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Understanding phenotype variability in frontotemporal lobar degeneration due to granulin mutation
Neurobiology of Aging 35 (2014) 1206e1211
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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Understanding phenotype variability in frontotemporal lobar degeneration due to granulin mutation Cristian Bonvicini a, Elena Milanesi a, Andrea Pilotto b, Nadia Cattane c, Enrico Premi b, Silvana Archetti d, Alessandro Padovani b, Massimo Gennarelli a, c, Barbara Borroni b, * a
Genetic Unit, IRCCS S.Giovanni di Dio, Fatebenefratelli, Brescia, Italy Centre for Neurodegenerative Disorders, Neurology Unit, University of Brescia, Brescia, Italy Departement of Molecular and Translational Medicine, University of Brescia, Brescia, Italy d Department of Laboratories, III Laboratory of Analysis, Brescia Hospital, Brescia, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2013 Received in revised form 5 October 2013 Accepted 28 October 2013 Available online 1 November 2013
Phenotype in patients with granulin (GRN) mutations is unpredictable, ranging from behavioral variant frontotemporal dementia (bvFTD) to agrammatic variant of primary progressive aphasia (avPPA). To date the wide clinical variability of FTLD-GRN remains unexplained. The aim of the study was to identify genetic pathways differentiating phenotypic expression in patients carrying GRN mutations. Patients carrying the same GRNT272SfsX10 mutation were enrolled, a careful clinical assessment was carried out, and the diagnosis of either bvFTD (n ¼ 10, age ¼ 63.9 9.4) or avPPA (n ¼ 6, age ¼ 58.8 4.7) was done. Microarray gene expression analysis on leukocytes was performed. Genes differentially expressed between the groups were validated by real time polymerase chain reaction considering an age-matched healthy controls group (n ¼ 16, age ¼ 58.4 10.7). We further considered a group of FTD with no GRN mutations (GRN-) (n ¼ 21, 13 bvFTD, and 8 avPPA) for comparisons. Real-time polymerase chain reaction (PCR) confirmed a significant decrease in leukocytes mRNA messenger RNA (mRNA) levels of RAP1GAP in bvFTD patients as compared with avPPA (p ¼ 0.049). This finding was specific for patients with GRN mutations, as we did not observe this pattern in FTD GRN-patients (p ¼ 0.99). The alteration of RAP1GAP mRNA levels may explain the clinical variability of GRN-FTLD patients. This is the first report linking a molecular pathway to specific phenotype expression in FTLD-GRN. To understand the clinical relevance of our early results it will be mandatory to extend the observation to other clinical and neuropathological series. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Frontotemporal dementia GRN mutations Phenotype variability bvFTD avPPA Genetic modulation
1. Introduction Frontotemporal lobar degeneration (FTLD) is a clinically, pathologically, and genetically heterogenous disorder. Three prototypical variants have been described, namely the behavioral variant frontotemporal dementia (bvFTD), the semantic variant of primary progressive aphasia, and the agrammatic variant of primary progressive aphasia (avPPA) (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). In most cases, at autopsy either FTLD with tau-positive inclusions or FTLD with TDP-43-positive aggregates may be variably found over the clinical spectrum of FTLD cases (Mackenzie et al., 2009, 2011). Despite the consistent progress in carefully characterizing the clinical phenotypes and in defining neuroimaging correlates, * Corresponding author at: Neurology Unit, University of Brescia, Piazza Spedali Civili 1, Brescia 25125, Italy. Tel.: þ39 0303995632; fax: þ39 0303995027. E-mail address: [email protected] (B. Borroni). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.10.097
there is no unique correspondence with neuropathological substrates. The mismatch between neuropathological and clinical features is still evident when monogenic FTLD is considered. Granulin (GRN) gene has been identified as a major cause of autosomal dominant FTLD, leading to TDP-43 inclusions by a haploinsufficiency mechanism (Cruts et al., 2006; Toh et al., 2011). FTLD patients carrying GRN mutations clinically present unpredictable phenotypic variability even within families carrying the same mutation (Beck et al., 2008; Bruni et al., 2007; Chen-Plotkin et al., 2011; Kelley et al., 2009; Larner, 2012; Le Ber 2008; Li et al., 2008; Moreno et al., 2009; Pickering-Brown et al., 2008; Rademakers et al., 2007; van Swieten and Heutink, 2008; Yu et al., 2010), and bvFTD and avPPA represent the most frequent pictures. Aim of the present study was to dissect clinical heterogeneity in patients carrying the same GRN mutation. To this, a coalescent cohort of patients with GRN Thr272fs mutation was considered, and whole gene expression analysis on leukocytes was carried
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211
out to compare the expression profiles in the different clinical phenotypes (bvFTD vs. avPPA).
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for each procedure. The work was conformed to the Helsinki Declaration and approved by local ethics committee. 2.2. Granulin sequencing
2. Methods 2.1. Subjects Two hundred fifty FTLD patients, recruited from the Centre for Ageing Brain and Neurodegenerative Disorders, at University of Brescia (Brescia, Italy), were screened for pathogenic GRN and MAPT mutations and C9orf72 expansion; those carrying GRN Thr272fs mutation were enrolled in the present study. In these patients, diagnosis of either bvFTD or avPPA was made according to the current clinical criteria (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). All FTLD patients carrying GRN Thr272fs mutation were part of a genetically homogenous population, as these patients harbor a common ancestor as previously reported (Borroni et al., 2011), making our experimental design well controlled. All patients were subjected to an extensive neurologic and neuropsychological evaluation, as previously described (Borroni et al., 2010), a routine laboratory examination, and a conventional brain magnetic resonance imaging before entering this study. We further enrolled a group of 21 FTD patients without GRN mutations, matched for age and gender, for comparisons and we sub-grouped them according to clinical phenotype, that is, 8 avPPA (age 69.4 9.6) and 13 bvFTD (age 67.1 6.2). A group of healthy controls, matched for age and gender was also considered (see Table 1). FTD patients, with or without GRN mutations, and healthy controls did not report significant medical problems. None was under treatment for cognitive disturbances, but 7 (3 FTD GRN and 4 GRN-) were under psychotropic drugs for behavioral disturbance control. Each subject underwent venous blood sampling for biological analyses. Written informed consent (from the subject or from the responsible guardian if the subject was incapable) was obtained,
Genomic DNA was extracted from peripheral blood using a standard procedure. All the 12 exons plus exon 0 of GRN, and at least 30 base pairs of their flanking introns were evaluated by polymerase chain reaction (PCR) and subsequent sequencing. GRN Thr272fs (g.1977_1980 delCACT) was tested as previously described (Borroni et al., 2008). 2.3. RNA isolation and microarray gene expression procedures Blood samples were obtained by venipuncture using PaxGene Tubes (Qiagen, Manchester, UK). RNA isolation was performed by PaxGene Blood RNA Kit (Qiagen) according to the manufacturer protocols. RNA quality and integrity were assessed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Total RNA (250 ng) from each sample were reverse transcribed to complementary DNA (cDNA), followed by overnight in vitro transcription to generate complementary RNA, which was reverse transcribed, and the 7.5 mg of sense cDNA were fragmented and labeled. The quality of cDNA and fragmented cDNA was assessed using Agilent bioanalyzer (Agilent Technology, TX, USA). Hybridization was performed on human gene 1.1 ST array strips (Affymetrix, Inc, Santa Clara, CA). The reactions of hybridation, fluidics, and imaging were performed on the Affymetrix Gene Atlas instrument according to the manufactured protocol. 2.4. Microarray data analysis Gene expression data were imported from the Gene Atlas instrument into Partek Genomics Suite 6.0 (Partek, St Louis, Mo) software as CEL files using default parameters. Principalcomponent analysis was performed to identify outliers. Differential expression analysis between bvFTD and avPPA phenotypes was performed by the analysis of variance (ANOVA) and a gene list was created using a cutoff of p < 0.05 and 1.6-fold change.
