- Email: [email protected]
One novel GRN null mutation, two different aphasia phenotypes
Journal Pre-proof One novel GRN null mutation, two different aphasia phenotypes Cinzia Coppola, Mariano Oliva, Dario Saracino, Sabina Pappatà, Emilia Zampella, Sara Cimini, Martina Ricci, Giorgio Giaccone, Giuseppe Di Iorio, Giacomina Rossi PII:
S0197-4580(19)30397-5
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.11.008
Reference:
NBA 10715
To appear in:
Neurobiology of Aging
Received Date: 9 July 2018 Revised Date:
4 September 2019
Accepted Date: 6 November 2019
Please cite this article as: Coppola, C., Oliva, M., Saracino, D., Pappatà, S., Zampella, E., Cimini, S., Ricci, M., Giaccone, G., Di Iorio, G., Rossi, G., One novel GRN null mutation, two different aphasia phenotypes, Neurobiology of Aging (2019), doi: https://doi.org/10.1016/j.neurobiolaging.2019.11.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
One novel GRN null mutation, two different aphasia phenotypes
Cinzia Coppolaa, Mariano Olivaa, Dario Saracinoa, Sabina Pappatàb, Emilia Zampellac, Sara Ciminid, Martina Riccid, Giorgio Giacconed, Giuseppe Di Iorioa, Giacomina Rossid
a
Second Division of Neurology, University of Campania “Luigi Vanvitelli” – Naples, Italy
b
Institute of Biostructure and Bioimaging, National Council of Research – Naples, Italy
c
Department of Advanced Biomedical Sciences, “Federico II” University – Naples, Italy
d
Division of Neurology V - Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta – Milan,
Italy
*Correspondence to: Cinzia Coppola, Second Division of Neurology, University of Campania “Luigi Vanvitelli”, Isola 8 – Edificio 10 Policlinico “Federico II” via Pansini 5, 80131 Naples, Italy. Tel.: +39 0815666811; Fax: +39 0815666805; Email: [email protected]
Abstract Progranulin gene (GRN) mutations are among the leading causes of Fronto-temporal lobar degeneration (FTLD), a group of neurodegenerative diseases characterized by remarkable clinical heterogeneity. In this paper we report the new GRN 708+4A>T splicing mutation, identified in two siblings of a family with several members affected by cognitive, behavioral and motor disorders. Plasma progranulin dosage and GRN expression analysis, together with in silico prediction studies, supported the pathogenicity of the mutation. Both the patients displayed a clinical syndrome in which language impairment was largely predominant. However, motor speech deficits were the major feature in one case, diagnosed as progressive non fluent aphasia, whereas marked semantic alterations were present in the other, whose clinical phenotype was in favor of a mixed aphasia. The profile of neuroanatomical alterations from imaging studies was in line with the clinical phenotypes. Therefore, also this novel GRN mutation is associated with haploinsufficiency and phenotypic heterogeneity, which are both typical features of progranulinopathies.
Keywords: Fronto-temporal lobar degeneration, GRN, progranulin protein, mutation, progressive non fluent aphasia, primary progressive aphasia.
1. Introduction Fronto-temporal lobar degeneration (FTLD) comprises a heterogeneous group of diseases that share predominant impairment of behavioural, executive and language functions. It accounts for about 515% of all cases of dementia (Rademakers et al., 2012), and is the second cause of cognitive decline in presenile age, its onset typically ranging between the 5th and the 7th decade (Bird et al., 2003). FTLD is characterized by a remarkable heterogeneity from a clinical, pathological and genetic point of view. Clinical presentations of FTLD include behavioral variant of fronto-temporal dementia (bvFTD) (Rascovsky et al., 2011), and two variants of primary progressive aphasia (PPA), progressive non fluent aphasia (PNFA) and semantic dementia (SD) (Gorno-Tempini et al., 2011; Leyton et al., 2011). In addition, there are some cases exhibiting an overlap with motor neuron disease/amyotrophic lateral sclerosis (FTD/ALS), and others associated with parkinsonism, namely progressive supranuclear palsy (PSP) and cortico-basal syndrome (CBS) (Rabinovici et al., 2010). As for the genetic aspects, the genes coding for progranulin (GRN), microtubule-associated protein tau (MAPT), and chromosome 9 open reading frame 72 (C9orf72) account for the majority of familial cases (Bang et al., 2015; Benussi et al., 2015). Other genes, such as those coding for valosin containing protein (VCP) or charged multivesicular body protein 2B (CHMP2B), are observed only in a small number of cases (Skibinski et al., 2005; Watts et al., 2004). Progranulin is a highly conserved glycoprotein coded by the GRN gene, located on the chromosome 17q21, in proximity to the MAPT locus (Ahmed et al., 2007). Its biological roles, mediated by either the whole protein or its proteolytic cleavage products, called granulins, include cell proliferation, inflammation, lysosome function, neurite outgrowth, neuronal survival (Cenik et al., 2012), and metabolic homeostasis (Korolczuk et al., 2017). GRN mutations account for 5-10% of FTLD cases, but this proportion increases to 22% in patients with positive family history (Seelar et al., 2011). To date, more than 170 GRN mutations have been described (Stenson et al., 2017). Most of pathogenic mutations are frameshift, non-sense or splicing mutations resulting in a premature termination
codon, degradation of mutated mRNA and consequent haploinsufficiency (Baker et al., 2006; Petkau et al., 2014). GRN mutations are characterized by a wide inter- and intra-familial clinical heterogeneity, which include bvFTD, PNFA and CBS (Le Ber et al., 2007; Davion et al., 2007), while the association with SD has been reported in rare instances (Cerami et al., 2013; Van Mossevelde et al., 2016; Wauters et al., 2018). Early appearance of memory impairment, apraxia and sometimes visuospatial disturbances is considered to be evocative of this underlying genetic background (Le Ber et al., 2008). Structural and functional neuroimaging studies may also highlight some suggestive aspects: cortical atrophy is generally asymmetric and involves frontal, temporal and parietal lobes (Benussi et al., 2015), while 18F-fluoro-2-deoxy-glucose (18FDG) positron emission tomography (PET) often reveals asymmetrical frontal and temporal hypometabolism often extending to the parietal regions (Meeter et al., 2017). The involvement of parietal and posterior cingulate cortices usually differentiates FTLD associated with GRN mutations from sporadic cases (Coppola et al., 2017; Milan et al., 2017). In this paper we describe the clinical and neuroradiological features of two related patients carrying the novel GRN c.708+4A>T splicing mutation. Both in silico predictions and expression analysis demonstrated that the mutation affects the splicing site, leading to progranulin haploinsufficiency.
