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Familial frontotemporal dementia with neuronal intranuclear inclusions is not a polyglutamine expansion… Mackenzie, Ian R; Butland, Stefanie L; Devon, Rebecca S; Dwosh, Emily; Feldman, Howard; Lindholm, Caroline; Neal, Scott J; Ouellette, Francis B; Leavitt, Blair R Aug 31, 2006

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ralssBioMed CentBMC NeurologyOpen AcceResearch articleFamilial frontotemporal dementia with neuronal intranuclear inclusions is not a polyglutamine expansion diseaseIan R Mackenzie1, Stefanie L Butland2, Rebecca S Devon3, Emily Dwosh4, Howard Feldman4, Caroline Lindholm4, Scott J Neal5, BF Francis Ouellette2 and Blair R Leavitt*5Address: 1Department of Pathology, University of British Columbia, Vancouver, BC, Canada, 2UBC Bioinformatics Centre, Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada, 3Medical Genetics Section, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Edinburgh, UK, 4Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, BC, Canada and 5Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, BC, CanadaEmail: Ian R Mackenzie -; Stefanie L Butland -; Rebecca S Devon -; Emily Dwosh -; Howard Feldman -; Caroline Lindholm -; Scott J Neal -; BF Francis Ouellette -; Blair R Leavitt* -* Corresponding author    AbstractBackground: Many cases of frontotemporal dementia (FTD) are familial, often with an autosomaldominant pattern of inheritance. Some are due to a mutation in the tau- encoding gene, onchromosome 17, and show an accumulation of abnormal tau in brain tissue (FTDP-17T). Most ofthe remaining familial cases do not exhibit tau pathology, but display neuropathology similar topatients with dementia and motor neuron disease, characterized by the presence of ubiquitin-immunoreactive (ub-ir), dystrophic neurites and neuronal cytoplasmic inclusions in the neocortexand hippocampus (FTLD-U). Recently, we described a subset of patients with familial FTD withautopsy-proven FTLD-U pathology and with the additional finding of ub-ir neuronal intranuclearinclusions (NII). NII are a characteristic feature of several other neurodegenerative conditions forwhich the genetic basis is abnormal expansion of a polyglutamine-encoding trinucleotide repeatregion. The genetic basis of familial FTLD-U is currently not known, however the presence of NIIsuggests that a subset of cases may represent a polyglutamine expansion disease.Methods: We studied DNA and post mortem brain tissue from 5 affected members of 4 differentfamilies with NII and one affected individual with familial FTLD-U without NII. Patient DNA wasscreened for CAA/CAG trinucleotide expansion in a set of candidate genes identified using agenome-wide computational approach. Genes containing CAA/CAG trinucleotide repeatsencoding at least five glutamines were examined (n = 63), including the nine genes currently knownto be associated with human disease. CAA/CAG tract sizes were compared with published normalvalues (where available) and with those of healthy controls (n = 94). High-resolution agarose gelelectrophoresis was used to measure allele size (number of CAA/CAG repeats). For any allelesestimated to be equal to or larger than the maximum measured in the control population, the CAA/Published: 31 August 2006BMC Neurology 2006, 6:32 doi:10.1186/1471-2377-6-32Received: 27 June 2006Accepted: 31 August 2006This article is available from:© 2006 Mackenzie et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 7(page number not for citation purposes)CAG tract length was confirmed by capillary electrophoresis. In addition, immunohistochemistryBMC Neurology 2006, 6:32 a monoclonal antibody that recognizes proteins containing expanded polyglutamines (1C2)was performed on sections of post mortem brain tissue from