Table 1 Demographic and clinical characteristics of FTLD-GRN patients according to clinical diagnosis Variable
FTD-GRN all (N ¼ 16)
bvFTD-GRN (N ¼ 10)
avPPA-GRN (N ¼ 6)
Controls (N ¼ 16)
p (bvFTD vs. avPPA)
Age at evaluation, years Gender, female% Family history, % Age at onset, years Education, years Serum PGRN, pg/ml FTLD-modified CDR MMSE UPDRS-III Short story Raven matrices Rey figure, copy Rey figure, recall Phonological fluency Semantic fluency Digit span, backward Token test Trail making test, A Trail making test, B NPI, total score FBI, total score
64.2 8.5 62.5 100 59.3 8.6 6.8 2.2 48.2 21.4 7.3 5.6 22.5 5.4 10.0 16.7 8.2 5.5 15.9 7.2 18.5 10.1 6.4 4.8 14.0 10.5 20.3 5.4 4.3 0.6 24.5 5.8 71.0 25.6 380.7 162.0 18.5 13.0 18.0 11.1
65.8 9.9 60 100 61.4 9.3 6.1 2.3 50.6 23.6 8.4 6.3 22.6 4.5 14.2 20.1 8.7 6.2 14.2 4.5 16.0 8.2 4.0 3.2 17.3 10.5 20.6 4.9 4.3 0.5 25.2 4.9 85.0 16.4 350 166.7 20.8 13.3 21.8 12.1
61.5 4.9 66 100 55.8 6.4 8.0 1.7 43.4 18.5 5.9 4.8 22.3 6.1 3.0 4.3 6.6 3.8 17.2 9.2 22.1 12.5 9.3 5.1 9.6 9.6 20.0 6.6 4.2 0.8 23.1 7.7 52.3 25.5 456.0 62.4 14.8 12.7 12.2 7.1
58.4 10.7 56 0 8.4 3.8 141.7 42.5
0.35 0.78 1.00 0.04 0.03 0.47 0.37 0.85 0.04 0.77 0.15 0.14 0.09 0.12 0.85 0.83 0.57 0.16 0.16 0.57 0.18
Key: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant of frontotemporal dementia; FBI, frontal behavioral inventory; FTD-GRN, frontotemporal patients carrying Granulin Thr272fs mutation; FTLD-modified CDR, clinical dementia rating scale modified for frontotemporal dementia, sum of boxes values; MMSE, Mini Mental State Examination; NPI, neuropsychiatric inventory; UPDRS-III, Unified Parkinson’s Disease rating Scale.
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2.5. Reverse transcription and quantitative real time PCR
3.2. Microarray gene expression profiling
RNA quality and quantity were assessed using NanoDrop (NanoDrop Technologies, Wilmington, DE). Two micrograms of total RNA were used for cDNA synthesis using random hexamers primers (Invitrogen-Life Technologies, CA, USA) and superscript II reverse transcriptase (Invitrogen). Real time quantitative RT-PCR analyses to determinate mRNA levels of selected genes were performed using StepOnePlus Real Time System (Applied Biosystem Foster City, CA, USA). Target gene levels were normalized on the geometric means of 2 housekeeping genes: glyceraldehyde-3phosphate dehydrogenase (GAPDH; Hs99999905_m1) and cytochrome c1 (Cyc1; Hs00357717_m1). All reactions were performed in double. For each gene the mean of mRNA expression levels was calculated using 2DDCT method (Schmittgen and Livak, 2008), normalizing the single values with the mean of control group mRNA levels.
Applying the cut-off scores as reported in the method section, we obtained a gene list of 12 genes (see Table 2). Among them, we considered the most significant fold change value and those genes expressed in the brain, and we selected the 2 best up-regulated genes (HLA-DQA1, SLC14A1), and the best 2 down-regulated (GTF2H2, RAP1GAP). Further, among those genes expressed in the brain, we selected other 2 genes with promising biological significance (GYPA and CA1), as they are involved in neuroinflammation and neurodegeneration processes. Thus, HLA-DQA1, SLC14A1, GYPA, CA1, GTF2H2, RAP1GAP were validated by real time PCR using TaqMan assay (Applied Biosystem).
2.6. Statistical analysis Statistical analysis was performed using SPSS version 17.0 statistic software (SPSS Inc, Chicago, IL). T test and c2 tests were used to test significance, as appropriate, and the results were reported as means SD. ANOVA has been used to analyze the differences of mRNA levels among the 3 groups and multiple comparisons have been adjusted using Sidak post hoc correction. p-values <0.05 were considered statistically significant.
3.3. Real time PCR validation The mean mRNA expression values of selected genes and the comparisons between groups are shown in Table 3. As shown in Fig. 1, a significant decrease in leukocytes mRNA levels of RAP1GAP in GRN-bvFTD phenotype as compared with GRN-avPPA (p ¼ 0.049 by post-hoc analysis with Sidak correction) was confirmed (p ¼ 0.050 by ANOVA with 3 groups: GRN-bvFTD, GRNavPPA, and controls). In FTD GRN-patients, we did not find any difference of RAP1GAP mRNA levels between bvFTD, avPPA (p ¼ 0.99 after post-hoc analysis with Sidak correction). 4. Discussion
3. Results 3.1. Subjects Demographic and clinical characteristics of patients carrying GRN mutation according to clinical diagnosis, that is, bvFTD and avPPA, and age-matched controls are reported in Table 1. bvFTD and avPPA patients subgroup were comparable for demographic and clinical characteristics, but not for age at onset and education (see Table 1). Serum progranulin levels were comparable between the 2 subgroups, markedly reduced as compared with control subjects. Neuropsychological profiles were in keeping with the clinical diagnosis, with comparable cognitive impairments as measured by frontal clinical dementia rating scale (see Table 1 for details).
Predicting the heterogeneity of clinical phenotype in FTLD is an intriguing issue, with still unclear answer. This uncertainty is more striking when we consider the monogenic forms of FTD. Patients with the same GRN mutation and belonging to the same family may be variably present either in bvFTD or avPPA. In the present study we faced this issue, and we hypothesized that regulator genes might drive clinical picture and the related regional brain damage in patients with GRN mutation. We thus investigated for the first time transcriptomic profiles of GRN patients to identify the molecular pathways differentiating bvFTD from avPPA cases. To minimize the high inter-population variability of gene expression, we enrolled only cases, which shared a common genetic background because of the presence of a common founder (Borroni et al., 2011).
Table 2 Gene list of transcripts differentially expressed between bvFTD and avPPA patients carrying GRN mutation Column ID
RefSeq
Gene ID
Brain expression
p
FC
Gene expression assay
8118556 8021081 7911229 8102998 8023466 8016044 7911218 8151592 7898894 7909164 8105970 7913385
NM_002122 NM_001128588 NM_001001957 NM_002099 NM_001012515 NM_000419 NM_015431 NM_001738 NM_020362 NM_001910 NM_001515 NM_001145657
HLA-DQA1 SLC14A1 OR2W3 GYPA FECH ITGA2B TRIM58 CA1 C1orf128 CTSE GTF2H2 RAP1GAP
D D D þ þ þ D þ D D
0,020842 0,010013 0,008209 0,043108 0,011117 0,023293 0,010233 0,021816 0,017807 0,019054 0,025731 0,039176
2,25 1,98 1,95 1,87 1,75 1,72 1,71 1,70 1,69 1,61 L1,65 L1,68
Hs00863321_g1 Hs00998197_m1 Hs00266777_m1
Hs01100176_m1
Hs00230979_m1 Hs00182299_m1
Genes selected for RT-PCR are shown in bold. Key: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; CA1, carbonic anhydrase I; C1orf128, PITHD1 PITH (Cterminal proteasome-interacting domain of thioredoxin-like) domain containing 1; CTSE, cathepsin E; FC, fold change; FECH, ferrochelatase; GTF2H2D, general transcription factor IIH, polypeptide 2D; GTF2H2, general transcription factor IIH, polypeptide 2, 44 kDa; GYPA, glycophorin A (MNS blood group); HLA-DQA1, major histocompatibility complex, class II, DQ alpha 1; ITGA2B, integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41); RAP1GAP, RAP1 GTPase activating protein; SLC14A1, solute carrier family 14 (urea transporter), member 1; TRIM58, tripartite motif containing 58; TSPAN5, tetraspanin 5; OR2W3, olfactory receptor, family 2, subfamily W, member 3.
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211 Table 3 Mean of mRNA expression value for all genes validated by real time PCR in bvFTD and avPPA patients Gene name
SLC14A1 GTF2H2 GYPA RAP1GAP HLA-DQA1 CA1
GRN mutation carriers bvFTD (N ¼ 10)
avPPA (N ¼ 6)
FC
SEM
FC
SEM
4.7 3.4 5.0 3.1 4.1 3.4
1.3 1.0 1.3 1.6 1.1 0.7
2.5 3.4 3.4 5.9 3.3 1.2
0.4 0.7 1.0 3.7 2.6 1.1
p
0.66 1 0.76 0.049 1 0.93
Values are expressed as Fold Change (FC) SEM using 2DDCT method. p value was calculated by ANOVA after Sidak post hoc correction. In bold we highlighted the selected gene names. Key: ANOVA, analysis of variance; avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; FC, fold change; mRNA, messenger RNA; PCR, polymerasse chain reaction; SEM, standard error of the mean.
