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• W2039134883 abstract "To the Editor: Sheridan and colleagues recently reported that mutations in the tubulin gene TUBA8 result in polymicrogyria with optic nerve hypoplasia (PMGOH [MIM 613180]).1Abdollahi M.R. Morrison E. Sirey T. Molnar Z. Hayward B.E. Carr I.M. Springell K. Woods C.G. Ahmed M. Hattingh L. et al.Mutation of the variant alpha-tubulin TUBA8 results in polymicrogyria with optic nerve hypoplasia.Am. J. Hum. Genet. 2009; 85: 737-744Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar This conclusion is based on the mapping of two consanguineous families of Pakistani origin to a 7.42 Mb region on chromosome 22q11.2 that contains ∼230 genes including TUBA8. Drawing on our previous finding that mutations in TUBA1A cause lissencephaly2Keays D.A. Tian G. Poirier K. Huang G.J. Siebold C. Cleak J. Oliver P.L. Fray M. Harvey R.J. Molnar Z. et al.Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans.Cell. 2007; 128: 45-57Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar and that mutations in TUBB2B cause asymmetric polymicrogyria,3Jaglin X.H. Poirier K. Saillour Y. Buhler E. Tian G. Bahi-Buisson N. Fallet-Bianco C. Phan-Dinh-Tuy F. Kong X.P. Bomont P. et al.Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria.Nat. Genet. 2009; 41: 746-752Crossref PubMed Scopus (260) Google Scholar Sheridan and colleagues sequenced TUBA8 and found a 14 bp deletion in intron 1 that affects splicing. They provide further evidence that TUBA8 is involved in the disease state by analyzing its expression in the developing mouse brain by in situ hybridization. They report that Tuba8 is widely expressed in developing neural structures, with strongest expression in the cortical plate at E15.5 and E18.5 and in the cortical plate, subplate, and hippocampus at P0. A meaningful analysis of individual tubulin gene expression by in situ hybridization requires the use of probes that avoid cross-hybridization among the highly conserved coding regions, relying exclusively on either the variant 5′ or 3′ untranslated regions. The probe employed by Sheridan and colleagues was 443 nucleotides in length, of which 415 correspond to sequences contained within the conserved coding region. Consequently, this probe shares a very high sequence homology with other α-tubulins.4Khodiyar V.K. Maltais L.J. Sneddon K.M. Smith J.R. Shimoyama M. Cabral F. Dumontet C. Dutcher S.K. Harvey R.J. Lafanechere L. et al.A revised nomenclature for the human and rodent alpha-tubulin gene family.Genomics. 2007; 90: 285-289Crossref PubMed Scopus (40) Google Scholar An Ensembl BLAST search with the Sheridan probe against total mouse cDNA results in six other hits, each being at least 300 nucleotides in length with at least 80% sequence identity. Each of these hits corresponds to one of the six other members of the α-tubulin family and includes a 374 nucleotide stretch that shares 84.2% identity with the coding sequence of Tuba1a, a gene that is highly expressed in the developing CNS.5Gloster A. El-Bizri H. Bamji S.X. Rogers D. Miller F.D. Early induction of Talpha1 alpha-tubulin transcription in neurons of the developing nervous system.J. Comp. Neurol. 1999; 405: 45-60Crossref PubMed Scopus (74) Google Scholar To establish whether the results reported by Sheridan and colleagues are a consequence of cross-hybridization, we conducted in situ hybridization on the developing (E14.5, E16.5, and P0) and adult mouse brain employing their probe and two others that we designed. We first confirmed the sequence of Tuba8 mRNA by amplifying and cloning it from a C57BL/6 mouse adult olfactory bulb cDNA library (AK032157, GU591980) (Figure S1 available online). Next we designed two probes, a short probe (224 base pairs in length [nt 1569–1792]) and a long probe (545 base pairs in length [nt 1367–1911]) targeted to the highly variant 3′ UTR of Tuba8 (GU591980). These probes, along with the probe employed by Sheridan, were cloned into a pCRII-TOPO vector (Invitrogen, 45-0640), and the sequence was confirmed by Big Dye sequencing with a 3730 DNA Analyzer (Applied Biosystems). We checked the specificity of our probes by conducting an Ensembl