subjects with NII.Results: No significant polyglutamine-encoding repeat expansions were identified in the DNAfrom any of our FTLD-U patients. NII in the FTLD-U cases showed no 1C2 immunoreactivity.Conclusion: We find no evidence to suggest that autosomal dominant FTLD-U with NII is apolyglutamine expansion disease.BackgroundFrontotemporal dementia (FTD, OMIM: 600274) is aneurodegenerative disease characterized by abnormalitiesin personality, behaviour and language with relative earlypreservation of episodic memory [1,2]. The pathologyunderlying clinical FTD is heterogeneous [3]. In somecases, post mortem examination discloses abnormal accu-mulations of the microtubule associated protein tau inneurons and/or glial cells. However, several recent studieshave demonstrated that the most common pathologyassociated with clinical FTD is the presence of dystrophicneurites and neuronal cytoplasmic inclusions in the cere-bral cortex and hippocampus that are immunoreactive forubiquitin (ub-ir) but negative for tau, synuclein and inter-mediate filament proteins (FTLD-U) [4,5].FTD is often familial, usually with an autosomal domi-nant pattern of inheritance. Various studies have demon-strated genetic linkage to loci on chromosomes 3, 9 and17 [6-11]. The gene on chromosome 3 and one of thegenes on chromosome 9 have recently been identified(chmp2B and valosin-containing protein, respectively)[12,13]. Some cases that show linkage to chromosome 17are found to have mutations in the gene for tau (MAPT)and all such cases show tau pathology [14,15]. FamilialFTLD-U has been linked to several different loci on chro-mosome 9 and to chromosome 17q21 [6,7,10,11,16].Interestingly, the 17q21 locus contains MAPT, and yetthese cases do not have any tau pathology and have nothad any tau mutations identified [6,7,11]. It remainsunclear whether these cases are due to some, as yet, unrec-ognized abnormality in MAPT or whether there is anothergene on 17q21 that is responsible for familial FTLD-U.Recently, we reported that a subset of patients with famil-ial FTLD-U have the additional post mortem finding ofunusual, lentiform, ub-ir neuronal intranuclear inclusions(NII) [11,17]. At least some of the cases of familial FTLD-U with NII are ones that have shown linkage to the 17q21locus [6,7,11]. NII are uncommon in neurodegenerativedisease in general, but are a characteristic pathological fea-ture of several conditions for which the genetic basis isabnormal expansion of a polyglutamine-encoding CAGebellar ataxia (SCA) [18-20]. The NII observed in thesediseases are composed in part of aggregates of theexpanded-repeat proteins. In this study, we explore thepossibility that autosomal dominant FTLD-U with NII is apolyglutamine expansion disease.MethodsStudy cohortSix patients with familial FTD, from five different families,were included in the study. In all families studied here, thepattern of inheritance suggested an autosomal dominanttrait with a high degree of penetrance as previouslydescribed [11,17]. There was no evidence of genetic antic-ipation in any of the families. Five of the study patientswere deceased and each was from a different family. Post-mortem neuropathological examination confirmed thepresence of ub-ir neuronal cytoplasmic inclusions charac-teristic of FTLD-U [Figure 1 a,b] and an absence of signif-icant tau and α-synuclein pathology (not shown). In fourdeceased patients (cases 1–4), numerous ub-ir NII werealso observed. NII had a characteristic lentiform shapeand were most numerous in small neurons of the fronto-temporal neocortex and striatum [Figure 1c] [17]. Thefifth deceased patient had FTLD-U type pathology but noNII (case 6). One living patient with FTD was alsoincluded in the study; he was the cousin of one of thedeceased study patients with FTLD-U and NII (case 