The microarray analysis and the subsequent real time validation highlighted significant differences in mRNA expression levels of RAP1GAP, increased in avPPA patients as compared with bvFTD. Interestingly, RAP1GAP is highly expressed in cortical neurons in fronto, temporal, and parietal lobes, which are the brain regions mostly affected in FTLD and in GRN-dementia in particular (http://human.brain-map.org/). In the brain, the activation of Rap GTPases cascade is important in neurogenesis, dendrite, and neurite outgrowths (Chen et al., 2005; Hisata et al., 2007; Li et al., 2008) and regulates structural and functional processes associated with synaptic plasticity (McAvoy et al., 2009; Spilker and Kreutz, 2010). Thus, RAP1GAP may be potentially involved in the molecular modulation of GRN-related neurodegeneration. In our model, RAP1GAP should be considered the most promising gene, because its levels are significantly different in bvFTD and avPPA, when considering the control group (see Fig. 1). In this theoretical model, as reported in Fig. 2, GRN mutations lead to FTLD via a haploinsufficiency mechanism, but the clinical expression may be further modified by regulatory genes that are able to differently affect regional cerebral damage, and drive the clinical picture into
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the 2 clinical entities (see Fig. 2). This model might be applied to the other monogenic FTLD, which are characterized by a wide body of presenting symptoms, and the study of modifier genes may be of help for predicting clinical phenotype and likely disease course. Indeed, the next intriguing step will be the identification of the molecular link between RAP1GAP and progranulin. On the other hand, it will be important to understand if molecular modifiers driving clinical presentation might be detected also in not inherited FTLD with a shared neuropathological background. To understand whether RAP1GAP level differences were specific for GRN inherited disorder or depend on regional brain vulnerability independently of the underneath pathologic process, we further investigated patients not carrying GRN mutations. We did not find the same pattern, suggesting that RAP1GAP may drive clinical phenotype in GRN disease only. Indeed, patients without GRN mutations may present different neuropathological hallmarks, thus being a heterogenous group of comparisons. To clarify this point it would be interesting to test our hypothesis in other monogenic forms of TDP-FTD such as C9orf72 expansion or TARDBP mutations or in autopsy-confirmed samples. We acknowledge that caution is needed to address a conclusive remark, as our sample is relatively small and we studied changes in peripheral tissues (Sullivan et al., 2006). Leukocytes have been used as peripheral model in different neurodegenerative and psychiatric diseases, but the transcriptional profile is often different from brain to other tissues. Compared with other works on sporadic neurodegenerative disorders, our investigation have less bias, considering only monogenic patients. We also preferred the inclusion of patients with the same mutation to avoid confounds, and confirmatory analysis on a larger sample and in other GRN mutations would be warranted. Although much remains to be done, our work generates important preliminary findings in elucidating mechanisms underlying the phenotypic variability in FTLD GRN Thr272fs mutation carriers. To date, sporadic FTLD and GRN associated patients are still orphan for any evidence based treatment approach. The haploinsufficency mechanism of GRN-FTD neurodegeneration offers a promising opportunity for intervention. It has been recently found that alkalizing agents were able to rescue haploinsufficiency in cellular models of FTD-GRN (Capell et al., 2011)
Fig. 1. Messenger RNA (mRNA) expression levels of RAP1GAP obtained by microarray gene expression analysis (Panels A) and confirmation real-time PCR (Panels B). In Panels B 0 represents the mean controls value. Bars represent the fold change mean value standard error of the mean (SEM). * p < 0.05. Abbreviations: mRNA, messenger RNA; SEM, standard error of the mean.
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Fig. 2. Schematic panel explaining the GRN-related phenotype variability. Granulin mutations lead to progranulin haploinsufficency, causing in adulthood, frontotemporal dementia (1), characterized by frontal and temporo-parietal involvement RAP1GAP expression augmentation and/or reduction appear to be the molecular shift (2) between agrammatic variant of primary progressive aphasia (avPPA) and behavioral variant frontotemporal dementia (bvFTD) variants, characterized by different and specific clinical and neuroimaging presentation (panels AeB). Panels A, B are obtained by voxel based morphometry comparison analysis between the same GRN-FTLD patients and 12 healthy control subjects, considering age and gender as nuisance variables. The threshold was set at false discovery rate (FDR) p < 0.001 with a voxel threshold ¼ 100, age-gender corrected. Abbreviations: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; GRN-FTLD, granulin-frontotemporal lobar degeneration.
but no disease-modifying therapy is to date available. The molecular pathways underpinning the monogenic GRN-FTLD variability is the first step to decipher this high-complex disorder. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgement The authors wish to acknowledge the helpful and generous collaboration of the patients and their family members, which was essential for this study. References Beck, J., Rohrer, J.D., Campbell, T., Isaacs, A., Morrison, K.E., Goodall, E.F., Warrington, E.K., Stevens, J., Revesz, T., Holton, J., Al-Sarraj, S., King, A., Scahill, R., Warren, J.D., Fox, N.C., Rossor, M.N., Collinge, J., Mead, S., 2008. A distinct clinical, neuropsychological and radiological phenotype is associated with progranulin gene mutations in a large UK series. Brain 131, 706e720. Borroni, B., Archetti, S., Alberici, A., Agosti, C., Gennarelli, M., Bigni, B., Bonvicini, C., Ferrari, M., Bellelli, G., Galimberti, D., Scarpini, E., Di, L.D., Caimi, L., Caltagirone, C., Di, L.M., Padovani, A., 2008. Progranulin genetic variations in frontotemporal lobar degeneration: evidence for low mutation frequency in an Italian clinical series. Neurogenetics 9, 197e205. Borroni, B., Grassi, M., Agosti, C., Premi, E., Archetti, S., Alberici, A., Bellelli, G., Caimi, L., Di, L.M., Padovani, A., 2010. Establishing short-term prognosis in frontotemporal lobar degeneration spectrum: role of genetic background and clinical phenotype. Neurobiol. Aging 31, 270e279. Borroni, B., Bonvicini, C., Galimberti, D., Tremolizzo, L., Papetti, A., Archetti, S., Turla, M., Alberici, A., Agosti, C., Premi, E., Appollonio, I., Rainero, I., Ferrarese, C.,