BLAST search against total mouse cDNA. This revealed no other hits that could potentially form a stable hybrid with either our short or long UTR probes. After linearization of the probes, a T7 or SP6 promoter-driven in vitro transcription reaction was done in the presence of a DIG labeling mix (Roche, 11175025910). The denatured probes (sense or antisense) were then applied to pretreated coronal sections and hybridized overnight in a custom-built chamber.6Murtaugh L.C. Chyung J.H. Lassar A.B. Sonic hedgehog promotes somitic chondrogenesis by altering the cellular response to BMP signaling.Genes Dev. 1999; 13: 225-237Crossref PubMed Scopus (232) Google Scholar Staining was visualized by incubating sections with an alkaline phosphatase conjugated digoxygenin antibody (1:2000, 4°C, overnight) (Roche, 11093274910), followed by the application of BM-Purple AP (Roche, 1144207400). When we used Sheridan's probe, we observed strong staining at E14.5, E16.5, and P0, particularly in the cortical plate, and to a lesser extent in the intermediate and ventricular zones (Figure 1). In contrast, we did not observe any signal at any of these time points with our Tuba8 3′UTR-specific probes, although we did observe positive staining in the Purkinje cell layer in the cerebellum and in the mitral and granule cell layers of the olfactory bulbs in the adult brain (Figure S2). Because there is evidence for an alternatively spliced variant of Tuba8 with a shorter UTR, we confirmed our in situ hybridization results by undertaking real-time quantitative PCR (qPCR) with primers targeted to the coding region (Table S1).7Stanchi F. Corso V. Scannapieco P. Ievolella C. Negrisolo E. Tiso N. Lanfranchi G. Valle G. TUBA8: A new tissue-specific isoform of alpha-tubulin that is highly conserved in human and mouse.Biochem. Biophys. Res. Commun. 2000; 270: 1111-1118Crossref PubMed Scopus (43) Google Scholar When designing intron-spanning primers for this task, we took care to ensure that each primer pair was specific, undertaking BLAST searches with multiple search engines (Ensemble, UCSC, and NCBI). In addition, we only used primer pairs with an efficiency between 95% and 105% that produced a melt curve indicative of a single product. To prepare templates for amplification, we dissected and flash froze embryonic brains (E10.5 [head], E12.5, E14.5, E16.5, E18.5), postnatal brains (P0, P6), various regions of the adult brain (8 weeks) and adult organs (C57BL/6), prior to RNA extraction (QIAGEN, 74804, 74704). Three independent RNA samples from each developmental time point, brain region, or organ were then quantitated and pooled for reverse transcription (Invitrogen, 45-0640). Quantitative PCR followed on a BioRad Cycler with SYBR green, alongside reverse transcriptase (RT) and cDNA negative controls. All reactions were performed in triplicate and normalized to the geometric mean of three reference genes (Pgk1, Tfrc, and Hprt).8Boda E. Pini A. Hoxha E. Parolisi R. Tempia F. Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain.J. Mol. Neurosci. 2009; 37: 238-253Crossref PubMed Scopus (89) Google Scholar, 9Vandesompele J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol. 2002; 3 (RESEARCH0034)Crossref PubMed Google Scholar We compared the expression of Tuba8 to those tubulin genes (Tuba1a [MIM 611603], Tubb2b [MIM 610031], Tubb3 [MIM 600638]) that have been shown to cause distinct neurodevelopmental diseases.10Tischfield M.A. Baris H.N. Wu C. Rudolph G. Van Maldergem L. He W. Chan W.M. Andrews C. Demer J.L. Robertson R.L. et al.Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance.Cell. 2010; 140: 74-87Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar We found that Tuba 1a, Tubb2b, and Tubb3 are highly expressed in the developing mouse brain from E12.5 to P6, whereas Tuba8 is expressed at low levels (Figure 2A ). For instance, at E14.5, a peak time for neuronal migration, Tuba1a is expressed at ∼94 times the control gene level, whereas Tuba8 is barely detectable, expressed at just 0.03 times the control gene level. We next analyzed expression levels of Tuba8 in different regions of the adult mouse brain by qPCR, again comparing it to Tuba1a, Tubb2b, and