Nucleotide Binding to ARL2 in the TBCD∙ARL2∙ß-Tubulin Complex Drives Conformational Changes in ß-Tubulin.

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αß-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and ß-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•ß-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not ß-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•ß-tubulin complex represents a functional intermediate in the ß-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the ß-tubulin in the trimer is also shown, with implications to modeling the pathway.

Microcephaly, intractable seizures and developmental delay caused by biallelic variants in TBCD: further delineation of a new chaperone-mediated tubulinopathy.

Microtubule dynamics play a crucial role in neuronal development and function, and several neurodevelopmental disorders have been linked to mutations in genes encoding tubulins and functionally related proteins. Most recently, variants in the tubulin cofactor D (TBCD) gene, which encodes one of the five co-chaperones required for assembly and disassembly of α/ß-tubulin heterodimer, were reported to underlie a recessive neurodevelopmental/neurodegenerative disorder. We report on five patients from three unrelated families, who presented with microcephaly, intellectual disability, intractable seizures, optic nerve pallor/atrophy, and cortical atrophy with delayed myelination and thinned corpus callosum on brain imaging. Exome sequencing allowed the identification of biallelic variants in TBCD segregating with the disease in the three families. TBCD protein level was significantly reduced in cultured fibroblasts from one patient, supporting defective TBCD function as the event underlying the disorder. Such reduced expression was associated with accelerated microtubule re-polymerization. Morpholino-mediated TBCD knockdown in zebrafish recapitulated several key pathological features of the human disease, and TBCD overexpression in the same model confirmed previous studies documenting an obligate dependency on proper TBCD levels during development. Our findings confirm the link between inactivating TBCD variants and this newly described chaperone-associated tubulinopathy, and provide insights into the phenotype of this disorder.

Higher order signaling: ARL2 as regulator of both mitochondrial fusion and microtubule dynamics allows integration of 2 essential cell functions.

ARL2 is among the most highly conserved proteins, predicted to be present in the last eukaryotic common ancestor, and ubiquitously expressed. Genetic screens in multiple model organisms identified ARL2, and its cytosolic binding partner cofactor D (TBCD), as important in tubulin folding and microtubule dynamics. Both ARL2 and TBCD also localize to centrosomes, making it difficult to dissect these effects. A growing body of evidence also has found roles for ARL2 inside mitochondria, as a regulator of mitochondrial fusion. Other studies have revealed roles for ARL2, in concert with its closest paralog ARL3, in the traffic of farnesylated cargos between membranes and specifically to cilia and photoreceptor cells. Details of each of these signaling processes continue to emerge. We summarize those data here and speculate about the potential for cross-talk or coordination of cell regulation, termed higher order signaling, based upon the use of a common GTPase in disparate cell functions.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.

Emerging roles for tubulin folding cofactors at the centrosome.

Despite its fundamental role in centrosome biology, procentriole formation, both in the canonical and in the de novo replication pathways, remains poorly understood, and the molecular components that are involved in human cells are not well established. We found that one of the tubulin cofactors, TBCD, is localized at centrosomes and the midbody, and is required for spindle organization, cell abscission, centriole formation and ciliogenesis. Our studies have established a molecular link between the centriole and the midbody, demonstrating that this cofactor is also necessary for microtubule retraction during cell abscission. TBCD is the first centriolar protein identified that plays a role in the assembly of both "centriolar rosettes" during early ciliogenesis, and at the procentriole budding site by S/G(2), a discovery that directly implicates tubulin cofactors in the cell division, cell migration and cell signaling research fields.