Gennarelli, M., Scarpini, E., Padovani, A., 2011. Founder effect and estimation of the age of the progranulin Thr272fs mutation in 14 Italian pedigrees with frontotemporal lobar degeneration. Neurobiol. Aging 32, 555e558. Bruni, A.C., Momeni, P., Bernardi, L., Tomaino, C., Frangipane, F., Elder, J., Kawarai, T., Sato, C., Pradella, S., Wakutani, Y., Anfossi, M., Gallo, M., Geracitano, S., Costanzo, A., Smirne, N., Curcio, S.A., Mirabelli, M., Puccio, G., Colao, R., Maletta, R.G., Kertesz, A., St George-Hyslop, P., Hardy, J., Rogaeva, E., 2007. Heterogeneity within a large kindred with frontotemporal dementia: a novel progranulin mutation. Neurology 69, 140e147. Capell, A., Liebscher, S., Fellerer, K., Brouwers, N., Willem, M., Lammich, S., Gijselinck, I., Bittner, T., Carlson, A.M., Sasse, F., Kunze, B., Steinmetz, H., Jansen, R., Dormann, D., Sleegers, K., Cruts, M., Herms, J., Van Broeckhoven, C., Haass, C., 2011. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J. Neurosci. 31, 1885e1894. Chen, Y., Wang, P.Y., Ghosh, A., 2005. Regulation of cortical dendrite development by Rap1 signaling. Mol. Cell Neurosci. 28, 215e228. Chen-Plotkin, A.S., Martinez-Lage, M., Sleiman, P.M., Hu, W., Greene, R., Wood, E.M., Bing, S., Grossman, M., Schellenberg, G.D., Hatanpaa, K.J., Weiner, M.F., White, C.L., Brooks, W.S., Halliday, G.M., Kril, J.J., Gearing, M., Beach, T.G., GraffRadford, N.R., Dickson, D.W., Rademakers, R., Boeve, B.F., Pickering-Brown, S.M., Snowden, J., van Swieten, J.C., Heutink, P., Seelaar, H., Murrell, J.R., Ghetti, B., Spina, S., Grafman, J., Kaye, J.A., Woltjer, R.L., Mesulam, M., Bigio, E., Lladó, A., Miller, B.L., Alzualde, A., Moreno, F., Rohrer, J.D., Mackenzie, I.R., Feldman, H.H., Hamilton, R.L., Cruts, M., Engelborghs, S., De Deyn, P.P., Van Broeckhoven, C., Bird, T.D., Cairns, N.J., Goate, A., Frosch, M.P., Riederer, P.F., Bogdanovic, N., Lee, V.M., Trojanowski, J.Q., Van Deerlin, V.M., 2011. Genetic and clinical features of progranulin-associated frontotemporal lobar degeneration. Arch. Neurol. 68, 488e497. Cruts, M., Gijselinck, I., van der Zee, J., Engelborghs, S., Wils, H., Pirici, D., Rademakers, R., Vandenberghe, R., Dermaut, B., Martin, J.J., van Duijn, C., Peeters, K., Sciot, R., Santens, P., De Pooter, T., Mattheijssens, M., Van den Broeck, M., Cuijt, I., Vennekens, K., De Deyn, P.P., Kumar-Singh, S., Van Broeckhoven, C., 2006. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 442, 920e924. Gorno-Tempini, M.L., Hillis, A.E., Weintraub, S., Gorno-Tempini, M.L., Hillis, A.E., Weintraub, S., Kertesz, A., Mendez, M., Cappa, S.F., Ogar, J.M., Rohrer, J.D.,
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211 Black, S., Boeve, B.F., Manes, F., Dronkers, N.F., Vandenberghe, R., Rascovsky, K., Patterson, K., Miller, B.L., Knopman, D.S., Hodges, J.R., Mesulam, M.M., Grossman, M., 2011. Classification of primary progressive aphasia and its variants. Neurology 76, 1006e1014. Hisata, S., Sakisaka, T., Baba, T., Yamada, T., Aoki, K., Matsuda, M., Takai, Y., 2007. Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth. J. Cell Biol. 178, 843e860. Kelley, B.J., Haidar, W., Boeve, B.F., Baker, M., Graff-Radford, N.R., Krefft, T., Frank, A.R., Jack Jr., C.R., Shiung, M., Knopman, D.S., Josephs, K.A., Parashos, S.A., Rademakers, R., Hutton, M., Pickering-Brown, S., Adamson, J., Kuntz, K.M., Dickson, D.W., Parisi, J.E., Smith, G.E., Ivnik, R.J., Petersen, R.C., 2009. Prominent phenotypic variability associated with mutations in progranulin. Neurobiol. Aging 30, 739e751. Larner, A.J., 2012. Intrafamilial clinical phenotypic heterogeneity with progranulin gene p.Glu498fs mutation. J. Neurol. Sci. 316, 189e190. Le Ber, I., Camuzat, A., Hannequin, D., Pasquier, F., Guedj, E., Rovelet-Lecrux, A., HahnBarma, V., van der Zee, J., Clot, F., Bakchine, S., Puel, M., Ghanim, M., Lacomblez, L., Mikol, J., Deramecourt, V., Lejeune, P., de la Sayette, V., Belliard, S., Vercelletto, M., Meyrignac, C., Van, B.C., Lambert, J.C., Verpillat, P., Campion, D., Habert, M.O., Dubois, B., Brice, A., 2008. Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study. Brain 131, 732e746. Li, Y.H., Werner, H., Puschel, A.W., 2008. Rheb and mTOR regulate neuronal polarity through Rap1B. J. Biol. Chem. 283, 33784e33792. Mackenzie, I.R., Neumann, M., Bigio, E.H., Cairns, N.J., Alafuzoff, I., Kril, J., Kovacs, G.G., Ghetti, B., Halliday, G., Holm, I.E., Ince, P.G., Kamphorst, W., Revesz, T., Rozemuller, A.J., Kumar-Singh, S., Akiyama, H., Baborie, A., Spina, S., Dickson, D.W., Trojanowski, J.Q., Mann, D.M., 2009. Nomenclature for neuropathologic subtypes of frontotemporal lobar degeneration: consensus recommendations. Acta Neuropathol. 117, 15e18. Mackenzie, I.R., Neumann, M., Baborie, A., Sampathu, D.M., Du, P.D., Jaros, E., Perry, R.H., Trojanowski, J.Q., Mann, D.M., Lee, V.M., 2011. A harmonized classification system for FTLD-TDP pathology. Acta Neuropathol. 122, 111e113. McAvoy, T., Zhou, M.M., Greengard, P., Nairn, A.C., 2009. Phosphorylation of Rap1GAP, a striatally enriched protein, by protein kinase A controls Rap1 activity and dendritic spine morphology. Proc. Natl. Acad. Sci. U.S.A 106, 3531e3536. Moreno, F., Indakoetxea, B., Barandiaran, M., Alzualde, A., Gabilondo, A., Estanga, A., Ruiz, J., Ruibal, M., Bergareche, A., Martí-Massó, J.F., López de Munain, A., 2009. “Frontotemporoparietal” dementia: clinical phenotype associated with the c.709-1G>A PGRN mutation. Neurology 73, 1367e1374. Pickering-Brown, S.M., Rollinson, S., Du, P.D., Morrison, K.E., Varma, A., Richardson, A.M., Neary, D., Snowden, J.S., Mann, D.M., 2008. Frequency and clinical characteristics of progranulin mutation carriers in the Manchester
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frontotemporal lobar degeneration cohort: comparison with patients with MAPT and no known mutations. Brain 131, 721e731. Rademakers, R., Baker, M., Gass, J., Adamson, J., Huey, E.D., Momeni, P., Spina, S., Coppola, G., Karydas, A.M., Stewart, H., Johnson, N., Hsiung, G.Y., Kelley, B., Kuntz, K., Steinbart, E., Wood, E.M., Yu, C.E., Josephs, K., Sorenson, E., Womack, K.B., Weintraub, S., Pickering-Brown, S.M., Schofield, P.R., Brooks, W.S., Van Deerlin, V.M., Snowden, J., Clark, C.M., Kertesz, A., Boylan, K., Ghetti, B., Neary, D., Schellenberg, G.D., Beach, T.G., Mesulam, M., Mann, D., Grafman, J., Mackenzie, I.R., Feldman, H., Bird, T., Petersen, R., Knopman, D., Boeve, B., Geschwind, D.H., Miller, B., Wszolek, Z., Lippa, C., Bigio, E.H., Dickson, D., GraffRadford, N., Hutton, M., 2007. Phenotypic variability associated with progranulin haploinsufficiency in patients with the common 1477C–>T (Arg493X) mutation: an international initiative. Lancet Neurol. 6, 857e868. Rascovsky, K., Hodges, J.R., Knopman, D., Mendez, M.F., Kramer, J.H., Neuhaus, J., van Swieten, J.C., Seelaar, H., Dopper, E.G., Onyike, C.U., Hillis, A.E., Josephs, K.A., Boeve, B.F., Kertesz, A., Seeley, W.W., Rankin, K.P., Johnson, J.K., GornoTempini, M.L., Rosen, H., Prioleau-Latham, C.E., Lee, A., Kipps, C.M., Lillo, P., Piguet, O., Rohrer, J.D., Rossor, M.N., Warren, J.D., Fox, N.C., Galasko, D., Salmon, D.P., Black, S.E., Mesulam, M., Weintraub, S., Dickerson, B.C., DiehlSchmid, J., Pasquier, F., Deramecourt, V., Lebert, F., Pijnenburg, Y., Chow, T.W., Manes, F., Grafman, J., Cappa, S.F., Freedman, M., Grossman, M., Miller, B.L., 2011. Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 134, 2456e2477. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101e1108. Spilker, C., Kreutz, M.R., 2010. RapGAPs in brain: multipurpose players in neuronal Rap signalling. Eur. J. Neurosci. 32, 1e9. Sullivan, P.F., Fan, C., Perou, C.M., 2006. Evaluating the comparability of gene expression in blood and brain. Am. J. Med. Genet. B Neuropsychiatr. Genet. 141B, 261e268. Toh, H., Chitramuthu, B.P., Bennett, H.P., Bateman, A., 2011. Structure, function, and mechanism of progranulin; the brain and beyond. J. Mol. Neurosci. 45, 538e548. van Swieten, J.C., Heutink, P., 2008. Mutations in progranulin (GRN) within the spectrum of clinical and pathological phenotypes of frontotemporal dementia. Lancet Neurol. 7, 965e974. Yu, C.E., Bird, T.D., Bekris, L.M., Montine, T.J., Leverenz, J.B., Steinbart, E., Galloway, N.M., Feldman, H., Woltjer, R., Miller, C.A., Wood, E.M., Grossman, M., McCluskey, L., Clark, C.M., Neumann, M., Danek, A., Galasko, D.R., Arnold, S.E., Chen-Plotkin, A., Karydas, A., Miller, B.L., Trojanowski, J.Q., Lee, V.M., Schellenberg, G.D., Van Deerlin, V.M., 2010. The spectrum of mutations in progranulin: a collaborative study screening 545 cases of neurodegeneration. Arch. Neurol. 67, 161e170.