2. Patients and Methods Pedigree of the family carrying the GRN c.708+4A>T splicing mutation is depicted in Figure 1. 2.1 Proband The proband (III-8) was a 70-year-old patient whose past medical history included arterial hypertension and hypertriglyceridemia. This patient had presented language disturbances with phonetic paraphasias and effortful speech with frequent interruptions since the age of 69, and also showed progressive behavioral disorders mainly characterized by apathy. These symptoms had been slowly worsening. Mini Mental State Examination (MMSE) score was 16/30. The extensive
neuropsychological examination showed multiple language impairments (phonological and semantic fluencies, writing, repetition, calculation, auditory and visual comprehension), executive dysfunctions and frontal behavioral disturbances (Supplementary Table S1). Clinical Dementia Rating (CDR) scale was 2, being communication abilities, interpersonal interactions and personal care among the most compromised domains. EEG showed widespread dysregulation in cerebral activity. Brain MRI revealed diffuse cortical atrophy prevailing in the left hemisphere, multiple T2hyperintense foci in subcortical and deep periventricular white matter and an empty sella (Supplementary Figure S1 A-C). Brain FDG-PET evidenced a reduction of glucose uptake in the anterior cingulate, frontal/insular cortex, especially on the left side, with a milder relative hypometabolism in left temporal and parietal cortices and striatum (Figure 2 A, B). Cerebro-spinal fluid (CSF) biomarkers were within reference ranges. The clinical diagnosis was Progressive Non Fluent Aphasia (PNFA). Familial history was positive for cognitive disorders in in one of the parents (II-3) and in one sibling (III-7). Moreover, one of the parent’s siblings (II-5) had likely been affected by an atypical parkinsonism since the age of 70 and died at the age of 86, while another sibling (II-7) had been affected by a behavioral disorder since the age of 68 and died at the age of 83. 2.2 Case III-7 A sibling of the proband was a 72-year-old patient whose medical antecedents were prostate cancer and cerebrovascular disease. He had insidiously manifested progressive language impairments (comprehension problems, anomia, semantic paraphasias and neologisms) and mild behavioral disorders (irritability, perseveration, disinhibition) since the age of 68. MMSE score was 23/30. The extensive neuropsychological examination confirmed the language impairment, with alteration of phonological and semantic fluencies, writing, repetition, denomination and comprehension of phrases. Short-term memory and executive functions were also impaired (Supplementary Table S1). EEG showed widespread dysregulation in cerebral activity. CDR scale was 1, mainly due to minor behavioral changes, problem solving difficulties and moderately impaired comprehension during
conversation. Brain MRI revealed multiple T2-hyperintense foci in white matter and diffuse cortical atrophy with left prevalence (Supplementary Figure S1 D-F). Brain FDG-PET evidenced reduced cortical and subcortical relative glucose metabolism in the left cerebral hemisphere, mainly involving the lateral and medial temporal cortex and, to a lesser extent, the parietal, posterior cingulate, frontal lateral/medial and anterior cingulate cortices and striatum (Figure 2 C, D). CSF biomarkers were within reference ranges. The clinical diagnosis was mixed aphasia with prevalent aspects of semantic impairment overlapping with a frontal behavioral disorder. The patients and their relatives expressed their informed consent to undergo genetic testing and to participate to research studies. The present study has been approved by the local ethical committee (University of Campania “Luigi Vanvitelli”, Naples). 2.3 Plasma progranulin dosage In the diagnostic procedure of our Laboratory, the first analysis we perform in FTLD cases is plasma progranulin dosage. If the progranulin level is below the reported cut-off (Ghidoni 2012), we sequence GRN gene; otherwise we analyze C9orf72 and sequence MAPT genes. To determine the level of plasma progranulin, an ELISA kit (Human Progranulin ELISA kit; Adipogen) was used, according to manufacturer’s instructions. 2.4 Genetic analysis 2.4.1 DNA analysis Genomic DNA was extracted from peripheral blood lymphocytes (PBL) of proband and subject III7. For the proband, all coding exons, flanking intronic sequences and 5' and 3' untranslated regions of GRN gene were amplified using previously published methods (Baker et al., 2006, Cruts et al., 2006), and amplified fragments were sequenced using the Big Dye terminator v 3.1 cycle sequencing kit (Applied Biosystems, Life Technologies) and analyzed on an ABI 3100 gene analyzer (Applied Biosystems, Life Technologies). Only a region containing the GRN splicing site c.708+4A was amplified and sequenced for subject III-7.
In order to exclude mutations from other known FTD causative genes, we carried out a next generation sequencing (NGS) analysis on the proband and subject III-7. Nextera Rapid Capture system for enrichment (Illumina) coupled with gene-specific probes (Integrated DNA Technologies) was used to sequence a panel containing the following genes: APP, PSEN1, PSEN2; PRNP; GRN, MAPT, CHMP2B, FUS, TARDBP, VCP, TREM2, CSF1R, TBK1. Sequencing was performed on the Illumina MiSeq instrument and MiSeq Reporter software was used for alignment and variant calling. Alignment was performed against the human genome UCSC hg19 and variants annotated with Variant Studio software (Illumina). Low-quality variants were filtered out using the Illumina Qscore threshold of 30; in addition, variants with a minimum allele frequency higher than 1% in GnomAD (Genome Aggregation Database, http://gnomad.broadinstitute.org/) were filtered out. Most of the genes were sequenced by NGS with 100% coverage (read depth ≥ 20X). The few exons not covered by NGS were sequenced by standard Sanger technique, to give a 100% final sequencing. 2.4.2 RNA analysis Total RNA was isolated from blood samples of proband, patient III-7, two wild-type controls and 1 patient carrying the GRN A199V mutation (positive control), using the PAXgene® Blood RNA Kit (PreAnalytiX, Qiagen). cDNA was synthesized using cloned AMV First-strand synthesis kit (Invitrogen). cDNA was subjected to quantitative real-time PCR using premade TaqMan gene expression assays: Hs00173570_m1 for GRN and Hs99999903_m1 for β-actin as an endogenous control for sample normalization (Applied Biosystems, Life Technologies). The cDNA was amplified in triplicate and run on a ViiA7 Real-time PCR system (Applied Biosystems, Life Technologies). The relative transcription levels of GRN mRNA were calculated by using the 2-∆∆Ct method (Livak et al., 2001), where ∆∆Ct = ∆Ctpatient –∆Ctcontrols and ∆Ct = CtGRN – Ctβactin. 2.5 Bioinformatics analysis
We used the in silico tools Netgene2 (www.cbs.dtu.dk/services/NetGene2/), BDGP splice Site Prediction (www.fruitfly.org/seq_tools/splice.html) and Human Splicing Finder (www.umd.be/HSF3/) to evaluate the effect of the GRN c.708+4A>T mutation on splice site.
3. Results 3.1 Plasma progranulin dosage Plasma progranulin levels were 37.5 and 26.8 ng/mL for proband and patient III-7, respectively, below the calculated cut-off level for GRN mutation prediction in FTLD patients (61.55 ng/mL; Ghidoni et al., 2012). 3.2 Genetic analysis 3.2.1 DNA analysis Sequencing of DNA disclosed the GRN c.708+4A>T base substitution (References: (i) chr17:42,428,172A>T, UCSC Genome Browser on GRCh37/hg19 assembly; (ii) g.101705A>T, reverse complement of GenBank AC003043.1 starting at nucleotide 1; (iii) NM_002087.3:c.708+4A>T, starting at the translation initiation site; (iv) exon 6/intron 6 boundary site) in the proband and subject III-7 (Figure 3). This is a splicing mutation predicting the exon 6 skipping, a frameshift of the coding region and a premature termination codon in exon 7, leading to the degradation of mutant mRNA (Gass et al.,2006). This mutation is not present in the Genome Aggregation Database (gnomAD) data set, which includes 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals. Thus, it is a new mutation. No pathogenic mutation was found in any of the genes sequenced by NGS panel, except the GRN mutation previously disclosed by traditional sequencing. 3.2.2 RNA analysis Quantitative real-time PCR demonstrated mRNA expression decreased to 56% and 42% in proband and patient III-7, respectively, compared with control samples (Table 1). 3.3 Bioinformatic analysis
Netgene2, BDGP splice Site Prediction and Human Splicing Finder all predicted the loss of splice site in presence of the base substitution GRN c.708+4A>T (data not shown).
4. Discussion GRN mutations show a wide clinical variability, the most frequent phenotypes being bvFTD, PNFA and CBS (Le Ber et al. 2008; Rossi et al., 2011). The association between GRN mutations and SD remains more elusive. SD was described in one familial case carrying the GRN T409M mutation, whose pathological nature is uncertain being a missense and not a null mutation; moreover, no progranulin dosage was available (Cerami et al., 2013). Additional SD cases were signaled in Belgian kindreds. Four patients were reported into a clinical-pathological cohort of 52 GRN-mutated FTLD patients (Van Mossevelde et al., 2016), and two were identified within a large family carrying the IVS1+5 G>C mutation (Wauters et al., 2018). Conversely, the SD phenotype was not reported in association with GRN mutations in the largest series of genetic PPA patients described so far (Ramos et al., 2019). Finally, FTD/ALS phenotype was described in one sporadic case carrying the GRN S120Y mutation (Schymick et al., 2007), which was also described in a case of sporadic ALS (Del Bo et al., 2011). The pathogenicity is unclear as the amino acid is not highly conserved across species and is not localized in a region critical to progranulin function; in addition, it is reported in gnomAD, although with a low frequency (0.1%). Our family shows a relevant phenotypic variability, not only considering different generations, but also within the same generation. The mode of onset and the extensive neuropsychological evaluation were strongly suggestive of PNFA in the proband, being reduced verbal fluency and apraxia of speech the most relevant impairments, in association with milder behavioral disorders and dysexecutive aspects. The sibling, patient III-7, displayed language impairments too, but their pattern hampers a precise classification according to currently adopted criteria (Gorno-Tempini et al., 2011). There were significant elements in favor of a semantic variant of PPA, in particular
naming deficits, comprehension impairment, and semantic paraphasias. However, those aspects occurred together with deficits in phonological encoding and repetition of sentences. For that reason, we formulated the diagnosis of mixed aphasia, associated with a frontal cognitive and behavioral syndrome. In both cases brain MRI showed a diffuse cortical atrophy with left prevalence and a significant burden of white matter lesions; this latter aspect has been frequently reported in GRN-related FTD (Caroppo et al., 2014). Likewise, brain 18FDG-PET also evidenced a similar pattern of hypometabolism between the two patients, namely a left frontal and temporoparietal hypometabolism, with mild involvement of the basal ganglia. The asymmetric anterior and posterior cortical relative hypometabolism has been often reported in cohorts or single cases of GRN mutations (Spina et al., 2007; Coppola et al., 2017; Milan et al., 2017). Interestingly, in our patient III.7 with prevalent semantic language disturbance the hypometabolism involved mainly the left anterior and lateral temporal lobe, whereas in the proband affected by PNFA the hypometabolism was more marked in the left fronto-insular cortex, suggesting an association between metabolic and cognitive phenotypes. A remarkable intra-familial phenotypic heterogeneity, even considering family members belonging to the same generation, appears to be a recurrent characteristic of GRN mutations. Notably the age at onset, the clinical phenotype and the duration of disease represent the main aspects of that variability (Rossi et al., 2011; Hosaka et al., 2017). The identification of genetic factors that may account for this heterogeneity is currently an active field of research (Chitramuthu et al., 2017; Wauters et al., 2017). Our two cases are also in line with the previous observation that GRN-associated PPA has specific aspects that do not always allow a proper classification (Rohrer et al., 2010). Phonological disturbances and difficulties in repetition were shared by both our two patients, and they are indeed two features often occurring with GRN mutations (Rohrer et al., 2010). However, the overall clinical pictures were quite divergent, given the presence of motor speech impairment in one case, and of semantic deficits in the other.