5).Therefore, the six study patients represented four familieswith FTLD-U with NII (cases 1–5) and one family withFTLD-U without NII (case 6).ImmunohistochemistryPost mortem brain tissue from the four deceased studypatients with previously confirmed NII was evaluated.Formalin fixed, paraffin-embedded tissue sections fromfrontal cortex and striatum (areas with maximal numbersof NII), were immunostained using the Ventana ES auto-mated system (Ventana, Tuscon, AZ) with primary anti-bodies against ubiquitin (DAKO, anti-ubiquitin; 1:500,following microwave antigen retrieval) and proteins withexpanded polyglutamine domains (Chemicon, 1C2;1:100 following formic acid pre-treatment). Tissue sec-tions from two patients with known polyglutaminePage 2 of 7(page number not for citation purposes)trinucleotide repeat within the gene (examples includeHuntington's disease (HD) and several types of spinocer-expansion diseases (one HD and one SCA1) wereincluded as positive controls.BMC Neurology 2006, 6:32 analysis of polyglutamine-encoding genesUsing a computational approach, we have previouslyidentified 63 genes in the human genome that containtracts of CAG/CAA trinucleotide repeats which encode atleast five consecutive glutamines (Butland et al., in sub-mission). FTLD-U patient DNA was screened for expan-sions in these trinucleotide repeat tracts (n = 64),including the nine tracts whose expansion is currentlyknown to cause neurodegenerative disorders in humans.One gene (PCQAP) contains two distinct polyglutamine-encoding tracts that were screened separately, hence thedisparity in tract number versus gene number.Gene-specific PCR primers were used to amplify therepeat-containing loci from the patient DNA, and theamplicons were subjected to high-resolution gel electro-phoresis to measure allele size. Metaphor agarose gels(3% w/v, Mandel Scientific) were run overnight at lowvoltage in re-circulated buffer. Under such conditions, it ispossible to resolve small allelic differences [see Additionalfile 1]. Gel data were digitised and interpreted withIMAGE® software, and product sizing was estimated to beaccurate within 6 base pairs. The length of CAG/CAAtracts was inferred from the overall allele length based oncalibration data we obtained by directly sequencing sev-eral alleles from each locus. In no case did we observeallele length differences that arose from sequence changesoutside of the repeat tract. Thus, all allele size changes cor-respond to differences in the repeat tract alone. From thepresent data we cannot, however, determine where in therepeat tract, such as in the longest contiguous CAG tract,the expansion is occurring.The data presented herein refer to the longest uninter-rupted tract of CAG and CAA trinucleotides in the givengene. These values were compared to published data (forknown disease loci) and to data obtained from a controlgroup (n = 94) of unaffected individuals (Butland et al., insubmission). Based on the above resolution limit, all alle-les estimated to be 6 or more b.p. longer than the longestcontrol allele were subjected to confirmatory capillaryelectrophoresis on an ABI 3700 DNA Analyzer (AppliedBiosystems) and subsequent analysis using GeneMappersoftware (Applied Biosystems).This research was carried out in compliance with the Hel-sinki Declaration, and ethical approval was provided bythe Clinical Research Ethics Board of the University ofBritish Columbia (certificate C03-0449).ResultsNeuropathologic analysis of FTLD-U brain tissueUbiquitin-positive neurites and neuronal cytoplasmicNeuropathologic analysis of brain tissue from FTLD-Upa-tientsFig re 1Neuropathologic analysis of brain tissue from FTLD-Upatients. Ubiquitin immunohistochemistry in cases of familial FTLD-U demonstrates staining of (a) neurites and neuronal cytoplasmic inclusions in