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Understanding phenotype variability in frontotemporal lobar degeneration due to granulin mutation Cristian Bonvicini a, Elena Milanesi a, Andrea Pilotto b, Nadia Cattane c, Enrico Premi b, Silvana Archetti d, Alessandro Padovani b, Massimo Gennarelli a, c, Barbara Borroni b, * a
Genetic Unit, IRCCS S.Giovanni di Dio, Fatebenefratelli, Brescia, Italy Centre for Neurodegenerative Disorders, Neurology Unit, University of Brescia, Brescia, Italy Departement of Molecular and Translational Medicine, University of Brescia, Brescia, Italy d Department of Laboratories, III Laboratory of Analysis, Brescia Hospital, Brescia, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 April 2013 Received in revised form 5 October 2013 Accepted 28 October 2013 Available online 1 November 2013
Phenotype in patients with granulin (GRN) mutations is unpredictable, ranging from behavioral variant frontotemporal dementia (bvFTD) to agrammatic variant of primary progressive aphasia (avPPA). To date the wide clinical variability of FTLD-GRN remains unexplained. The aim of the study was to identify genetic pathways differentiating phenotypic expression in patients carrying GRN mutations. Patients carrying the same GRNT272SfsX10 mutation were enrolled, a careful clinical assessment was carried out, and the diagnosis of either bvFTD (n ¼ 10, age ¼ 63.9 9.4) or avPPA (n ¼ 6, age ¼ 58.8 4.7) was done. Microarray gene expression analysis on leukocytes was performed. Genes differentially expressed between the groups were validated by real time polymerase chain reaction considering an age-matched healthy controls group (n ¼ 16, age ¼ 58.4 10.7). We further considered a group of FTD with no GRN mutations (GRN-) (n ¼ 21, 13 bvFTD, and 8 avPPA) for comparisons. Real-time polymerase chain reaction (PCR) confirmed a significant decrease in leukocytes mRNA messenger RNA (mRNA) levels of RAP1GAP in bvFTD patients as compared with avPPA (p ¼ 0.049). This finding was specific for patients with GRN mutations, as we did not observe this pattern in FTD GRN-patients (p ¼ 0.99). The alteration of RAP1GAP mRNA levels may explain the clinical variability of GRN-FTLD patients. This is the first report linking a molecular pathway to specific phenotype expression in FTLD-GRN. To understand the clinical relevance of our early results it will be mandatory to extend the observation to other clinical and neuropathological series. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Frontotemporal dementia GRN mutations Phenotype variability bvFTD avPPA Genetic modulation
1. Introduction Frontotemporal lobar degeneration (FTLD) is a clinically, pathologically, and genetically heterogenous disorder. Three prototypical variants have been described, namely the behavioral variant frontotemporal dementia (bvFTD), the semantic variant of primary progressive aphasia, and the agrammatic variant of primary progressive aphasia (avPPA) (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). In most cases, at autopsy either FTLD with tau-positive inclusions or FTLD with TDP-43-positive aggregates may be variably found over the clinical spectrum of FTLD cases (Mackenzie et al., 2009, 2011). Despite the consistent progress in carefully characterizing the clinical phenotypes and in defining neuroimaging correlates, * Corresponding author at: Neurology Unit, University of Brescia, Piazza Spedali Civili 1, Brescia 25125, Italy. Tel.: þ39 0303995632; fax: þ39 0303995027. E-mail address: [email protected] (B. Borroni). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2013.10.097
there is no unique correspondence with neuropathological substrates. The mismatch between neuropathological and clinical features is still evident when monogenic FTLD is considered. Granulin (GRN) gene has been identified as a major cause of autosomal dominant FTLD, leading to TDP-43 inclusions by a haploinsufficiency mechanism (Cruts et al., 2006; Toh et al., 2011). FTLD patients carrying GRN mutations clinically present unpredictable phenotypic variability even within families carrying the same mutation (Beck et al., 2008; Bruni et al., 2007; Chen-Plotkin et al., 2011; Kelley et al., 2009; Larner, 2012; Le Ber 2008; Li et al., 2008; Moreno et al., 2009; Pickering-Brown et al., 2008; Rademakers et al., 2007; van Swieten and Heutink, 2008; Yu et al., 2010), and bvFTD and avPPA represent the most frequent pictures. Aim of the present study was to dissect clinical heterogeneity in patients carrying the same GRN mutation. To this, a coalescent cohort of patients with GRN Thr272fs mutation was considered, and whole gene expression analysis on leukocytes was carried
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211
out to compare the expression profiles in the different clinical phenotypes (bvFTD vs. avPPA).
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for each procedure. The work was conformed to the Helsinki Declaration and approved by local ethics committee. 2.2. Granulin sequencing
2. Methods 2.1. Subjects Two hundred fifty FTLD patients, recruited from the Centre for Ageing Brain and Neurodegenerative Disorders, at University of Brescia (Brescia, Italy), were screened for pathogenic GRN and MAPT mutations and C9orf72 expansion; those carrying GRN Thr272fs mutation were enrolled in the present study. In these patients, diagnosis of either bvFTD or avPPA was made according to the current clinical criteria (Gorno-Tempini et al., 2011; Rascovsky et al., 2011). All FTLD patients carrying GRN Thr272fs mutation were part of a genetically homogenous population, as these patients harbor a common ancestor as previously reported (Borroni et al., 2011), making our experimental design well controlled. All patients were subjected to an extensive neurologic and neuropsychological evaluation, as previously described (Borroni et al., 2010), a routine laboratory examination, and a conventional brain magnetic resonance imaging before entering this study. We further enrolled a group of 21 FTD patients without GRN mutations, matched for age and gender, for comparisons and we sub-grouped them according to clinical phenotype, that is, 8 avPPA (age 69.4 9.6) and 13 bvFTD (age 67.1 6.2). A group of healthy controls, matched for age and gender was also considered (see Table 1). FTD patients, with or without GRN mutations, and healthy controls did not report significant medical problems. None was under treatment for cognitive disturbances, but 7 (3 FTD GRN and 4 GRN-) were under psychotropic drugs for behavioral disturbance control. Each subject underwent venous blood sampling for biological analyses. Written informed consent (from the subject or from the responsible guardian if the subject was incapable) was obtained,
Genomic DNA was extracted from peripheral blood using a standard procedure. All the 12 exons plus exon 0 of GRN, and at least 30 base pairs of their flanking introns were evaluated by polymerase chain reaction (PCR) and subsequent sequencing. GRN Thr272fs (g.1977_1980 delCACT) was tested as previously described (Borroni et al., 2008). 2.3. RNA isolation and microarray gene expression procedures Blood samples were obtained by venipuncture using PaxGene Tubes (Qiagen, Manchester, UK). RNA isolation was performed by PaxGene Blood RNA Kit (Qiagen) according to the manufacturer protocols. RNA quality and integrity were assessed using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Total RNA (250 ng) from each sample were reverse transcribed to complementary DNA (cDNA), followed by overnight in vitro transcription to generate complementary RNA, which was reverse transcribed, and the 7.5 mg of sense cDNA were fragmented and labeled. The quality of cDNA and fragmented cDNA was assessed using Agilent bioanalyzer (Agilent Technology, TX, USA). Hybridization was performed on human gene 1.1 ST array strips (Affymetrix, Inc, Santa Clara, CA). The reactions of hybridation, fluidics, and imaging were performed on the Affymetrix Gene Atlas instrument according to the manufactured protocol. 2.4. Microarray data analysis Gene expression data were imported from the Gene Atlas instrument into Partek Genomics Suite 6.0 (Partek, St Louis, Mo) software as CEL files using default parameters. Principalcomponent analysis was performed to identify outliers. Differential expression analysis between bvFTD and avPPA phenotypes was performed by the analysis of variance (ANOVA) and a gene list was created using a cutoff of p < 0.05 and 1.6-fold change.
Table 1 Demographic and clinical characteristics of FTLD-GRN patients according to clinical diagnosis Variable
FTD-GRN all (N ¼ 16)
bvFTD-GRN (N ¼ 10)
avPPA-GRN (N ¼ 6)
Controls (N ¼ 16)
p (bvFTD vs. avPPA)
Age at evaluation, years Gender, female% Family history, % Age at onset, years Education, years Serum PGRN, pg/ml FTLD-modified CDR MMSE UPDRS-III Short story Raven matrices Rey figure, copy Rey figure, recall Phonological fluency Semantic fluency Digit span, backward Token test Trail making test, A Trail making test, B NPI, total score FBI, total score
64.2 8.5 62.5 100 59.3 8.6 6.8 2.2 48.2 21.4 7.3 5.6 22.5 5.4 10.0 16.7 8.2 5.5 15.9 7.2 18.5 10.1 6.4 4.8 14.0 10.5 20.3 5.4 4.3 0.6 24.5 5.8 71.0 25.6 380.7 162.0 18.5 13.0 18.0 11.1
65.8 9.9 60 100 61.4 9.3 6.1 2.3 50.6 23.6 8.4 6.3 22.6 4.5 14.2 20.1 8.7 6.2 14.2 4.5 16.0 8.2 4.0 3.2 17.3 10.5 20.6 4.9 4.3 0.5 25.2 4.9 85.0 16.4 350 166.7 20.8 13.3 21.8 12.1
61.5 4.9 66 100 55.8 6.4 8.0 1.7 43.4 18.5 5.9 4.8 22.3 6.1 3.0 4.3 6.6 3.8 17.2 9.2 22.1 12.5 9.3 5.1 9.6 9.6 20.0 6.6 4.2 0.8 23.1 7.7 52.3 25.5 456.0 62.4 14.8 12.7 12.2 7.1
58.4 10.7 56 0 8.4 3.8 141.7 42.5
0.35 0.78 1.00 0.04 0.03 0.47 0.37 0.85 0.04 0.77 0.15 0.14 0.09 0.12 0.85 0.83 0.57 0.16 0.16 0.57 0.18
Key: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant of frontotemporal dementia; FBI, frontal behavioral inventory; FTD-GRN, frontotemporal patients carrying Granulin Thr272fs mutation; FTLD-modified CDR, clinical dementia rating scale modified for frontotemporal dementia, sum of boxes values; MMSE, Mini Mental State Examination; NPI, neuropsychiatric inventory; UPDRS-III, Unified Parkinson’s Disease rating Scale.
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2.5. Reverse transcription and quantitative real time PCR
3.2. Microarray gene expression profiling
RNA quality and quantity were assessed using NanoDrop (NanoDrop Technologies, Wilmington, DE). Two micrograms of total RNA were used for cDNA synthesis using random hexamers primers (Invitrogen-Life Technologies, CA, USA) and superscript II reverse transcriptase (Invitrogen). Real time quantitative RT-PCR analyses to determinate mRNA levels of selected genes were performed using StepOnePlus Real Time System (Applied Biosystem Foster City, CA, USA). Target gene levels were normalized on the geometric means of 2 housekeeping genes: glyceraldehyde-3phosphate dehydrogenase (GAPDH; Hs99999905_m1) and cytochrome c1 (Cyc1; Hs00357717_m1). All reactions were performed in double. For each gene the mean of mRNA expression levels was calculated using 2DDCT method (Schmittgen and Livak, 2008), normalizing the single values with the mean of control group mRNA levels.
Applying the cut-off scores as reported in the method section, we obtained a gene list of 12 genes (see Table 2). Among them, we considered the most significant fold change value and those genes expressed in the brain, and we selected the 2 best up-regulated genes (HLA-DQA1, SLC14A1), and the best 2 down-regulated (GTF2H2, RAP1GAP). Further, among those genes expressed in the brain, we selected other 2 genes with promising biological significance (GYPA and CA1), as they are involved in neuroinflammation and neurodegeneration processes. Thus, HLA-DQA1, SLC14A1, GYPA, CA1, GTF2H2, RAP1GAP were validated by real time PCR using TaqMan assay (Applied Biosystem).
2.6. Statistical analysis Statistical analysis was performed using SPSS version 17.0 statistic software (SPSS Inc, Chicago, IL). T test and c2 tests were used to test significance, as appropriate, and the results were reported as means SD. ANOVA has been used to analyze the differences of mRNA levels among the 3 groups and multiple comparisons have been adjusted using Sidak post hoc correction. p-values <0.05 were considered statistically significant.
3.3. Real time PCR validation The mean mRNA expression values of selected genes and the comparisons between groups are shown in Table 3. As shown in Fig. 1, a significant decrease in leukocytes mRNA levels of RAP1GAP in GRN-bvFTD phenotype as compared with GRN-avPPA (p ¼ 0.049 by post-hoc analysis with Sidak correction) was confirmed (p ¼ 0.050 by ANOVA with 3 groups: GRN-bvFTD, GRNavPPA, and controls). In FTD GRN-patients, we did not find any difference of RAP1GAP mRNA levels between bvFTD, avPPA (p ¼ 0.99 after post-hoc analysis with Sidak correction). 4. Discussion
3. Results 3.1. Subjects Demographic and clinical characteristics of patients carrying GRN mutation according to clinical diagnosis, that is, bvFTD and avPPA, and age-matched controls are reported in Table 1. bvFTD and avPPA patients subgroup were comparable for demographic and clinical characteristics, but not for age at onset and education (see Table 1). Serum progranulin levels were comparable between the 2 subgroups, markedly reduced as compared with control subjects. Neuropsychological profiles were in keeping with the clinical diagnosis, with comparable cognitive impairments as measured by frontal clinical dementia rating scale (see Table 1 for details).
Predicting the heterogeneity of clinical phenotype in FTLD is an intriguing issue, with still unclear answer. This uncertainty is more striking when we consider the monogenic forms of FTD. Patients with the same GRN mutation and belonging to the same family may be variably present either in bvFTD or avPPA. In the present study we faced this issue, and we hypothesized that regulator genes might drive clinical picture and the related regional brain damage in patients with GRN mutation. We thus investigated for the first time transcriptomic profiles of GRN patients to identify the molecular pathways differentiating bvFTD from avPPA cases. To minimize the high inter-population variability of gene expression, we enrolled only cases, which shared a common genetic background because of the presence of a common founder (Borroni et al., 2011).
Table 2 Gene list of transcripts differentially expressed between bvFTD and avPPA patients carrying GRN mutation Column ID
RefSeq
Gene ID
Brain expression
p
FC
Gene expression assay
8118556 8021081 7911229 8102998 8023466 8016044 7911218 8151592 7898894 7909164 8105970 7913385
NM_002122 NM_001128588 NM_001001957 NM_002099 NM_001012515 NM_000419 NM_015431 NM_001738 NM_020362 NM_001910 NM_001515 NM_001145657
HLA-DQA1 SLC14A1 OR2W3 GYPA FECH ITGA2B TRIM58 CA1 C1orf128 CTSE GTF2H2 RAP1GAP
D D D þ þ þ D þ D D
0,020842 0,010013 0,008209 0,043108 0,011117 0,023293 0,010233 0,021816 0,017807 0,019054 0,025731 0,039176
2,25 1,98 1,95 1,87 1,75 1,72 1,71 1,70 1,69 1,61 L1,65 L1,68
Hs00863321_g1 Hs00998197_m1 Hs00266777_m1
Hs01100176_m1
Hs00230979_m1 Hs00182299_m1
Genes selected for RT-PCR are shown in bold. Key: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; CA1, carbonic anhydrase I; C1orf128, PITHD1 PITH (Cterminal proteasome-interacting domain of thioredoxin-like) domain containing 1; CTSE, cathepsin E; FC, fold change; FECH, ferrochelatase; GTF2H2D, general transcription factor IIH, polypeptide 2D; GTF2H2, general transcription factor IIH, polypeptide 2, 44 kDa; GYPA, glycophorin A (MNS blood group); HLA-DQA1, major histocompatibility complex, class II, DQ alpha 1; ITGA2B, integrin, alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41); RAP1GAP, RAP1 GTPase activating protein; SLC14A1, solute carrier family 14 (urea transporter), member 1; TRIM58, tripartite motif containing 58; TSPAN5, tetraspanin 5; OR2W3, olfactory receptor, family 2, subfamily W, member 3.
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211 Table 3 Mean of mRNA expression value for all genes validated by real time PCR in bvFTD and avPPA patients Gene name
SLC14A1 GTF2H2 GYPA RAP1GAP HLA-DQA1 CA1
GRN mutation carriers bvFTD (N ¼ 10)
avPPA (N ¼ 6)
FC
SEM
FC
SEM
4.7 3.4 5.0 3.1 4.1 3.4
1.3 1.0 1.3 1.6 1.1 0.7
2.5 3.4 3.4 5.9 3.3 1.2
0.4 0.7 1.0 3.7 2.6 1.1
p
0.66 1 0.76 0.049 1 0.93
Values are expressed as Fold Change (FC) SEM using 2DDCT method. p value was calculated by ANOVA after Sidak post hoc correction. In bold we highlighted the selected gene names. Key: ANOVA, analysis of variance; avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; FC, fold change; mRNA, messenger RNA; PCR, polymerasse chain reaction; SEM, standard error of the mean.
The microarray analysis and the subsequent real time validation highlighted significant differences in mRNA expression levels of RAP1GAP, increased in avPPA patients as compared with bvFTD. Interestingly, RAP1GAP is highly expressed in cortical neurons in fronto, temporal, and parietal lobes, which are the brain regions mostly affected in FTLD and in GRN-dementia in particular (http://human.brain-map.org/). In the brain, the activation of Rap GTPases cascade is important in neurogenesis, dendrite, and neurite outgrowths (Chen et al., 2005; Hisata et al., 2007; Li et al., 2008) and regulates structural and functional processes associated with synaptic plasticity (McAvoy et al., 2009; Spilker and Kreutz, 2010). Thus, RAP1GAP may be potentially involved in the molecular modulation of GRN-related neurodegeneration. In our model, RAP1GAP should be considered the most promising gene, because its levels are significantly different in bvFTD and avPPA, when considering the control group (see Fig. 1). In this theoretical model, as reported in Fig. 2, GRN mutations lead to FTLD via a haploinsufficiency mechanism, but the clinical expression may be further modified by regulatory genes that are able to differently affect regional cerebral damage, and drive the clinical picture into
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the 2 clinical entities (see Fig. 2). This model might be applied to the other monogenic FTLD, which are characterized by a wide body of presenting symptoms, and the study of modifier genes may be of help for predicting clinical phenotype and likely disease course. Indeed, the next intriguing step will be the identification of the molecular link between RAP1GAP and progranulin. On the other hand, it will be important to understand if molecular modifiers driving clinical presentation might be detected also in not inherited FTLD with a shared neuropathological background. To understand whether RAP1GAP level differences were specific for GRN inherited disorder or depend on regional brain vulnerability independently of the underneath pathologic process, we further investigated patients not carrying GRN mutations. We did not find the same pattern, suggesting that RAP1GAP may drive clinical phenotype in GRN disease only. Indeed, patients without GRN mutations may present different neuropathological hallmarks, thus being a heterogenous group of comparisons. To clarify this point it would be interesting to test our hypothesis in other monogenic forms of TDP-FTD such as C9orf72 expansion or TARDBP mutations or in autopsy-confirmed samples. We acknowledge that caution is needed to address a conclusive remark, as our sample is relatively small and we studied changes in peripheral tissues (Sullivan et al., 2006). Leukocytes have been used as peripheral model in different neurodegenerative and psychiatric diseases, but the transcriptional profile is often different from brain to other tissues. Compared with other works on sporadic neurodegenerative disorders, our investigation have less bias, considering only monogenic patients. We also preferred the inclusion of patients with the same mutation to avoid confounds, and confirmatory analysis on a larger sample and in other GRN mutations would be warranted. Although much remains to be done, our work generates important preliminary findings in elucidating mechanisms underlying the phenotypic variability in FTLD GRN Thr272fs mutation carriers. To date, sporadic FTLD and GRN associated patients are still orphan for any evidence based treatment approach. The haploinsufficency mechanism of GRN-FTD neurodegeneration offers a promising opportunity for intervention. It has been recently found that alkalizing agents were able to rescue haploinsufficiency in cellular models of FTD-GRN (Capell et al., 2011)
Fig. 1. Messenger RNA (mRNA) expression levels of RAP1GAP obtained by microarray gene expression analysis (Panels A) and confirmation real-time PCR (Panels B). In Panels B 0 represents the mean controls value. Bars represent the fold change mean value standard error of the mean (SEM). * p < 0.05. Abbreviations: mRNA, messenger RNA; SEM, standard error of the mean.
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C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211
Fig. 2. Schematic panel explaining the GRN-related phenotype variability. Granulin mutations lead to progranulin haploinsufficency, causing in adulthood, frontotemporal dementia (1), characterized by frontal and temporo-parietal involvement RAP1GAP expression augmentation and/or reduction appear to be the molecular shift (2) between agrammatic variant of primary progressive aphasia (avPPA) and behavioral variant frontotemporal dementia (bvFTD) variants, characterized by different and specific clinical and neuroimaging presentation (panels AeB). Panels A, B are obtained by voxel based morphometry comparison analysis between the same GRN-FTLD patients and 12 healthy control subjects, considering age and gender as nuisance variables. The threshold was set at false discovery rate (FDR) p < 0.001 with a voxel threshold ¼ 100, age-gender corrected. Abbreviations: avPPA, agrammatic variant of primary progressive aphasia; bvFTD, behavioral variant frontotemporal dementia; GRN-FTLD, granulin-frontotemporal lobar degeneration.
but no disease-modifying therapy is to date available. The molecular pathways underpinning the monogenic GRN-FTLD variability is the first step to decipher this high-complex disorder. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgement The authors wish to acknowledge the helpful and generous collaboration of the patients and their family members, which was essential for this study. References Beck, J., Rohrer, J.D., Campbell, T., Isaacs, A., Morrison, K.E., Goodall, E.F., Warrington, E.K., Stevens, J., Revesz, T., Holton, J., Al-Sarraj, S., King, A., Scahill, R., Warren, J.D., Fox, N.C., Rossor, M.N., Collinge, J., Mead, S., 2008. A distinct clinical, neuropsychological and radiological phenotype is associated with progranulin gene mutations in a large UK series. Brain 131, 706e720. Borroni, B., Archetti, S., Alberici, A., Agosti, C., Gennarelli, M., Bigni, B., Bonvicini, C., Ferrari, M., Bellelli, G., Galimberti, D., Scarpini, E., Di, L.D., Caimi, L., Caltagirone, C., Di, L.M., Padovani, A., 2008. Progranulin genetic variations in frontotemporal lobar degeneration: evidence for low mutation frequency in an Italian clinical series. Neurogenetics 9, 197e205. Borroni, B., Grassi, M., Agosti, C., Premi, E., Archetti, S., Alberici, A., Bellelli, G., Caimi, L., Di, L.M., Padovani, A., 2010. Establishing short-term prognosis in frontotemporal lobar degeneration spectrum: role of genetic background and clinical phenotype. Neurobiol. Aging 31, 270e279. Borroni, B., Bonvicini, C., Galimberti, D., Tremolizzo, L., Papetti, A., Archetti, S., Turla, M., Alberici, A., Agosti, C., Premi, E., Appollonio, I., Rainero, I., Ferrarese, C.,