Tubb3 (Figure 2B). We observed regional differences in the expression levels of these genes, but generally Tuba1a was most highly expressed, followed by Tubb3 and Tubb2b. Tuba8 was again expressed at very low levels, with the exception of the olfactory bulbs and the cerebellum, where its modest level of expression was concordant with the results from our in situ hybridization experiments. We extended our expression analysis beyond the adult brain to the major adult organs in the mouse (Figure S3). We found that the level of Tuba8 expression is highest in testis (∼12 times control gene expression), followed by skeletal and heart muscle, confirming the results of Stanchi and colleagues who studied TUBA8 expression in adult human tissues.7Stanchi F. Corso V. Scannapieco P. Ievolella C. Negrisolo E. Tiso N. Lanfranchi G. Valle G. TUBA8: A new tissue-specific isoform of alpha-tubulin that is highly conserved in human and mouse.Biochem. Biophys. Res. Commun. 2000; 270: 1111-1118Crossref PubMed Scopus (43) Google Scholar Finally, we investigated the expression of TUBA1A, TUBB2B, TUBB3, and TUBA8 in human fetal brain at GW13 (Biochain, C1244051) and GW22 (Biochain, C1244035), again employing three reference genes for normalization (HPRT, PGK1, TBP). Consistent with our mouse data, we observed very high levels of expression of TUBA1A, TUBB2B, and TUBB3, but only low levels of TUBA8 (Figure 2C). For instance, in the frontal lobe at GW13, a peak time for neuronal migration in humans,11Jaglin X.H. Chelly J. Tubulin-related cortical dysgeneses: microtubule dysfunction underlying neuronal migration defects.Trends Genet. 2009; 25: 555-566Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar expression of TUBA1A is ∼224 times the control level, whereas TUBA8 is expressed at just 0.18 times the control gene level. We replicated our qPCR results with alternative sets of primers (in both mouse and human), confirming that our qPCR results are not due to alternative splicing (data not shown). The data presented here show that Tuba8 is expressed at a low level in the developing mouse and human brain. This raises a puzzling question: How is it that a gene that is preferentially expressed in testis, heart, and skeletal muscle presents as a rare brain malformation? This question is particularly pertinent given that those tubulin genes known to cause neurodevelopmental disorders are highly expressed in the developing CNS, whereas Tuba8 is not. We suggest three possible explanations. First, it is plausible that an unidentified mutation lies in one of the 230 unsequenced genes in the 7.42 Mb candidate interval that contributes to, or is responsible for, the observed phenotype. Second, it is conceivable that despite its low expression level, Tuba8 serves some unique function that is essential to the developing brain and that is not vital in other tissues. Third, one might imagine a scenario in which the deletion described by Sheridan results in the expression of a truncated tubulin polypeptide and that this might confer some deleterious effect. The generation of appropriate transgenic mouse models or the identification of additional genetically unrelated patients harboring mutations in TUBA8 would help to resolve these issues. This work was supported by the Oxford Partnership Comprehensive Biomedical Research Centre with funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. N.J.C. acknowledges the receipt of a Grant (R01HD057028) from the NIH. D.A.K. is supported by an FWF project grant (P21092-B09). Download .pdf (.09 MB) Help with pdf files Document S1. Three Figures and One Table The URL for data presented herein is as follow:Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ Response to Braun et al.Sheridan et al.The American Journal of Human GeneticsMay 14, 2010In BriefTo the Editor: We note with interest the discordance between the TUBA8 (MIM 605742 ) expression data obtained by Keays et al. and those we previously presented.1 We accept that there is a possibility of cross-hybridization of the probes we used with other members of the α-tubulin family. Full-Text PDF Open Archive" @default.
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