the superficial cerebral neocortex, (b) neuronal cytoplasmic inclusions in hippocam-pal dentate granule cells, and (c) neuronal intranuclear inclu-sions in the cerebral neocortex (arrows). Scale bar; (a) and (b) 40 µm, (c) 25 µm, insert 6 µm.Page 3 of 7(page number not for citation purposes)inclusions were identified in the superficial cerebral neo-cortex of the FTLD-U patients (Figure 1a), and similarBMC Neurology 2006, 6:32 1: Genetic analysis of FTLD-U DNA samples reveals no significant CAG/CAA repeat expansions in candidate polyglutamine-encoding genes.Gene Name (associated polyQ dis-ease, CAA/CAG tract length of small-est disease-cuasing allele) aControl Samples (n = 94) estimated CAA/CAG tract lengthFTD Patients estimated CAA/CAG tract lengthcase 1b case 2b case 3b case 4b case 5b case 6cA AR (SBMA, 38) 10–37 22 19 20 21, 23 19 24ATN1 (DRPLA, 48) 10–39 18, 23 18, 20 20, 23 19 15, 19 15ATXN1 (SCA1, 39) 6–39 15, 20 15, 20 16 15, 20 15, 20 14, 21ATXN2 (SCA2, 37) 25–41 22 22 22 22 22 22ATXN3 (SCA3/MJD, 51) 14–42 15 9 9 9 17 naATXN7 (SCA7, 36) 4–35 10 11 10 10 10 10CACNA1A (SCA6, 20) 4–18 12, 21d 13, 16 15 14 14 13HD (HD, 36) 10–35 14, 15 12, 17 17, 21 15, 17 12, 14 12TBP (SCA17, 49) 38–61 39, 44 38, 41 38, 43 36, 39 38, 47 38, 42B ARID3B 11–13 12 12 12, 16 12, 16 13, 16 13ASCL1 5–24 13, 17 13 13, 17 13, 18 13, 18 12BMP2K 22–31 26 26 26 26 26 26C14orf4 19–24 22, 25 22, 25 23 23 24 23CXorf6 11–12 12 12 12 11 11 12DCP1B 10–12 12 na 12 11, 12 11, 12 11, 12KCNN3 10–43 18 17, 18 na 19, 21 18, 21 18, 20MED12 26 27 26 26 26 26 26MEF2A 9–16 12, 15 11 12 10, 11 11, 14 11MINK1 5–6 6 6 6 6 6 7MLL2 6–9 9 9 9 9 9 9NCOA3 25–34 26, 29 28 26, 28 29 28, 29 28NCOA6 23–26 25 25 na 24 25 25NCOR2 13–22 17, 19 17 17 16, 17 13, 17 17, 18NFAT5 18–21 19 18 18 18 na naNM 014856 13–17 16 17 14, 16 17 16 14, 16NUMBL 26 18, 20 18 20 18, 20 18, 20 20PCQAPa 10–18 11 11 11 10 10 11PCQAPb 11–22 17 16 16 15 16 16PHLDA1 15–19 15 15 16 15 15 15POLG 13–16 13 14 13 16, 17 13 13POU3F2 20–21 15 21, 22 21 21 21 21POU6F2 7–11 10 10 10 10 10 10PRDM10 7–8 8 8 8 8 8 8PRKCBP1 7–11 8 8 7 8 8 8RAI1 8–20 14 14 14 14 14 10, 14RUNX2 26–53 22 21 22 23 23 21SATB1 12–21 15 15 15 15 15 15SMARCA2 18–25 22 22 22 22 22 22SOCS7 7–22 8, 12 8 8 8 8 8TFEB 9–15 12 9 9 9 10 10TNRC4 14–17 15 15 15 15 15 15TNRC6B 7–10 9, 10 9 9 9 8 8TNS 8–11 10, 12 10 9 10 9 10ZNF161 8–22 16, 17 14, 20 14 na 14 14ZNF384 11–20 15 16 15 15 20 15C ARID1B 16–23 18 18 19 19 18 18BAIAP1 16–21 20, 24 19, 25 18, 19 19 20 17, 19BRD4 8–9 8 8 8 8 8 8C9orf43 8–9 9 9, 12 8 8 9, 12 8CHERP 12 12 12 12 12 12 12CIZ1 6 6 6 6 6 6 6Page 4 of 7(page number not for citation purposes)CREBBP 18 18 18 18 18 18 18EP400 28–31 30 30, 34 30 30 31 30, 34BMC Neurology 2006, 6:32 cytoplasmic inclusions were found in hippoc-ampal dentate granule cells (Figure 1b). Ub-ir NII werefound in the cerebral neocortex (arrows, Figure 1c). NII inthe control cases with known polyglutamine disease (HDand SCA1) were immunoreactive for both ubiquitin and1C2. In contrast, NII in FTLD-U patients were ub-ir, butwere 1C2 negative (data not shown).Genetic analysis of FTLD-U DNA samples for CAG/CAA repeat expansions in polyglutamine-encoding genesFTLD-U patient DNA was screened for expansions in theCAA/CAG trinucleotide repeat tracts of our candidategenes (Table 1), and the repeat sizes from the study sub-jects were compared with published normal values andwith those of healthy controls (n = 94) (Butland et al., insubmission). Sixteen small putative expanded alleles weredetected, while the vast majority of alleles from our FTDpatients fell within the range of normal alleles from ourunaffected controls. At least one allele was identified ineach patient that fell just outside the range observed inour controls samples, and these alleles arose from eightgenes (CACNA1A, ARID3B, BAIAP1, C9orf43, EP400,FOXP2, MAML2, PAXIP1L). The largest putative expan-sion identified consisted of a 15 b.p. (~ 5 CAG/CAA)increase over the largest control allele (case 2, MAML2).The exact CAG repeat size of all the putative expandedalleles was confirmed by capillary electrophoresis sizingand/or direct DNA sequencing, and none of these specificDiscussionWe have adapted an inexpensive, high-resolution agarosegel electrophoresis method for the precise sizing of tar-geted polyglutamine-encoding repeat tracts in the humangenome. Using this method, the normal distribution ofCAG repeat lengths for known disease loci comparedfavourably with previously published data and matchedthe results obtained by direct sequencing of specific alle-les, thus confirming the reliability of our method. No sig-nificant CAA/CAG repeat expansions were detected at thenine known disease loci in any of the FTLD-U patients.The initial analysis of the data suggested a putative minorexpansion in the CACNA1A gene (case 1) by gel electro-phoresis that was subsequently refuted by capillary elec-trophoresis. This method was subsequently applied to 55candidate loci that we identified as part of our computa-tional screen for polyglutamine-encoding tracts in thehuman genome (Butland et al., in submission). We iden-tified 15 alleles from seven genes in our FTLD-U patientsthat were 6 or more b.p. longer than the longest controlallele (Table 1, allele sizes in bold text). None of thesesmall expansions consistently correlated with affectedFTLD-U status in multiple individuals. An allele ofMAML2 harboured the largest putative expansion (15b.p., case 2). However, according to the annotation of thereference genome, the trinucleotide repeat tract passesthrough a proposed intron. Thus, it is unclear if in fact thisallele would encode an expanded polyglutamine tract inFOXP2 34–40 41 na 41 41 42 42KIAA1817 26–27 27 28 28 28 27 27KIAA2018 11–16 13 14 14 14 12, 17 12MAML2 27–31 29 32, 36 28 28 28, 31 28MAML3b 18 19 19 19 19 19 19MN1 26–30 28 28 28 28 28 28, 29PAXIP1L 7 9 9 9 8 8 8PHC1 13–15 15 15 15 15 15 15ST6GALNAC5 12–14 12 12 12 12 12 12THAP11 18–30 24 24 24, 33 24 24, 26 24, 28TNRC6A 4–8 4, 8 5, 8 8 8 5, 8 8a Genes identified in a computational analysis of polyglutamine repeat-containing genes in the human genome. A) Genes known to cause disease via polyglutamine expansion. B) Patient and control samples were both assessed by high resolution agarose gel electrophoresis. C) Control samples were analyzed by capillary electrophoresis (Butland et al., in submission) and patient samples analyzed by high resolution agarose gel electrophoresis.b FTDL-U patients with NII.c FTDL-U patient without NII.d Alleles 6 or more b.p. longer than the largest contol allele (putative expanded alleles) appear in bold type.Samples of FTLD-U patient DNA with NII (Cases 1–5) and without NII (Case 6) were screened for expansions in the CAG/CAA trinucleotide repeat tracts of our candidate genes including known disease genes (A). The CAG/CAA repeat lengths from the FTLD-U subjects were compared with published normal values and with those of healthy controls (n = 94), assessed by high resolution agarose gel electrophoresis (B), and/or capillary electrophoresis (C). For the few alleles estimated to be equal to or slightly larger than the maximum measured in our control samples (bold), the CAG/CAA repeat length was confirmed by capillary electrophoresis on an ABI 3700 sequencer using GeneMapper software. Using this approach, no clinically significant CAG/CAA repeat expansions were identified in the DNA from any of our FTLD-U patients.Table 1: Genetic analysis of FTLD-U DNA samples reveals no significant CAG/CAA repeat expansions in candidate polyglutamine-encoding genes. (Continued)alleles was found to be expanded in all of the