5. Conclusions In summary, in this work we describe the novel GRN c.708+4A>T mutation, whose pathogenic nature is evidenced by: (i) the segregation with the disease within the family; (ii) the results of the GRN expression analyses, showing decreased production of mRNA, predicted also by the bioinformatics analysis; (iii) the reduced levels of plasma progranulin in both subjects. An interesting aspect associated with this mutation is the variability of the profile of linguistic impairment evidenced in two mutation carriers, with an unclassifiable aphasia phenotype, not fitting the clinical and neurolinguistic phenotypes proposed by current PPA criteria, in one case. Finally, our report underlines an already well-established concept, namely the remarkable phenotypic heterogeneity occurring with GRN mutations even among family members belonging to the same generation.
Acknowledgements The authors have no conflicts of interest to disclose.
References Ahmed Z, Mackenzie IR, Hutton ML, Dickson DW. Progranulin in frontotemporal lobar degeneration and neuroinflammation. J Neuroinflammation 2007;4(1):7. Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Berger Z, Eriksen J, Robinson T, Zehr C, Dickey CA, Crook R, McGowan E, Mann D, Boeve B, Feldman H, Hutton M. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 2006;442(7105):916-9.
Bang J, Spina S, Miller BL. Frontotemporal dementia. Lancet 2015;386(10004):1672-82. Benussi A, Padovani A, Borroni B. Phenotypic heterogeneity of monogenic frontotemporal dementia. Front Aging Neurosci 2015;7:171. Bird T, Knopman D, VanSwieten J, Rosso S, Feldman H, Tanabe H, Graff-Raford N, Geschwind D, Verpillat P, Hutton M. Epidemiology and genetics of frontotemporal dementia/Pick’s disease. Ann Neurol 2003;54(Suppl 5):S29–31. Caroppo P, Le Ber I, Camuzat A, Clot F, Naccache L, Lamari F, De Septenville A, Bertrand A, Belliard S, Hannequin D, Colliot O, Brice A. Extensive white matter involvement in patients with frontotemporal lobar degeneration: think progranulin. JAMA Neurol 2014;71:1562-6. Cenik B, Sephton CF, Cenik BK, Herz J, Yu G. Progranulin: a proteolytically processed protein at the crossroads of inflammation and neurodegeneration. J Biol Chem 2012;287(39):32298-306. Cerami C, Marcone A, Galimberti D, Villa C, Fenoglio C, Scarpini E, Cappa SF. Novel missense progranulin gene mutation associated with the semantic variant of primary progressive aphasia. J Alzheimers Dis 2013;36(3):415-20. Chitramuthu BP, Bennett HPJ, Bateman A. Progranulin: a new avenue towards the understanding and treatment of neurodegenerative disease. Brain. 2017;140(12):3081-104. Coppola C, Saracino D, Puoti G, Lus G, Dato C, Le Ber I, Pariente J, Caroppo P, Piccoli E, Tagliavini F, Di Iorio G, Rossi G. A cluster of progranulin C157KfsX97 mutations in Southern Italy: clinical characterization and genetic correlations. Neurobiol Aging 2017;49:219.e5-219.e13. Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Randenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, Kumar-Singh S, Van
Broeckhoven C. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 2006;442(7105):920-4. Davion S, Johnson N, Weintraub S, Mesulam MM, Engberg A, Mishra M, Baker M, Adamson J, Hutton M, Rademakers R, Bigio EH. Clinicopathologic correlation in PGRN mutations. Neurology. 2007;69(11):1113-21. Del Bo R, Corti S, Santoro D, Ghione I, Fenoglio C, Ghezzi S, Ranieri M, Galimberti D, Mancuso M, Siciliano G, Briani C, Murri L, Scarpini E, Schymick JC, Traynor BJ, Bresolin N, Comi GP. No major progranulin genetic variability contribution to disease etiopathogenesis in an ALS Italian cohort. Neurobiol Aging 2011;32(6):1157-8. Gass J, Cannon A, Mackenzie IR, Boeve B, Baker M, Adamson J, Crook R, Melquist S, Kuntz K, Petersen R, Josephs K, Pickering-Brown SM, Graff-Radford N, Uitti R, Dickson D, Wszolek Z, Gonzalez J, Beach TG, Bigio E, Johnson N, Weintraub S, Mesulam M, White CL 3rd, Woodruff B, Caselli R, Hsiung GY, Feldman H, Knopman D, Hutton M, Rademakers R. Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration. Hum Mol Genet 2006;15(20):2988-3001. Ghidoni R, Stoppani E, Rossi G, Piccoli E, Albertini V, Paterlini A, Glionna M, Pegoiani E, Agnati LF, Fenoglio C, Scarpini E, Galimberti D, Morbin M, Tagliavini F, Binetti G, Benussi L. Optimal plasma progranulin cutoff value for predicting null progranulin mutations in neurodegenerative diseases: A multicenter Italian study. Neurodegener Dis 2012;9(3):121-7. Gorno-Tempini ML, Hillis AE, Weintraub S, Kertesz A, Mendez M, Cappa SF, Ogar JM, Rohrer JD, Black S, Boeve BF, Manes F, Dronkers NF, Vandenberghe R, Rascovsky K, Patterson K, Miller BL, Knopman DS, Hodges JR, Mesulam MM, Grossman M. Classification of primary progressive aphasia and its variants. Neurology 2011;76(11):1006-14.
Hosaka T, Ishii K, Miura T, Mezaki N , Kasuga K, Ikeuchi T, Tamaoka A. A novel frameshift GRN mutation results in frontotemporal lobar degeneration with a distinct clinical phenotype in two siblings: case report and literature review. BMC Neurol. 2017;17(1):182. Korolczuk A, Bełtowski J. Progranulin, a new adipokine at the crossroads of metabolic syndrome, diabetes, dyslipidemia and hypertension. Curr Pharm Des 2017;23(10):1533-9. Le Ber I, van der Zee J, Hannequin D, Gijselinck I, Campion D, Puel M, Laquerrie`re A, De Pooter T, Camuzat A, Van den Broeck M, Dubois B, Sellal F, Lacomblez L, Vercelletto M, ThomasAntérion C, Michel BF, Golfier V, Didic M, Salachas F, Duyckaerts C, Cruts M, Verpillat P, Van Broeckhoven C, Brice A, French Research Network on FTD/FTD-MND. Progranulin null mutations in both sporadic and familial frontotemporal dementia. Human Mutat 2007;28:846-55. Le Ber, I, Camuzat, A, Hannequin, D, Pasquier, F, Guedj, E, Rovelet-Lecrux, A, Hahn-Barma, 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 Broeckhoven, C, Lambert, JC, Verpillat, P, Campion, D, Habert, MO, Dubois, B, Brice, A, French research network on FTD/FTD-MND. Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study. Brain 2008;131:732-46. Leyton CE, Villemagne VL, Savage S, Pike KE, Ballard KJ, Piguet O, Burrell JR, Rowe CC, Hodges JR. Subtypes of progressive aphasia: application of the International Consensus Criteria and validation using β-amyloid imaging. Brain 2011;134:3030-43. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-8. Meeter LH, Kaat LD, Rohrer JD, van Swieten JC. Imaging and fluid biomarkers in frontotemporal dementia. Nat Rev Neurol 2017;13(7):406-19.