Gennarelli, M., Scarpini, E., Padovani, A., 2011. Founder effect and estimation of the age of the progranulin Thr272fs mutation in 14 Italian pedigrees with frontotemporal lobar degeneration. Neurobiol. Aging 32, 555e558. Bruni, A.C., Momeni, P., Bernardi, L., Tomaino, C., Frangipane, F., Elder, J., Kawarai, T., Sato, C., Pradella, S., Wakutani, Y., Anfossi, M., Gallo, M., Geracitano, S., Costanzo, A., Smirne, N., Curcio, S.A., Mirabelli, M., Puccio, G., Colao, R., Maletta, R.G., Kertesz, A., St George-Hyslop, P., Hardy, J., Rogaeva, E., 2007. Heterogeneity within a large kindred with frontotemporal dementia: a novel progranulin mutation. Neurology 69, 140e147. Capell, A., Liebscher, S., Fellerer, K., Brouwers, N., Willem, M., Lammich, S., Gijselinck, I., Bittner, T., Carlson, A.M., Sasse, F., Kunze, B., Steinmetz, H., Jansen, R., Dormann, D., Sleegers, K., Cruts, M., Herms, J., Van Broeckhoven, C., Haass, C., 2011. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J. Neurosci. 31, 1885e1894. Chen, Y., Wang, P.Y., Ghosh, A., 2005. Regulation of cortical dendrite development by Rap1 signaling. Mol. Cell Neurosci. 28, 215e228. Chen-Plotkin, A.S., Martinez-Lage, M., Sleiman, P.M., Hu, W., Greene, R., Wood, E.M., Bing, S., Grossman, M., Schellenberg, G.D., Hatanpaa, K.J., Weiner, M.F., White, C.L., Brooks, W.S., Halliday, G.M., Kril, J.J., Gearing, M., Beach, T.G., GraffRadford, N.R., Dickson, D.W., Rademakers, R., Boeve, B.F., Pickering-Brown, S.M., Snowden, J., van Swieten, J.C., Heutink, P., Seelaar, H., Murrell, J.R., Ghetti, B., Spina, S., Grafman, J., Kaye, J.A., Woltjer, R.L., Mesulam, M., Bigio, E., Lladó, A., Miller, B.L., Alzualde, A., Moreno, F., Rohrer, J.D., Mackenzie, I.R., Feldman, H.H., Hamilton, R.L., Cruts, M., Engelborghs, S., De Deyn, P.P., Van Broeckhoven, C., Bird, T.D., Cairns, N.J., Goate, A., Frosch, M.P., Riederer, P.F., Bogdanovic, N., Lee, V.M., Trojanowski, J.Q., Van Deerlin, V.M., 2011. Genetic and clinical features of progranulin-associated frontotemporal lobar degeneration. Arch. Neurol. 68, 488e497. Cruts, M., Gijselinck, I., van der Zee, J., Engelborghs, S., Wils, H., Pirici, D., Rademakers, R., Vandenberghe, R., Dermaut, B., Martin, J.J., van Duijn, C., Peeters, K., Sciot, R., Santens, P., De Pooter, T., Mattheijssens, M., Van den Broeck, M., Cuijt, I., Vennekens, K., De Deyn, P.P., Kumar-Singh, S., Van Broeckhoven, C., 2006. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 442, 920e924. Gorno-Tempini, M.L., Hillis, A.E., Weintraub, S., Gorno-Tempini, M.L., Hillis, A.E., Weintraub, S., Kertesz, A., Mendez, M., Cappa, S.F., Ogar, J.M., Rohrer, J.D.,
C. Bonvicini et al. / Neurobiology of Aging 35 (2014) 1206e1211 Black, S., Boeve, B.F., Manes, F., Dronkers, N.F., Vandenberghe, R., Rascovsky, K., Patterson, K., Miller, B.L., Knopman, D.S., Hodges, J.R., Mesulam, M.M., Grossman, M., 2011. Classification of primary progressive aphasia and its variants. Neurology 76, 1006e1014. Hisata, S., Sakisaka, T., Baba, T., Yamada, T., Aoki, K., Matsuda, M., Takai, Y., 2007. Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth. J. Cell Biol. 178, 843e860. Kelley, B.J., Haidar, W., Boeve, B.F., Baker, M., Graff-Radford, N.R., Krefft, T., Frank, A.R., Jack Jr., C.R., Shiung, M., Knopman, D.S., Josephs, K.A., Parashos, S.A., Rademakers, R., Hutton, M., Pickering-Brown, S., Adamson, J., Kuntz, K.M., Dickson, D.W., Parisi, J.E., Smith, G.E., Ivnik, R.J., Petersen, R.C., 2009. Prominent phenotypic variability associated with mutations in progranulin. Neurobiol. Aging 30, 739e751. Larner, A.J., 2012. Intrafamilial clinical phenotypic heterogeneity with progranulin gene p.Glu498fs mutation. J. Neurol. Sci. 316, 189e190. Le Ber, I., Camuzat, A., Hannequin, D., Pasquier, F., Guedj, E., Rovelet-Lecrux, A., HahnBarma, V., van der Zee, J., Clot, F., Bakchine, S., Puel, M., Ghanim, M., Lacomblez, L., Mikol, J., Deramecourt, V., Lejeune, P., de la Sayette, V., Belliard, S., Vercelletto, M., Meyrignac, C., Van, B.C., Lambert, J.C., Verpillat, P., Campion, D., Habert, M.O., Dubois, B., Brice, A., 2008. Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study. Brain 131, 732e746. Li, Y.H., Werner, H., Puschel, A.W., 2008. Rheb and mTOR regulate neuronal polarity through Rap1B. J. Biol. Chem. 283, 33784e33792. Mackenzie, I.R., Neumann, M., Bigio, E.H., Cairns, N.J., Alafuzoff, I., Kril, J., Kovacs, G.G., Ghetti, B., Halliday, G., Holm, I.E., Ince, P.G., Kamphorst, W., Revesz, T., Rozemuller, A.J., Kumar-Singh, S., Akiyama, H., Baborie, A., Spina, S., Dickson, D.W., Trojanowski, J.Q., Mann, D.M., 2009. Nomenclature for neuropathologic subtypes of frontotemporal lobar degeneration: consensus recommendations. Acta Neuropathol. 117, 15e18. Mackenzie, I.R., Neumann, M., Baborie, A., Sampathu, D.M., Du, P.D., Jaros, E., Perry, R.H., Trojanowski, J.Q., Mann, D.M., Lee, V.M., 2011. A harmonized classification system for FTLD-TDP pathology. Acta Neuropathol. 122, 111e113. McAvoy, T., Zhou, M.M., Greengard, P., Nairn, A.C., 2009. Phosphorylation of Rap1GAP, a striatally enriched protein, by protein kinase A controls Rap1 activity and dendritic spine morphology. Proc. Natl. Acad. Sci. U.S.A 106, 3531e3536. Moreno, F., Indakoetxea, B., Barandiaran, M., Alzualde, A., Gabilondo, A., Estanga, A., Ruiz, J., Ruibal, M., Bergareche, A., Martí-Massó, J.F., López de Munain, A., 2009. “Frontotemporoparietal” dementia: clinical phenotype associated with the c.709-1G>A PGRN mutation. Neurology 73, 1367e1374. Pickering-Brown, S.M., Rollinson, S., Du, P.D., Morrison, K.E., Varma, A., Richardson, A.M., Neary, D., Snowden, J.S., Mann, D.M., 2008. Frequency and clinical characteristics of progranulin mutation carriers in the Manchester
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frontotemporal lobar degeneration cohort: comparison with patients with MAPT and no known mutations. Brain 131, 721e731. Rademakers, R., Baker, M., Gass, J., Adamson, J., Huey, E.D., Momeni, P., Spina, S., Coppola, G., Karydas, A.M., Stewart, H., Johnson, N., Hsiung, G.Y., Kelley, B., Kuntz, K., Steinbart, E., Wood, E.M., Yu, C.E., Josephs, K., Sorenson, E., Womack, K.B., Weintraub, S., Pickering-Brown, S.M., Schofield, P.R., Brooks, W.S., Van Deerlin, V.M., Snowden, J., Clark, C.M., Kertesz, A., Boylan, K., Ghetti, B., Neary, D., Schellenberg, G.D., Beach, T.G., Mesulam, M., Mann, D., Grafman, J., Mackenzie, I.R., Feldman, H., Bird, T., Petersen, R., Knopman, D., Boeve, B., Geschwind, D.H., Miller, B., Wszolek, Z., Lippa, C., Bigio, E.H., Dickson, D., GraffRadford, N., Hutton, M., 2007. Phenotypic variability associated with progranulin haploinsufficiency in patients with the common 1477C–>T (Arg493X) mutation: an international initiative. Lancet Neurol. 6, 857e868. Rascovsky, K., Hodges, J.R., Knopman, D., Mendez, M.F., Kramer, J.H., Neuhaus, J., van Swieten, J.C., Seelaar, H., Dopper, E.G., Onyike, C.U., Hillis, A.E., Josephs, K.A., Boeve, B.F., Kertesz, A., Seeley, W.W., Rankin, K.P., Johnson, J.K., GornoTempini, M.L., Rosen, H., Prioleau-Latham, C.E., Lee, A., Kipps, C.M., Lillo, P., Piguet, O., Rohrer, J.D., Rossor, M.N., Warren, J.D., Fox, N.C., Galasko, D., Salmon, D.P., Black, S.E., Mesulam, M., Weintraub, S., Dickerson, B.C., DiehlSchmid, J., Pasquier, F., Deramecourt, V., Lebert, F., Pijnenburg, Y., Chow, T.W., Manes, F., Grafman, J., Cappa, S.F., Freedman, M., Grossman, M., Miller, B.L., 2011. Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 134, 2456e2477. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101e1108. Spilker, C., Kreutz, M.R., 2010. RapGAPs in brain: multipurpose players in neuronal Rap signalling. Eur. J. Neurosci. 32, 1e9. Sullivan, P.F., Fan, C., Perou, C.M., 2006. Evaluating the comparability of gene expression in blood and brain. Am. J. Med. Genet. B Neuropsychiatr. Genet. 141B, 261e268. Toh, H., Chitramuthu, B.P., Bennett, H.P., Bateman, A., 2011. Structure, function, and mechanism of progranulin; the brain and beyond. J. Mol. Neurosci. 45, 538e548. van Swieten, J.C., Heutink, P., 2008. Mutations in progranulin (GRN) within the spectrum of clinical and pathological phenotypes of frontotemporal dementia. Lancet Neurol. 7, 965e974. Yu, C.E., Bird, T.D., Bekris, L.M., Montine, T.J., Leverenz, J.B., Steinbart, E., Galloway, N.M., Feldman, H., Woltjer, R., Miller, C.A., Wood, E.M., Grossman, M., McCluskey, L., Clark, C.M., Neumann, M., Danek, A., Galasko, D.R., Arnold, S.E., Chen-Plotkin, A., Karydas, A., Miller, B.L., Trojanowski, J.Q., Lee, V.M., Schellenberg, G.D., Van Deerlin, V.M., 2010. The spectrum of mutations in progranulin: a collaborative study screening 545 cases of neurodegeneration. Arch. Neurol. 67, 161e170.