affected the protein.Page 5 of 7(page number not for citation purposes)FTDL-U individuals.BMC Neurology 2006, 6:32 factors make it unlikely that the small putativeexpansions identified are pathogenic. First, there were nogenes for which the allele sizes in FTD patients with NII(cases 1–5) were consistently larger than those from theFTD patient without NII (case 6). Second, pathogenic alle-les of known human polyglutamine disorders typicallyencode more than 35 consecutive glutamines whereasnone of our putative expanded alleles encode this many.Our histopathological findings also support the conclu-sion than FTLD-U with NII is not a polyglutamine expan-sion disorder. All known polyglutamine expansionsdisorder include the presence of ub-ir NII which also labelwith the monoclonal antibody 1C2 [18-20]. The absenceof any 1C2 reactivity in our FTLD-U patient tissue thusmakes it unlikely that their NII contain proteins withexpanded polyglutamine tracts.Finally, although only a small number of such familieshave been reported to date, there is evidence that NII maybe a specific pathological marker for FTLD-U linked to thechromosome 17q21 locus [6,7,11]. Our computationalapproach did not identify any gene in the defined regionof interest that contains at least five CAG or CAA repeats,although ubiquitinated intranuclear inclusions areknown to occur in triplet repeat disorders encoding foramino acids other than glutamine (alanine), and in disor-ders caused by expansions in untranslated regions. Thisregion does contain 294 non-CAG trinucleotide repeattracts of minimum length 5; 11 of which are found withinthe coding sequences of 6 known and 3 hypotheticalgenes [21]. Thus, a non-glutamine encoding trinucleotiderepeat expansion could still be the basis of the observedphenotype.ConclusionIn summary, we find no evidence to suggest that auto-somal dominant FTLD-U with NII is a polyglutamineexpansion disease. We did not observe immunoreactivityfor expanded polyglutamines within FTLD-U brain, nordid we identify any alleles with large polyglutamine-encoding repeat expansions in our set of candidate genes,which comprises all of the predicted genes of interest withat least five polyglutamine-encoding CAA/CAG repeats inthe human genome. Furthermore, none of the slightlylonger alleles from FTLD-U subjects within the candidategenes identified by our computational approach arefound within the various linkage regions established inprior studies. Therefore, both our genetic analysis andimmunohistochemical data, suggest that the formation ofNII in FTLD-U is due to a mechanism other than accumu-lation of a protein with a polyglutamine expansion.Authors' contributionsSLB, RSD, BRL, BFFO, IRM conceived and designed theexperiments. HF, CL, ED contributed the genetic and clin-ical materials. SLB, RSD, IRM performed the experiments.SLB, SJN, IRM analysed the data. SLB, SJN, IRM and BRLwrote the paper and all other authors provided com-ments.Note added in proofThe conclusions of this manuscript have recently beenverified by two manuscripts: Baker M et al, Mutations inprogranulin cause tau-negative frontotemporal dementialinked to chromosome 17. Nature. 2006 Aug24;442(7105):916–9, and Cruts M, et al. Null mutationsin progranulin cause ubiquitin-positive frontotemporaldementia linked to chromosome 17q21. Nature. 2006Aug 24;442(7105):920–4. This work identified non-poly-glutamine encoding mutations in the gene encoding pro-granulin as the cause of FTLD-U in the subjects westudied.Additional materialAcknowledgementsThe authors wish to thank Soo Sen Lee, Anna Wilkinson, Ashvinder Bhogal, Macaire Man St. Yuen for technical assistance, sample management, and database management, Ian Bosdet and Jacquie Schein for early technology development. 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