Milan G, Napoletano S, Pappatà S, Gentile MT, Colucci-D'Amato L, Della Rocca G, Maciag A, Rossetti CP, Fucci L, Puca A, Grossi D, Postiglione A, Vitale E. GRN deletion in familial frontotemporal dementia showing association with clinical variability in 3 familial cases. Neurobiol Aging 2017;53:193.e9-193.e16. Petkau TL, Leavitt BR. Progranulin in neurodegenerative disease. Trends Neurosci. 2014;37(7):388-98. Rabinovici GD, Miller BL. Frontotemporal lobar degeneration: epidemiology, pathophysiology, diagnosis and management. CNS Drugs 2010;24(5):375-98. Rademakers R, Neumann M, Mackenzie IR. Advances in the molecular basis of frontotemporal dementia. Nat Rev Neurol 2012;8(8):423-34. Ramos EM, Dokuru DR, Van Berlo V, Wojta K, Wang Q, Huang AY, Miller ZA, Karydas AM, Bigio EH, Rogalski E, Weintraub S, Rader B, Miller BL, Gorno-Tempini ML, Mesulam MM, Coppola G. Genetic screen in a large series of patients with primary progressive aphasia. Alzheimers Dement 2019;15(4):553-60. Rascovsky K, Hodges JR, Knopman D, Mendez MF, Kramer JH, Neuhaus J, van Swieten JC, Seelaar H, Dopper EG, Onyike CU, Hillis AE, Josephs KA, Boeve BF, Kertesz A, Seeley WW, Rankin KP, Johnson JK, Gorno-Tempini ML, Rosen H, Prioleau-Latham CE, Lee A, Kipps CM, Lillo P, Piguet O, Rohrer JD, Rossor MN, Warren JD, Fox NC, Galasko D, Salmon DP, Black SE, Mesulam M, Weintraub S, Dickerson BC, Diehl-Schmid J, Pasquier F, Deramecourt V, Lebert F, Pijnenburg Y, Chow TW, Manes F, Grafman J, Cappa SF, Freedman M, Grossman M, Miller BL. Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 2011;134:2456-77.
Rohrer JD, Crutch SJ, Warrington EK, Warren JD. Progranulin-associated primary progressive aphasia: a distinct phenotype? Neuropsychologia 2010;48(1):288-97. Rossi G, Piccoli E, Benussi L, Caso F, Redaelli V, Magnani G, Binetti G, Ghidoni R, Perani D, Giaccone G, Tagliavini F. A novel progranulin mutation causing frontotemporal lobar degeneration with heterogeneous phenotypic expression. J Alzheimers Dis 2011;23(1):7-12. Schymick JC, Yang Y, Andersen PM, Vonsattel JP, Greenway M, Momeni P, Elder J, Chiò A, Restagno G, Robberecht W, Dahlberg C, Mukherjee O, Goate A, Graff-Radford N, Caselli RJ, Hutton M, Gass J, Cannon A, Rademakers R, Singleton AB, Hardiman O, Rothstein J, Hardy J, Traynor BJ. Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes. J Neurol Neurosurg Psychiatry 2007;78(7):754-6. Seelaar H, Rohrer JD, Pijnenburg YA, Fox NC, van Swieten JC. Clinical, genetic and pathological heterogeneity of frontotemporal dementia: a review. J Neurol Neurosurg Psychiatry 2011;82(5):476-86. Skibinski G, Parkinson NJ, Brown JM, Chakrabarti L, Lloyd SL, Hummerich H, Nielsen JE, Hodges JR, Spillantini MG, Thusgaard T, Brandner S, Brun A, Rossor MN, Gade A, Johannsen P, Sørensen SA, Gydesen S, Fisher EM, Collinge J. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat Genet 2005;37:806-8. Spina S, Murrell JR, Huey ED, Wassermann EM, Pietrini P, Grafman J, Ghetti B. Corticobasal syndrome associated with the A9D progranulin mutation. J Neuropathol Exp Neurol 2007;66:892900. Stenson PD, Mort M, Ball EV, Evans K, Hayden M, Heywood S, Hussain M, Phillips AD, Cooper DN. The Human Gene Mutation Database: towards a comprehensive repository of inherited
mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet. 2017;136(6):665-77. Van Mossevelde S, van der Zee J, Gijselinck I, Engelborghs S, Sieben A, Van Langenhove T, De Bleecker J, Baets J, Vandenbulcke M, Van Laere K, Ceyssens S, Van den Broeck M, Peeters K, Mattheijssens M, Cras P, Vandenberghe R, De Jonghe P, Martin JJ, De Deyn PP, Cruts M, Van Broeckhoven C. Clinical features of TBK1 carriers compared with C9orf72, GRN and non-mutation carriers in a Belgian cohort. Brain. 2016;139(2):452-67. Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP, Kimonis VE. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 2004;36:377-81. Wauters E, Van Mossevelde S, Van der Zee J, Cruts M, Van Broeckhoven C. Modifiers of GRNAssociated Frontotemporal Lobar Degeneration. Trends Mol Med 2017;23(10):962-79. Wauters E, Van Mossevelde S, Sleegers K, van der Zee J, Engelborghs S, Sieben A, Vandenberghe R, Philtjens S, Van den Broeck M, Peeters K, Cuijt I, De Coster W, Van Langenhove T, Santens P, Ivanoiu A, Cras P, De Bleecker JL, Versijpt J, Crols R, De Klippel N, Martin JJ, De Deyn PP, Cruts M, Van Broeckhoven C; Belgian Neurology (BELNEU) Consortium. Clinical variability and onset age modifiers in an extended Belgian GRN founder family. Neurobiol Aging 2018;67:84-94.
Table 1. The percentage reduction of GRN mRNA as calculated with the 2−∆∆Ct method. Subjects Controls (mean) Proband Patient III-7 GRN A199V
∆Ct ± SD 8.85 ± 0.45 10.02 ± 0.45 9.63 ± 0.45 10.14 ± 0.41
2-∆∆Ct 0.44 0.58 0.41
% reduction 56 42 59
Figure legends Figure 1. Family pedigree. The proband is indicated by the arrow; black filled symbols represent affected subjects; diagonal lines indicate the deceased and asterisks the patients in whom the mutation has been demonstrated. Subjects’ gender has been masked for the sake of confidentiality. Abbreviations: AD, age of death; AO, age of onset; BD, behavioural disorder; CA, current age; HA, hallucinations; LDnos, language disorder not otherwise specified; mPPA, mixed primary progressive aphasia; Park, parkinsonian syndrome; PNFA, progressive non fluent aphasia. Figure 2. Transaxial PET images of 18FDG uptake at three different sections in patient III-8 (A, B) and III-7 (C, D). Both patients show at 18F-FDG-PET scans similar left relative corticalsubcortical hypometabolism involving the frontal, parietal, temporal cortices and the striatum (A, C). The results of SPM analysis (patient vs 16 healthy controls, age range of controls:35–70, age was considered in the statistical model of SPM as nuisance covariate) show the clusters of significant relative hypometabolism (statistical threshold of p<0.01 uncorrected for voxel height, cluster extent 50 voxels) in each patient as compared to controls superimposed on FDG-PET scans (B,D) and highlight the topography of the most affected cortical-subcortical regions on the left hemisphere. R=right, L=left. Figure 3. Sequencing chromatogram of GRN from the proband. The GRN c.708+4A>T splicing mutation is shown. Capital letters indicate the exon 6 while small letters the intron 6 sequences.
Supplementary Figure S1. MRI scans of the proband (A-C) and his sibling (D-F). In the proband, subject III-8, diffuse cortical atrophy prevailing in the left frontal, temporal and insular cortices is evident, along with multiple T2-hyperintense foci in subcortical and deep periventricular white matter. A: T1-weighted axial scan. B: FLAIR axial scan. C: T2-weighted coronal scan. In the sibling, subject III-7, cortical atrophy predominates in left fronto-temporal regions, with significant involvement of anterior and medial temporal cortex. T2-hyperintense lesions are present in periventricular white matter. D and E: FLAIR axial scans. F: T1-weighted sagittal scan.
1
I
1
2
3
4-6
1-3
3
3
mPPA AO: 68 CA: 72
*
7 7
PNFA AO: 69 CA: 70
BD AO: 68 AD: 83
Park AO: 70 AD: 86
*
8
9-11
3
17-18
12-16
5
7
6
5
4
BD, HA AO: 68 AD: 73
II
III
2
LDnos AO: 65 AD: 75
2
19-23
5
One novel GRN null mutation, two different aphasia phenotypes Verification
All the authors declare that they have no actual or potential conflicts of interest with this work. No authors’ institutions have contracts relating to this research through which they or any other organization may stand to gain financially now or in the future. There are no other agreements (of authors or their institutions) that could be seen as involving a financial interest in this work. We assure that the data contained in the manuscript being submitted have not been previously published, have not been submitted elsewhere and will not be submitted elsewhere while under consideration at Neurobiology of Aging. This study was approved by the local ethical committee (University of Campania “Luigi Vanvitelli” - Naples). All authors have reviewed the contents of the manuscript being submitted, approve of its contents and validate the accuracy of the data.
Yours sincerely, Cinzia Coppola Giacomina Rossi and all the co-authors
S0197-4580(19)30397-5
DOI:
https://doi.org/10.1016/j.neurobiolaging.2019.11.008
Reference:
NBA 10715
To appear in:
Neurobiology of Aging
Received Date: 9 July 2018 Revised Date:
4 September 2019
Accepted Date: 6 November 2019
Please cite this article as: Coppola, C., Oliva, M., Saracino, D., Pappatà, S., Zampella, E., Cimini, S., Ricci, M., Giaccone, G., Di Iorio, G., Rossi, G., One novel GRN null mutation, two different aphasia phenotypes, Neurobiology of Aging (2019), doi: https://doi.org/10.1016/j.neurobiolaging.2019.11.008. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
One novel GRN null mutation, two different aphasia phenotypes
Cinzia Coppolaa, Mariano Olivaa, Dario Saracinoa, Sabina Pappatàb, Emilia Zampellac, Sara Ciminid, Martina Riccid, Giorgio Giacconed, Giuseppe Di Iorioa, Giacomina Rossid
a
Second Division of Neurology, University of Campania “Luigi Vanvitelli” – Naples, Italy
b
Institute of Biostructure and Bioimaging, National Council of Research – Naples, Italy
c
Department of Advanced Biomedical Sciences, “Federico II” University – Naples, Italy
d
Division of Neurology V - Neuropathology, Fondazione IRCCS Istituto Neurologico Carlo Besta – Milan,
Italy
*Correspondence to: Cinzia Coppola, Second Division of Neurology, University of Campania “Luigi Vanvitelli”, Isola 8 – Edificio 10 Policlinico “Federico II” via Pansini 5, 80131 Naples, Italy. Tel.: +39 0815666811; Fax: +39 0815666805; Email: [email protected]
Abstract Progranulin gene (GRN) mutations are among the leading causes of Fronto-temporal lobar degeneration (FTLD), a group of neurodegenerative diseases characterized by remarkable clinical heterogeneity. In this paper we report the new GRN 708+4A>T splicing mutation, identified in two siblings of a family with several members affected by cognitive, behavioral and motor disorders. Plasma progranulin dosage and GRN expression analysis, together with in silico prediction studies, supported the pathogenicity of the mutation. Both the patients displayed a clinical syndrome in which language impairment was largely predominant. However, motor speech deficits were the major feature in one case, diagnosed as progressive non fluent aphasia, whereas marked semantic alterations were present in the other, whose clinical phenotype was in favor of a mixed aphasia. The profile of neuroanatomical alterations from imaging studies was in line with the clinical phenotypes. Therefore, also this novel GRN mutation is associated with haploinsufficiency and phenotypic heterogeneity, which are both typical features of progranulinopathies.
Keywords: Fronto-temporal lobar degeneration, GRN, progranulin protein, mutation, progressive non fluent aphasia, primary progressive aphasia.
1. Introduction Fronto-temporal lobar degeneration (FTLD) comprises a heterogeneous group of diseases that share predominant impairment of behavioural, executive and language functions. It accounts for about 515% of all cases of dementia (Rademakers et al., 2012), and is the second cause of cognitive decline in presenile age, its onset typically ranging between the 5th and the 7th decade (Bird et al., 2003). FTLD is characterized by a remarkable heterogeneity from a clinical, pathological and genetic point of view. Clinical presentations of FTLD include behavioral variant of fronto-temporal dementia (bvFTD) (Rascovsky et al., 2011), and two variants of primary progressive aphasia (PPA), progressive non fluent aphasia (PNFA) and semantic dementia (SD) (Gorno-Tempini et al., 2011; Leyton et al., 2011). In addition, there are some cases exhibiting an overlap with motor neuron disease/amyotrophic lateral sclerosis (FTD/ALS), and others associated with parkinsonism, namely progressive supranuclear palsy (PSP) and cortico-basal syndrome (CBS) (Rabinovici et al., 2010). As for the genetic aspects, the genes coding for progranulin (GRN), microtubule-associated protein tau (MAPT), and chromosome 9 open reading frame 72 (C9orf72) account for the majority of familial cases (Bang et al., 2015; Benussi et al., 2015). Other genes, such as those coding for valosin containing protein (VCP) or charged multivesicular body protein 2B (CHMP2B), are observed only in a small number of cases (Skibinski et al., 2005; Watts et al., 2004). Progranulin is a highly conserved glycoprotein coded by the GRN gene, located on the chromosome 17q21, in proximity to the MAPT locus (Ahmed et al., 2007). Its biological roles, mediated by either the whole protein or its proteolytic cleavage products, called granulins, include cell proliferation, inflammation, lysosome function, neurite outgrowth, neuronal survival (Cenik et al., 2012), and metabolic homeostasis (Korolczuk et al., 2017). GRN mutations account for 5-10% of FTLD cases, but this proportion increases to 22% in patients with positive family history (Seelar et al., 2011). To date, more than 170 GRN mutations have been described (Stenson et al., 2017). Most of pathogenic mutations are frameshift, non-sense or splicing mutations resulting in a premature termination
codon, degradation of mutated mRNA and consequent haploinsufficiency (Baker et al., 2006; Petkau et al., 2014). GRN mutations are characterized by a wide inter- and intra-familial clinical heterogeneity, which include bvFTD, PNFA and CBS (Le Ber et al., 2007; Davion et al., 2007), while the association with SD has been reported in rare instances (Cerami et al., 2013; Van Mossevelde et al., 2016; Wauters et al., 2018). Early appearance of memory impairment, apraxia and sometimes visuospatial disturbances is considered to be evocative of this underlying genetic background (Le Ber et al., 2008). Structural and functional neuroimaging studies may also highlight some suggestive aspects: cortical atrophy is generally asymmetric and involves frontal, temporal and parietal lobes (Benussi et al., 2015), while 18F-fluoro-2-deoxy-glucose (18FDG) positron emission tomography (PET) often reveals asymmetrical frontal and temporal hypometabolism often extending to the parietal regions (Meeter et al., 2017). The involvement of parietal and posterior cingulate cortices usually differentiates FTLD associated with GRN mutations from sporadic cases (Coppola et al., 2017; Milan et al., 2017). In this paper we describe the clinical and neuroradiological features of two related patients carrying the novel GRN c.708+4A>T splicing mutation. Both in silico predictions and expression analysis demonstrated that the mutation affects the splicing site, leading to progranulin haploinsufficiency.
2. Patients and Methods Pedigree of the family carrying the GRN c.708+4A>T splicing mutation is depicted in Figure 1. 2.1 Proband The proband (III-8) was a 70-year-old patient whose past medical history included arterial hypertension and hypertriglyceridemia. This patient had presented language disturbances with phonetic paraphasias and effortful speech with frequent interruptions since the age of 69, and also showed progressive behavioral disorders mainly characterized by apathy. These symptoms had been slowly worsening. Mini Mental State Examination (MMSE) score was 16/30. The extensive
neuropsychological examination showed multiple language impairments (phonological and semantic fluencies, writing, repetition, calculation, auditory and visual comprehension), executive dysfunctions and frontal behavioral disturbances (Supplementary Table S1). Clinical Dementia Rating (CDR) scale was 2, being communication abilities, interpersonal interactions and personal care among the most compromised domains. EEG showed widespread dysregulation in cerebral activity. Brain MRI revealed diffuse cortical atrophy prevailing in the left hemisphere, multiple T2hyperintense foci in subcortical and deep periventricular white matter and an empty sella (Supplementary Figure S1 A-C). Brain FDG-PET evidenced a reduction of glucose uptake in the anterior cingulate, frontal/insular cortex, especially on the left side, with a milder relative hypometabolism in left temporal and parietal cortices and striatum (Figure 2 A, B). Cerebro-spinal fluid (CSF) biomarkers were within reference ranges. The clinical diagnosis was Progressive Non Fluent Aphasia (PNFA). Familial history was positive for cognitive disorders in in one of the parents (II-3) and in one sibling (III-7). Moreover, one of the parent’s siblings (II-5) had likely been affected by an atypical parkinsonism since the age of 70 and died at the age of 86, while another sibling (II-7) had been affected by a behavioral disorder since the age of 68 and died at the age of 83. 2.2 Case III-7 A sibling of the proband was a 72-year-old patient whose medical antecedents were prostate cancer and cerebrovascular disease. He had insidiously manifested progressive language impairments (comprehension problems, anomia, semantic paraphasias and neologisms) and mild behavioral disorders (irritability, perseveration, disinhibition) since the age of 68. MMSE score was 23/30. The extensive neuropsychological examination confirmed the language impairment, with alteration of phonological and semantic fluencies, writing, repetition, denomination and comprehension of phrases. Short-term memory and executive functions were also impaired (Supplementary Table S1). EEG showed widespread dysregulation in cerebral activity. CDR scale was 1, mainly due to minor behavioral changes, problem solving difficulties and moderately impaired comprehension during
conversation. Brain MRI revealed multiple T2-hyperintense foci in white matter and diffuse cortical atrophy with left prevalence (Supplementary Figure S1 D-F). Brain FDG-PET evidenced reduced cortical and subcortical relative glucose metabolism in the left cerebral hemisphere, mainly involving the lateral and medial temporal cortex and, to a lesser extent, the parietal, posterior cingulate, frontal lateral/medial and anterior cingulate cortices and striatum (Figure 2 C, D). CSF biomarkers were within reference ranges. The clinical diagnosis was mixed aphasia with prevalent aspects of semantic impairment overlapping with a frontal behavioral disorder. The patients and their relatives expressed their informed consent to undergo genetic testing and to participate to research studies. The present study has been approved by the local ethical committee (University of Campania “Luigi Vanvitelli”, Naples). 2.3 Plasma progranulin dosage In the diagnostic procedure of our Laboratory, the first analysis we perform in FTLD cases is plasma progranulin dosage. If the progranulin level is below the reported cut-off (Ghidoni 2012), we sequence GRN gene; otherwise we analyze C9orf72 and sequence MAPT genes. To determine the level of plasma progranulin, an ELISA kit (Human Progranulin ELISA kit; Adipogen) was used, according to manufacturer’s instructions. 2.4 Genetic analysis 2.4.1 DNA analysis Genomic DNA was extracted from peripheral blood lymphocytes (PBL) of proband and subject III7. For the proband, all coding exons, flanking intronic sequences and 5' and 3' untranslated regions of GRN gene were amplified using previously published methods (Baker et al., 2006, Cruts et al., 2006), and amplified fragments were sequenced using the Big Dye terminator v 3.1 cycle sequencing kit (Applied Biosystems, Life Technologies) and analyzed on an ABI 3100 gene analyzer (Applied Biosystems, Life Technologies). Only a region containing the GRN splicing site c.708+4A was amplified and sequenced for subject III-7.
In order to exclude mutations from other known FTD causative genes, we carried out a next generation sequencing (NGS) analysis on the proband and subject III-7. Nextera Rapid Capture system for enrichment (Illumina) coupled with gene-specific probes (Integrated DNA Technologies) was used to sequence a panel containing the following genes: APP, PSEN1, PSEN2; PRNP; GRN, MAPT, CHMP2B, FUS, TARDBP, VCP, TREM2, CSF1R, TBK1. Sequencing was performed on the Illumina MiSeq instrument and MiSeq Reporter software was used for alignment and variant calling. Alignment was performed against the human genome UCSC hg19 and variants annotated with Variant Studio software (Illumina). Low-quality variants were filtered out using the Illumina Qscore threshold of 30; in addition, variants with a minimum allele frequency higher than 1% in GnomAD (Genome Aggregation Database, http://gnomad.broadinstitute.org/) were filtered out. Most of the genes were sequenced by NGS with 100% coverage (read depth ≥ 20X). The few exons not covered by NGS were sequenced by standard Sanger technique, to give a 100% final sequencing. 2.4.2 RNA analysis Total RNA was isolated from blood samples of proband, patient III-7, two wild-type controls and 1 patient carrying the GRN A199V mutation (positive control), using the PAXgene® Blood RNA Kit (PreAnalytiX, Qiagen). cDNA was synthesized using cloned AMV First-strand synthesis kit (Invitrogen). cDNA was subjected to quantitative real-time PCR using premade TaqMan gene expression assays: Hs00173570_m1 for GRN and Hs99999903_m1 for β-actin as an endogenous control for sample normalization (Applied Biosystems, Life Technologies). The cDNA was amplified in triplicate and run on a ViiA7 Real-time PCR system (Applied Biosystems, Life Technologies). The relative transcription levels of GRN mRNA were calculated by using the 2-∆∆Ct method (Livak et al., 2001), where ∆∆Ct = ∆Ctpatient –∆Ctcontrols and ∆Ct = CtGRN – Ctβactin. 2.5 Bioinformatics analysis
We used the in silico tools Netgene2 (www.cbs.dtu.dk/services/NetGene2/), BDGP splice Site Prediction (www.fruitfly.org/seq_tools/splice.html) and Human Splicing Finder (www.umd.be/HSF3/) to evaluate the effect of the GRN c.708+4A>T mutation on splice site.
3. Results 3.1 Plasma progranulin dosage Plasma progranulin levels were 37.5 and 26.8 ng/mL for proband and patient III-7, respectively, below the calculated cut-off level for GRN mutation prediction in FTLD patients (61.55 ng/mL; Ghidoni et al., 2012). 3.2 Genetic analysis 3.2.1 DNA analysis Sequencing of DNA disclosed the GRN c.708+4A>T base substitution (References: (i) chr17:42,428,172A>T, UCSC Genome Browser on GRCh37/hg19 assembly; (ii) g.101705A>T, reverse complement of GenBank AC003043.1 starting at nucleotide 1; (iii) NM_002087.3:c.708+4A>T, starting at the translation initiation site; (iv) exon 6/intron 6 boundary site) in the proband and subject III-7 (Figure 3). This is a splicing mutation predicting the exon 6 skipping, a frameshift of the coding region and a premature termination codon in exon 7, leading to the degradation of mutant mRNA (Gass et al.,2006). This mutation is not present in the Genome Aggregation Database (gnomAD) data set, which includes 123,136 exome sequences and 15,496 whole-genome sequences from unrelated individuals. Thus, it is a new mutation. No pathogenic mutation was found in any of the genes sequenced by NGS panel, except the GRN mutation previously disclosed by traditional sequencing. 3.2.2 RNA analysis Quantitative real-time PCR demonstrated mRNA expression decreased to 56% and 42% in proband and patient III-7, respectively, compared with control samples (Table 1). 3.3 Bioinformatic analysis
Netgene2, BDGP splice Site Prediction and Human Splicing Finder all predicted the loss of splice site in presence of the base substitution GRN c.708+4A>T (data not shown).
4. Discussion GRN mutations show a wide clinical variability, the most frequent phenotypes being bvFTD, PNFA and CBS (Le Ber et al. 2008; Rossi et al., 2011). The association between GRN mutations and SD remains more elusive. SD was described in one familial case carrying the GRN T409M mutation, whose pathological nature is uncertain being a missense and not a null mutation; moreover, no progranulin dosage was available (Cerami et al., 2013). Additional SD cases were signaled in Belgian kindreds. Four patients were reported into a clinical-pathological cohort of 52 GRN-mutated FTLD patients (Van Mossevelde et al., 2016), and two were identified within a large family carrying the IVS1+5 G>C mutation (Wauters et al., 2018). Conversely, the SD phenotype was not reported in association with GRN mutations in the largest series of genetic PPA patients described so far (Ramos et al., 2019). Finally, FTD/ALS phenotype was described in one sporadic case carrying the GRN S120Y mutation (Schymick et al., 2007), which was also described in a case of sporadic ALS (Del Bo et al., 2011). The pathogenicity is unclear as the amino acid is not highly conserved across species and is not localized in a region critical to progranulin function; in addition, it is reported in gnomAD, although with a low frequency (0.1%). Our family shows a relevant phenotypic variability, not only considering different generations, but also within the same generation. The mode of onset and the extensive neuropsychological evaluation were strongly suggestive of PNFA in the proband, being reduced verbal fluency and apraxia of speech the most relevant impairments, in association with milder behavioral disorders and dysexecutive aspects. The sibling, patient III-7, displayed language impairments too, but their pattern hampers a precise classification according to currently adopted criteria (Gorno-Tempini et al., 2011). There were significant elements in favor of a semantic variant of PPA, in particular
naming deficits, comprehension impairment, and semantic paraphasias. However, those aspects occurred together with deficits in phonological encoding and repetition of sentences. For that reason, we formulated the diagnosis of mixed aphasia, associated with a frontal cognitive and behavioral syndrome. In both cases brain MRI showed a diffuse cortical atrophy with left prevalence and a significant burden of white matter lesions; this latter aspect has been frequently reported in GRN-related FTD (Caroppo et al., 2014). Likewise, brain 18FDG-PET also evidenced a similar pattern of hypometabolism between the two patients, namely a left frontal and temporoparietal hypometabolism, with mild involvement of the basal ganglia. The asymmetric anterior and posterior cortical relative hypometabolism has been often reported in cohorts or single cases of GRN mutations (Spina et al., 2007; Coppola et al., 2017; Milan et al., 2017). Interestingly, in our patient III.7 with prevalent semantic language disturbance the hypometabolism involved mainly the left anterior and lateral temporal lobe, whereas in the proband affected by PNFA the hypometabolism was more marked in the left fronto-insular cortex, suggesting an association between metabolic and cognitive phenotypes. A remarkable intra-familial phenotypic heterogeneity, even considering family members belonging to the same generation, appears to be a recurrent characteristic of GRN mutations. Notably the age at onset, the clinical phenotype and the duration of disease represent the main aspects of that variability (Rossi et al., 2011; Hosaka et al., 2017). The identification of genetic factors that may account for this heterogeneity is currently an active field of research (Chitramuthu et al., 2017; Wauters et al., 2017). Our two cases are also in line with the previous observation that GRN-associated PPA has specific aspects that do not always allow a proper classification (Rohrer et al., 2010). Phonological disturbances and difficulties in repetition were shared by both our two patients, and they are indeed two features often occurring with GRN mutations (Rohrer et al., 2010). However, the overall clinical pictures were quite divergent, given the presence of motor speech impairment in one case, and of semantic deficits in the other.
5. Conclusions In summary, in this work we describe the novel GRN c.708+4A>T mutation, whose pathogenic nature is evidenced by: (i) the segregation with the disease within the family; (ii) the results of the GRN expression analyses, showing decreased production of mRNA, predicted also by the bioinformatics analysis; (iii) the reduced levels of plasma progranulin in both subjects. An interesting aspect associated with this mutation is the variability of the profile of linguistic impairment evidenced in two mutation carriers, with an unclassifiable aphasia phenotype, not fitting the clinical and neurolinguistic phenotypes proposed by current PPA criteria, in one case. Finally, our report underlines an already well-established concept, namely the remarkable phenotypic heterogeneity occurring with GRN mutations even among family members belonging to the same generation.
Acknowledgements The authors have no conflicts of interest to disclose.
References Ahmed Z, Mackenzie IR, Hutton ML, Dickson DW. Progranulin in frontotemporal lobar degeneration and neuroinflammation. J Neuroinflammation 2007;4(1):7. Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Berger Z, Eriksen J, Robinson T, Zehr C, Dickey CA, Crook R, McGowan E, Mann D, Boeve B, Feldman H, Hutton M. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 2006;442(7105):916-9.
Bang J, Spina S, Miller BL. Frontotemporal dementia. Lancet 2015;386(10004):1672-82. Benussi A, Padovani A, Borroni B. Phenotypic heterogeneity of monogenic frontotemporal dementia. Front Aging Neurosci 2015;7:171. Bird T, Knopman D, VanSwieten J, Rosso S, Feldman H, Tanabe H, Graff-Raford N, Geschwind D, Verpillat P, Hutton M. Epidemiology and genetics of frontotemporal dementia/Pick’s disease. Ann Neurol 2003;54(Suppl 5):S29–31. Caroppo P, Le Ber I, Camuzat A, Clot F, Naccache L, Lamari F, De Septenville A, Bertrand A, Belliard S, Hannequin D, Colliot O, Brice A. Extensive white matter involvement in patients with frontotemporal lobar degeneration: think progranulin. JAMA Neurol 2014;71:1562-6. Cenik B, Sephton CF, Cenik BK, Herz J, Yu G. Progranulin: a proteolytically processed protein at the crossroads of inflammation and neurodegeneration. J Biol Chem 2012;287(39):32298-306. Cerami C, Marcone A, Galimberti D, Villa C, Fenoglio C, Scarpini E, Cappa SF. Novel missense progranulin gene mutation associated with the semantic variant of primary progressive aphasia. J Alzheimers Dis 2013;36(3):415-20. Chitramuthu BP, Bennett HPJ, Bateman A. Progranulin: a new avenue towards the understanding and treatment of neurodegenerative disease. Brain. 2017;140(12):3081-104. Coppola C, Saracino D, Puoti G, Lus G, Dato C, Le Ber I, Pariente J, Caroppo P, Piccoli E, Tagliavini F, Di Iorio G, Rossi G. A cluster of progranulin C157KfsX97 mutations in Southern Italy: clinical characterization and genetic correlations. Neurobiol Aging 2017;49:219.e5-219.e13. Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Randenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, Kumar-Singh S, Van
Broeckhoven C. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 2006;442(7105):920-4. Davion S, Johnson N, Weintraub S, Mesulam MM, Engberg A, Mishra M, Baker M, Adamson J, Hutton M, Rademakers R, Bigio EH. Clinicopathologic correlation in PGRN mutations. Neurology. 2007;69(11):1113-21. Del Bo R, Corti S, Santoro D, Ghione I, Fenoglio C, Ghezzi S, Ranieri M, Galimberti D, Mancuso M, Siciliano G, Briani C, Murri L, Scarpini E, Schymick JC, Traynor BJ, Bresolin N, Comi GP. No major progranulin genetic variability contribution to disease etiopathogenesis in an ALS Italian cohort. Neurobiol Aging 2011;32(6):1157-8. Gass J, Cannon A, Mackenzie IR, Boeve B, Baker M, Adamson J, Crook R, Melquist S, Kuntz K, Petersen R, Josephs K, Pickering-Brown SM, Graff-Radford N, Uitti R, Dickson D, Wszolek Z, Gonzalez J, Beach TG, Bigio E, Johnson N, Weintraub S, Mesulam M, White CL 3rd, Woodruff B, Caselli R, Hsiung GY, Feldman H, Knopman D, Hutton M, Rademakers R. Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration. Hum Mol Genet 2006;15(20):2988-3001. Ghidoni R, Stoppani E, Rossi G, Piccoli E, Albertini V, Paterlini A, Glionna M, Pegoiani E, Agnati LF, Fenoglio C, Scarpini E, Galimberti D, Morbin M, Tagliavini F, Binetti G, Benussi L. Optimal plasma progranulin cutoff value for predicting null progranulin mutations in neurodegenerative diseases: A multicenter Italian study. Neurodegener Dis 2012;9(3):121-7. Gorno-Tempini ML, Hillis AE, Weintraub S, Kertesz A, Mendez M, Cappa SF, Ogar JM, Rohrer JD, Black S, Boeve BF, Manes F, Dronkers NF, Vandenberghe R, Rascovsky K, Patterson K, Miller BL, Knopman DS, Hodges JR, Mesulam MM, Grossman M. Classification of primary progressive aphasia and its variants. Neurology 2011;76(11):1006-14.
Hosaka T, Ishii K, Miura T, Mezaki N , Kasuga K, Ikeuchi T, Tamaoka A. A novel frameshift GRN mutation results in frontotemporal lobar degeneration with a distinct clinical phenotype in two siblings: case report and literature review. BMC Neurol. 2017;17(1):182. Korolczuk A, Bełtowski J. Progranulin, a new adipokine at the crossroads of metabolic syndrome, diabetes, dyslipidemia and hypertension. Curr Pharm Des 2017;23(10):1533-9. Le Ber I, van der Zee J, Hannequin D, Gijselinck I, Campion D, Puel M, Laquerrie`re A, De Pooter T, Camuzat A, Van den Broeck M, Dubois B, Sellal F, Lacomblez L, Vercelletto M, ThomasAntérion C, Michel BF, Golfier V, Didic M, Salachas F, Duyckaerts C, Cruts M, Verpillat P, Van Broeckhoven C, Brice A, French Research Network on FTD/FTD-MND. Progranulin null mutations in both sporadic and familial frontotemporal dementia. Human Mutat 2007;28:846-55. Le Ber, I, Camuzat, A, Hannequin, D, Pasquier, F, Guedj, E, Rovelet-Lecrux, A, Hahn-Barma, 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 Broeckhoven, C, Lambert, JC, Verpillat, P, Campion, D, Habert, MO, Dubois, B, Brice, A, French research network on FTD/FTD-MND. Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study. Brain 2008;131:732-46. Leyton CE, Villemagne VL, Savage S, Pike KE, Ballard KJ, Piguet O, Burrell JR, Rowe CC, Hodges JR. Subtypes of progressive aphasia: application of the International Consensus Criteria and validation using β-amyloid imaging. Brain 2011;134:3030-43. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-8. Meeter LH, Kaat LD, Rohrer JD, van Swieten JC. Imaging and fluid biomarkers in frontotemporal dementia. Nat Rev Neurol 2017;13(7):406-19.
Milan G, Napoletano S, Pappatà S, Gentile MT, Colucci-D'Amato L, Della Rocca G, Maciag A, Rossetti CP, Fucci L, Puca A, Grossi D, Postiglione A, Vitale E. GRN deletion in familial frontotemporal dementia showing association with clinical variability in 3 familial cases. Neurobiol Aging 2017;53:193.e9-193.e16. Petkau TL, Leavitt BR. Progranulin in neurodegenerative disease. Trends Neurosci. 2014;37(7):388-98. Rabinovici GD, Miller BL. Frontotemporal lobar degeneration: epidemiology, pathophysiology, diagnosis and management. CNS Drugs 2010;24(5):375-98. Rademakers R, Neumann M, Mackenzie IR. Advances in the molecular basis of frontotemporal dementia. Nat Rev Neurol 2012;8(8):423-34. Ramos EM, Dokuru DR, Van Berlo V, Wojta K, Wang Q, Huang AY, Miller ZA, Karydas AM, Bigio EH, Rogalski E, Weintraub S, Rader B, Miller BL, Gorno-Tempini ML, Mesulam MM, Coppola G. Genetic screen in a large series of patients with primary progressive aphasia. Alzheimers Dement 2019;15(4):553-60. Rascovsky K, Hodges JR, Knopman D, Mendez MF, Kramer JH, Neuhaus J, van Swieten JC, Seelaar H, Dopper EG, Onyike CU, Hillis AE, Josephs KA, Boeve BF, Kertesz A, Seeley WW, Rankin KP, Johnson JK, Gorno-Tempini ML, Rosen H, Prioleau-Latham CE, Lee A, Kipps CM, Lillo P, Piguet O, Rohrer JD, Rossor MN, Warren JD, Fox NC, Galasko D, Salmon DP, Black SE, Mesulam M, Weintraub S, Dickerson BC, Diehl-Schmid J, Pasquier F, Deramecourt V, Lebert F, Pijnenburg Y, Chow TW, Manes F, Grafman J, Cappa SF, Freedman M, Grossman M, Miller BL. Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 2011;134:2456-77.
Rohrer JD, Crutch SJ, Warrington EK, Warren JD. Progranulin-associated primary progressive aphasia: a distinct phenotype? Neuropsychologia 2010;48(1):288-97. Rossi G, Piccoli E, Benussi L, Caso F, Redaelli V, Magnani G, Binetti G, Ghidoni R, Perani D, Giaccone G, Tagliavini F. A novel progranulin mutation causing frontotemporal lobar degeneration with heterogeneous phenotypic expression. J Alzheimers Dis 2011;23(1):7-12. Schymick JC, Yang Y, Andersen PM, Vonsattel JP, Greenway M, Momeni P, Elder J, Chiò A, Restagno G, Robberecht W, Dahlberg C, Mukherjee O, Goate A, Graff-Radford N, Caselli RJ, Hutton M, Gass J, Cannon A, Rademakers R, Singleton AB, Hardiman O, Rothstein J, Hardy J, Traynor BJ. Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes. J Neurol Neurosurg Psychiatry 2007;78(7):754-6. Seelaar H, Rohrer JD, Pijnenburg YA, Fox NC, van Swieten JC. Clinical, genetic and pathological heterogeneity of frontotemporal dementia: a review. J Neurol Neurosurg Psychiatry 2011;82(5):476-86. Skibinski G, Parkinson NJ, Brown JM, Chakrabarti L, Lloyd SL, Hummerich H, Nielsen JE, Hodges JR, Spillantini MG, Thusgaard T, Brandner S, Brun A, Rossor MN, Gade A, Johannsen P, Sørensen SA, Gydesen S, Fisher EM, Collinge J. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat Genet 2005;37:806-8. Spina S, Murrell JR, Huey ED, Wassermann EM, Pietrini P, Grafman J, Ghetti B. Corticobasal syndrome associated with the A9D progranulin mutation. J Neuropathol Exp Neurol 2007;66:892900. Stenson PD, Mort M, Ball EV, Evans K, Hayden M, Heywood S, Hussain M, Phillips AD, Cooper DN. The Human Gene Mutation Database: towards a comprehensive repository of inherited
mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet. 2017;136(6):665-77. Van Mossevelde S, van der Zee J, Gijselinck I, Engelborghs S, Sieben A, Van Langenhove T, De Bleecker J, Baets J, Vandenbulcke M, Van Laere K, Ceyssens S, Van den Broeck M, Peeters K, Mattheijssens M, Cras P, Vandenberghe R, De Jonghe P, Martin JJ, De Deyn PP, Cruts M, Van Broeckhoven C. Clinical features of TBK1 carriers compared with C9orf72, GRN and non-mutation carriers in a Belgian cohort. Brain. 2016;139(2):452-67. Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP, Kimonis VE. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 2004;36:377-81. Wauters E, Van Mossevelde S, Van der Zee J, Cruts M, Van Broeckhoven C. Modifiers of GRNAssociated Frontotemporal Lobar Degeneration. Trends Mol Med 2017;23(10):962-79. Wauters E, Van Mossevelde S, Sleegers K, van der Zee J, Engelborghs S, Sieben A, Vandenberghe R, Philtjens S, Van den Broeck M, Peeters K, Cuijt I, De Coster W, Van Langenhove T, Santens P, Ivanoiu A, Cras P, De Bleecker JL, Versijpt J, Crols R, De Klippel N, Martin JJ, De Deyn PP, Cruts M, Van Broeckhoven C; Belgian Neurology (BELNEU) Consortium. Clinical variability and onset age modifiers in an extended Belgian GRN founder family. Neurobiol Aging 2018;67:84-94.
Table 1. The percentage reduction of GRN mRNA as calculated with the 2−∆∆Ct method. Subjects Controls (mean) Proband Patient III-7 GRN A199V
∆Ct ± SD 8.85 ± 0.45 10.02 ± 0.45 9.63 ± 0.45 10.14 ± 0.41
2-∆∆Ct 0.44 0.58 0.41
% reduction 56 42 59
Figure legends Figure 1. Family pedigree. The proband is indicated by the arrow; black filled symbols represent affected subjects; diagonal lines indicate the deceased and asterisks the patients in whom the mutation has been demonstrated. Subjects’ gender has been masked for the sake of confidentiality. Abbreviations: AD, age of death; AO, age of onset; BD, behavioural disorder; CA, current age; HA, hallucinations; LDnos, language disorder not otherwise specified; mPPA, mixed primary progressive aphasia; Park, parkinsonian syndrome; PNFA, progressive non fluent aphasia. Figure 2. Transaxial PET images of 18FDG uptake at three different sections in patient III-8 (A, B) and III-7 (C, D). Both patients show at 18F-FDG-PET scans similar left relative corticalsubcortical hypometabolism involving the frontal, parietal, temporal cortices and the striatum (A, C). The results of SPM analysis (patient vs 16 healthy controls, age range of controls:35–70, age was considered in the statistical model of SPM as nuisance covariate) show the clusters of significant relative hypometabolism (statistical threshold of p<0.01 uncorrected for voxel height, cluster extent 50 voxels) in each patient as compared to controls superimposed on FDG-PET scans (B,D) and highlight the topography of the most affected cortical-subcortical regions on the left hemisphere. R=right, L=left. Figure 3. Sequencing chromatogram of GRN from the proband. The GRN c.708+4A>T splicing mutation is shown. Capital letters indicate the exon 6 while small letters the intron 6 sequences.
Supplementary Figure S1. MRI scans of the proband (A-C) and his sibling (D-F). In the proband, subject III-8, diffuse cortical atrophy prevailing in the left frontal, temporal and insular cortices is evident, along with multiple T2-hyperintense foci in subcortical and deep periventricular white matter. A: T1-weighted axial scan. B: FLAIR axial scan. C: T2-weighted coronal scan. In the sibling, subject III-7, cortical atrophy predominates in left fronto-temporal regions, with significant involvement of anterior and medial temporal cortex. T2-hyperintense lesions are present in periventricular white matter. D and E: FLAIR axial scans. F: T1-weighted sagittal scan.
1
I
1
2
3
4-6
1-3
3
3
mPPA AO: 68 CA: 72
*
7 7
PNFA AO: 69 CA: 70
BD AO: 68 AD: 83
Park AO: 70 AD: 86
*
8
9-11
3
17-18
12-16
5
7
6
5
4
BD, HA AO: 68 AD: 73
II
III
2
LDnos AO: 65 AD: 75
2
19-23
5
One novel GRN null mutation, two different aphasia phenotypes Verification
All the authors declare that they have no actual or potential conflicts of interest with this work. No authors’ institutions have contracts relating to this research through which they or any other organization may stand to gain financially now or in the future. There are no other agreements (of authors or their institutions) that could be seen as involving a financial interest in this work. We assure that the data contained in the manuscript being submitted have not been previously published, have not been submitted elsewhere and will not be submitted elsewhere while under consideration at Neurobiology of Aging. This study was approved by the local ethical committee (University of Campania “Luigi Vanvitelli” - Naples). All authors have reviewed the contents of the manuscript being submitted, approve of its contents and validate the accuracy of the data.
Yours sincerely, Cinzia Coppola Giacomina Rossi and all the co-authors