Almost half of all individuals affected by intellectual disability (ID) remain undiagnosed. In the Solve-RD project, exome sequencing (ES) datasets from unresolved individuals with (syndromic) ID (n = 1,472 probands) are systematically reanalyzed, starting from raw sequencing files, followed by genome-wide variant calling and new data interpretation. This strategy led to the identification of a disease-causing de novo missense variant in TUBB3 in a girl with severe developmental delay, secondary microcephaly, brain imaging abnormalities, high hypermetropia, strabismus and short stature. Interestingly, the TUBB3 variant could only be identified through reanalysis of ES data using a genome-wide variant calling approach, despite being located in protein coding sequence. More detailed analysis revealed that the position of the variant within exon 5 of TUBB3 was not targeted by the enrichment kit, although consistent high-quality coverage was obtained at this position, resulting from nearby targets that provide off-target coverage. In the initial analysis, variant calling was restricted to the exon targets ± 200 bases, allowing the variant to escape detection by the variant calling algorithm. This phenomenon may potentially occur more often, as we determined that 36 established ID genes have robust off-target coverage in coding sequence. Moreover, within these regions, for 17 genes (likely) pathogenic variants have been identified before. Therefore, this clinical report highlights that, although compute-intensive, performing genome-wide variant calling instead of target-based calling may lead to the detection of diagnostically relevant variants that would otherwise remain unnoticed.

• Klíčová slova
• ERN ITHACA, Exome sequencing, Genome-wide variant calling, Solve-RD, TUBB3,
• MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• mikrocefalie genetika   MeSH
• missense mutace MeSH
• mladiství MeSH
• mozek abnormality   MeSH
• obličej abnormality   MeSH
• sekvenování exomu MeSH
• strabismus genetika   MeSH
• tubulin genetika   MeSH
• vývojové poruchy u dětí genetika   MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH

Class III beta-tubulin isotype (betaIII-tubulin) is widely regarded as a neuronal marker in developmental neurobiology and stem cell research. To test the specificity of this marker protein, we determined its expression and distribution in primary cultures of glial fibrillary acidic protein (GFAP)-expressing astrocytes isolated from the cerebral hemispheres of 2 human fetuses at 18 to 20 weeks of gestation. Cells were maintained as monolayer cultures for 1 to 21 days without differentiation induction. By immunofluorescence microscopy, coexpression of betaIII-tubulin and GFAP was detected in cells at all time points but in spatially distinct patterns. The numbers of GFAP+ cells gradually decreased from Days 1 to 21 in vitro, whereas betaIII-tubulin immunoreactivity was present in 100% of cells at all time points. beta-III-tubulin mRNA and protein expression were demonstrated in cultured cells by reverse-transcriptase-polymerase chain reaction and immunoblotting, respectively. Glial fibrillary acidic protein+/beta-III-tubulin-positive cells coexpressed nestin and vimentin but lacked neurofilament proteins, CD133, and glutamate-aspartate transporter. Weak cytoplasmic staining was detected with antibodies against microtubule-associated protein 2 isoforms. Confocal microscopy, performed on autopsy brain samples of human fetuses at 16 to 20 gestational weeks, revealed widespread colocalization of GFAP and betaIII-tubulin in cells of the ventricular/subventricular zones and the cortical plate. Our results indicate that in the midgestational human brain, betaIII-tubulin is not neuron specific because it is constitutively expressed in GFAP+/nestin+ presumptive fetal astrocytes.

• MeSH
• astrocyty metabolismus   MeSH
• fenotyp * MeSH
• gestační stáří * MeSH
• gliový fibrilární kyselý protein genetika   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mozková kůra cytologie   MeSH
• myši MeSH
• nestin MeSH
• plod cytologie   MeSH
• proteiny intermediálních filament genetika   metabolismus   MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• tubulin genetika   metabolismus   MeSH
• vimentin metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• gliový fibrilární kyselý protein MeSH
• messenger RNA MeSH
• NES protein, human MeSH Prohlížeč
• Nes protein, mouse MeSH Prohlížeč
• nestin MeSH
• proteiny intermediálních filament MeSH
• proteiny nervové tkáně MeSH
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH
• vimentin MeSH

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and βIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and βIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

• Klíčová slova
• DPYSL5, brain malformation, corpus callosum agenesis, de novo missense variants, dendrite branching, neurodevelopmental disorder, primary neuronal cultures,
• MeSH
• ageneze corpus callosum diagnostické zobrazování   genetika   MeSH
• dítě MeSH
• dospělí MeSH
• hydrolasy chemie   genetika   MeSH
• lidé MeSH
• mentální retardace diagnostické zobrazování   genetika   MeSH
• missense mutace genetika   MeSH
• mladý dospělý MeSH
• molekulární modely MeSH
• mozeček abnormality   diagnostické zobrazování   MeSH
• neurovývojové poruchy diagnostické zobrazování   genetika   MeSH
• předškolní dítě MeSH
• proteiny asociované s mikrotubuly chemie   genetika   metabolismus   MeSH
• tubulin metabolismus   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DPYSL5 protein, human MeSH Prohlížeč
• hydrolasy MeSH
• MAP2 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH

The aim of our work was to confirm an immunohistochemical profile of routine markers of epithelial and neuroendocrine differentiation in eleven cases of Merkel cell carcinoma, as well as to study the expression of two markers of early phases of neuronal differentiation, namely reelin and class III beta-tubulin, markers which have not yet been studied in Merkel cell carcinomas. In all the investigated tumours the characteristic "dot-like" pattern of cytokeratin 20 immunoexpression, as well as negative immunostaining for cytokeratin 7 and thyroid transcription factor 1 (TTF-1) were disclosed; all the tumours showed neuroendocrine differentiation, expressing either neuron specific enolase (NSE) or chromogranin A(CgA), or both. An interesting finding was observed when the anti-cytokeratin monoclonal antibody MNF 116 was used. The characteristic "dot-like" pattern was detected in high proportion of tumours, including two samples of local recurrence of one of the carcinomas, where neoplastic cells have lost the expression of cytokeratin 20. The majority (91%) of Merkel cell carcinomas included in our group showed positive immunodetection of class III beta-tubulin when TU-20 antibody was used, while TuJ-1 immunostaining was surprisingly negative in all the investigated tumours. Detection of reelin was negative in almost all the studied Merkel cell carcinomas except for cases, where neoplastic cells revealed weak focal immunostaining in a minor portion of neoplastic cells.

• MeSH
• extracelulární matrix - proteiny analýza   MeSH
• imunohistochemie MeSH
• keratiny analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Merkelův nádor chemie   patologie   MeSH
• molekuly buněčné adheze neuronové analýza   MeSH
• nádorové biomarkery analýza   MeSH
• nádory kůže chemie   patologie   MeSH
• protein reelin MeSH
• proteiny nervové tkáně analýza   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• serinové endopeptidasy analýza   MeSH
• tubulin analýza   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• extracelulární matrix - proteiny MeSH
• keratiny MeSH
• molekuly buněčné adheze neuronové MeSH
• nádorové biomarkery MeSH
• protein reelin MeSH
• proteiny nervové tkáně MeSH
• RELN protein, human MeSH Prohlížeč
• serinové endopeptidasy MeSH
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH

BACKGROUND & OBJECTIVE: Expression of class III beta-tubulin represents newly discovered marker of resistance to taxol-based chemotherapy in a wide spectra of carcinomas. However, very little is known about its expression in colorectal carcinomas. This study was done to determine class III beta-tubulin expression in a large series of colonic carcinomas, covering tumours with different degree of differentiation in order to evaluate its prospective significance in resistance to taxol-based chemotherapeutics and to compare the immunostaining profile of two widely used monoclonal antibodies, TU-20 and TuJ-1 METHODS: Sixty patients with colorectal carcinoma were enrolled; all of them were treated surgically by the resection. Twenty tumours were histologically assessed as G1, 20 as G2 and 20 as G3. Routine immunohistochemical procedure using TU-20 and TuJ-1 mouse monoclonal antibodies was applied to all 60 specimen and slides were evaluated using an optical microscope. RESULTS: Expression of class III beta-tubulin was detected in 14 tumours (23.3%), while remaining tumours were negative. Relatively higher frequency of class III beta-tubulin expression was observed in G3 tumours (10 cases) in comparison with G1 (3 cases) and G2 (1 case), respectively. Seven tumours displayed positive immunostaining with both tested antibodies TU-20 and TuJ-1. Six tumours showed expression of class III beta- tubulin in more than 1 per cent of neoplastic cell population. In remaining 8 tumours only individual scattered neoplastic cells exhibited class III beta-tubulin expression either with TU-20, or with TuJ-1 antibody. INTERPRETATION & CONCLUSION: Higher frequency of immunoreactivity was observed in poorly differentiated tumours. However, more than 90 per cent of neoplastic cell population did not express class III beta-tubulin in almost all tumours. These negative cells of colonic cancer could represent the potential target for taxane-based chemotherapy in the future. Our results indicate that TU-20 and TuJ-1 antibodies exhibit very similar immunoreactivity in neoplastic tissue.

• MeSH
• dospělí MeSH
• imunohistochemie MeSH
• kolorektální nádory metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky metabolismus   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tubulin metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• monoklonální protilátky MeSH
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH

Class III beta-tubulin has been discovered as a marker of early phases of neuronal differentiation in developmental conditions, as well as in different tumours of neuronal origin. More recently, the expression of class III beta-tubulin molecule has been described as a marker of different types of malignant epithelial tumours. This study attempts to compare the immunostaining features of two different mouse monoclonal antibodies TU-20 and TuJ-1, both detecting class III beta-tubulin, in a group of twenty bioptically evaluated carcinomas of various sites. The proposal that class III beta-tubulin expression can correlate with the degree of tumour differentiation and thus could be potentially used as predictive marker of prognosis has been previously done; one of aims of our study was to confirm this hypothesis. Our results showed that both TuJ- 1 and TU-20 antibodies displayed similar immunostaining profile and pattern within individual tumours. Surprisingly, we discovered that only 50% of tumours included in our group showed expression of class III beta-tubulin, however, positive immunoreaction did not correspond with the degree of differentiation of individual tumours. In our group of carcinomas, the class III beta-tubulin positivity was not related to the tumour site, histologic type of tumour or its grade.

• MeSH
• dospělí MeSH
• imunohistochemie MeSH
• karcinom diagnóza   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• tubulin imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• monoklonální protilátky MeSH
• TUBB3 protein, human MeSH Prohlížeč
• tubulin MeSH

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

• Klíčová slova
• Cell line characterization, Mass spectrometry, Neural differentiation, Neural stem cell, Protein marker, Selected reaction monitoring,
• MeSH
• biologické markery MeSH
• buněčná diferenciace * MeSH
• buněčné linie MeSH
• buněčný rodokmen genetika   MeSH
• hmotnostní spektrometrie MeSH
• imunohistochemie MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neuroglie MeSH
• neurony MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH