Frontotemporal dementia in a large Swedish family is caused by a progranulin null mutation

Neurogenetics (2009) 10:27–34 DOI 10.1007/s10048-008-0155-z

ORIGINAL ARTICLE

Frontotemporal dementia in a large Swedish family is caused by a progranulin null mutation Lena Skoglund & RoseMarie Brundin & Tommie Olofsson & Hannu Kalimo & Sofie Ingvast & Elin S. Blom & Vilmantas Giedraitis & Martin Ingelsson & Lars Lannfelt & Hans Basun & Anna Glaser

Received: 24 July 2008 / Accepted: 23 September 2008 / Published online: 15 October 2008 # Springer-Verlag 2008

Abstract Mutations in the progranulin (PGRN) gene have recently been identified in families with frontotemporal lobar degeneration and ubiquitin-positive brain inclusions linked to chromosome 17q21. We have previously described a Swedish family displaying frontotemporal dementia with rapid progression and linkage to chromosome 17q21. In this study, we performed an extended clinical and neuropathological investigation of affected members of the family and a genetic analysis of the PGRN gene. There was a large variation of the initial presenting symptoms in this family, but common clinical features were non-fluent aphasia and loss of spontaneous speech as well as personality and behavioural changes. Mean age at onset was 54 years with disease duration of close to 4 years. Neuropathological examination revealed frontotemporal neurodegeneration with

ubiquitin and TAR DNA binding protein-43 immunoreactive intraneuronal inclusions. Mutation screening of the PGRN gene identified a 1 bp deletion in exon 1 causing a frameshift of the coding sequence and introducing a premature termination codon in exon 2 (Gly35GlufsX19). Analysis of PGRN messenger RNA (mRNA) levels revealed a considerable decrease in lymphoblasts from mutation carriers and fragment size separation, and sequence analysis confirmed that the mutated mRNA allele was almost absent in these samples. In conclusion, the PGRN Gly35fs mutation causes frontotemporal dementia with variable clinical presentation in a large Swedish family, most likely through nonsense-mediated decay of mutant PGRN mRNA and resulting haploinsufficiency. Keywords Frontotemporal lobar degeneration . Frontotemporal dementia . Progranulin . Ubiquitin . TDP-43

Electronic supplementary material The online version of this article (doi:10.1007/s10048-008-0155-z) contains supplementary material, which is available to authorized users. L. Skoglund (*) : R. Brundin : S. Ingvast : E. Blom : V. Giedraitis : M. Ingelsson : L. Lannfelt : H. Basun : A. Glaser Department of Public Health and Caring Sciences, Uppsala University, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden e-mail: [email protected] T. Olofsson Department of Surgical Sciences, Uppsala University, Uppsala, Sweden H. Kalimo Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden H. Kalimo Department of Pathology, University of Helsinki, Helsinki, Finland

Introduction Frontotemporal lobar degeneration (FTLD) refers to a heterogeneous group of neurodegenerative disorders that account for up to 15% of all cases of dementia [1]. FTLD can be divided into three clinical subtypes, of which frontotemporal dementia (FTD) comprises the major group [2]. FTD is characterised by a progressive change in behaviour and personality often accompanied by language deficits. In some patients, language dysfunction presents as an initial symptom with behavioural changes appearing later in the course of the disease, further dividing FTLD into progressive non-fluent aphasia (PNFA) and semantic dementia. Neuropathologically, FTLD is characterised by severe neurodegeneration of the frontal and temporal cortices. Approximately one third of the FTLD cases

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present with intraneuronal protein inclusions consisting of the microtubule associated protein tau (MAPT) [3], while 40–65% of the cases are characterised by intraneuronal inclusions composed of ubiquitin and TAR DNA binding protein-43 (TDP-43) [4–6]. A positive family history of dementia can be found in approximately 40% of all FTLD patients [7–9]. Up to 20% of the familial cases are caused by mutations in the MAPT gene located on chromosome 17q21 [10–12]. These socalled FTDP-17 patients present clinically with disturbances in behaviour and personality, together with parkinsonian features. The neuropathology of FTDP-17 patients is characterised by abnormal accumulation of hyperphosphorylated tau in the cytoplasm of neurons and glial cells. After the discovery of mutations in the MAPT gene, it became evident that all families with conclusive linkage to chromosome 17 did not have mutations in the MAPT gene [13–20]. Neuropathologically, cases from these families lack tau pathology and are instead characterised by the presence of TDP-43-immunoreactive intraneuronal inclusions [21]. Recently, mutations in the progranulin (PGRN) gene were found among affected individuals in these families [22, 23]. To date, 56 PGRN mutations have been identified in FTLD patients worldwide (http://www.molgen. ua.ac.be/FTDMutations). Most mutations identified in the PGRN gene are nonsense, frameshift and splice-site mutations that create premature termination codons in the PGRN messenger RNA (mRNA) sequence. The mutant mRNA is degraded by nonsense-mediated decay (NMD),

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consistent with haploinsufficiency being the pathogenic mechanism of PGRN mutations [22, 23]. PGRN is a 593-amino acid glycoprotein composed of 7.5 tandem repeats of highly conserved 12-cysteine motifs, which can be cleaved to form a family of 6-kDa peptides called granulins [24]. PGRN is a growth factor involved in multiple processes including wound repair, inflammation, development and tumorigenesis [25]. The function of PGRN in the brain is not fully understood, but it might function as a neurotrophic factor enhancing axonal outgrowth and neuronal survival [26]. We previously described a Swedish family with rapidly progressive FTD with linkage to chromosome 17q21 and lacking mutations in the MAPT gene [14, 27]. In this paper, we report an extended clinical and neuropathological investigation of the family and provide evidence of the PGRN Gly35fs mutation as a cause of the disease in this family.

Materials and methods Subjects The pedigree of the Swedish family investigated is illustrated in Fig. 1. The family consisted of 39 individuals spanning four generations and included 14 affected family members. The study was approved by the Ethical Committee of Uppsala University, Uppsala, Sweden.

Fig. 1 Pedigree structure of a Swedish FTD family with a PGRN Gly35fs mutation. Affected individuals are indicated by filled symbols. Question marks indicate individuals with unknown disease status. Plus signs indicate mutation carriers and minus signs indicate non-mutation carriers

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Immunohistochemical analysis A consented brain autopsy was performed on patient IV-4. The right half of the brain and the spinal cord were fixed in phosphate-buffered 4% formaldehyde and selected specimens were routinely processed for embedding in paraffin, while the left half was sectioned and frozen at −80°C. Paraffin sections were stained with haematoxylin and eosin and modified Bielschowsky silver methods. Immunohistochemistry was carried out using antibodies directed against hyperphosphorylated tau (AT8, 1:1000; Innogenetics, Ghent, Belgium), amyloid-β (NCL-B-Amyloid, clone 6F/ 3D, 1:100; Novocastra Laboratories, Newcastle, UK), αsynuclein (mouse anti-α-synuclein antibody, 1:100; Zymed, San Francisco, CA, USA), ubiquitin (Polyclonal Rabbit Anti-Ubiquitin, 1:1500, Dako, Glostrup, Denmark) and TDP-43 (TARDBP monoclonal antibody (M01), clone 2E2-D3, 1:4000, Abnova, Taipei, Taiwan).

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assess mRNA levels of PGRN from two mutation carriers and two unaffected family members. All samples were run in triplicates. Data were normalised against mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. Inhibition of PGRN nonsense-mediated decay in lymphoblast cells Epstein–Barr virus-transformed lymphoblast cells from one unaffected and one affected family member were cultured in RPMI 1640 growth media (Invitrogen) supplemented with 10% fetal calf serum and antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin, Sigma-Aldrich, St. Louis, MO, USA). To inhibit NMD of the mutant allele, cells were treated with cycloheximide (CHX, 500 μM, Sigma-Aldrich). PCR fragment size analysis was performed to demonstrate mutant and wild-type PGRN alleles of lymphoblast cDNA with or without CHX treatment as described above.

Genomic analysis of PGRN DNA for genetic analysis was available from 21 family members (Fig. 1). Coding exons 1–12 with flanking intronic sequences as well as the non-coding exon 0 of the PGRN gene were amplified from genomic DNA by polymerase chain reaction (PCR) and sequenced using Big Dye terminator v3.1 sequencing chemistry (Applied Biosystems, Foster City, CA, USA). Identified sequence variants were analysed in 165 healthy control individuals. In order to confirm the identified 1-bp deletion, PCR fragment size analysis was performed using fluorescently labelled primers flanking PGRN exon 1. The PCR products were separated on an ABI 3700 (Applied Biosystems) and analysed using the GeneMapper program (Applied Biosystems). PGRN mRNA analysis Total RNA was isolated from lymphoblast cell lines established from two mutation carriers and two unaffected family members using Trizol Reagent (Invitrogen, Paisley, UK). First-strand complementary DNA (cDNA) was synthesised with Superscript First-Strand Synthesis system for RT-PCR (Invitrogen). PCR fragment size analysis was performed to study the presence of the PGRN wild-type and mutant alleles in lymphoblast cDNA from mutation carriers and unaffected family members using fluorescently labelled primers located in PGRN exons 1 and 2. To determine the relative amounts of wild-type and mutant mRNA, PGRN exons 0–2 were amplified from lymphoblast cDNA, and the resulting PCR products were sequenced using Big Dye terminator v3.1 sequencing chemistry (Applied Biosystems). Quantitative PCR (qPCR) using SYBR green Mastermix (Applied Biosystems) was used to

Results Clinical features The prominent clinical features of the family have been summarised in Table 1 using the characteristics from Boeve and Hutton [28]. Mean age at onset was 54 years, with the average duration of disease to death being close to 4 years. The clinical symptoms of four patients (IV-14, IV-16, IV-17 and IV-18) from this Swedish family have been reported previously [27]. Two patients presented with speech disturbances leading to a progressive non-fluent aphasia, whereas the third patient hade onset symptoms of leg apraxia followed by symptoms of parkinsonism in a later stage. The fourth patient presented with personality changes and demonstrated reckless driving as the initial symptom. Loss of spontaneous speech later developed in all patients and emotional bluntness in three of the patients. In six additional patients from the same pedigree (III-6, IV-1, IV3, IV-4, IV-5 and IV-9), the first presenting symptoms were personality and behavioural changes in two patients, language impairment in another two patients and memory impairment in the remaining two patients. Although there was a large variation of the initial symptoms in this family, a common pattern of clinical features could be observed: A non-fluent aphasia with loss of spontaneous speech was seen for all patients, and personality and behavioural changes were also common symptoms in addition to a rapid disease progression. Limb ataxia and parkinsonism were uncommon symptoms. Motor neuron disease was not observed in this family, although dysphagia was seen in three patients.

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Table 1 Summary of clinical features Patients

1

2

3

4

5

6

7

8

9

10

Sex Age at onset (years) Duration of disease (years) Personality/behavioural changes Executive function Language impairment Memory impairment Visuospatial impairment Limb apraxia Parkinsonism Motor neuron disease

F 52 3 + 0 + 0 0 0 0 02

F 59 7 + 0 + 0 0 + 0 02

M 58 61 + + + 0 0 0 0 0

F 55 5 + 0 + + 0 0 0 0

F 58 41 + 0 + + + 0 0 0

M 56 4 + 0 + 0 0 0 0 0

F 55 2 + 0 + 0 0 0 0 0

M 48 3 + 0 + + 0 0 0 02

M 46 4 0 0 + 0 0 + + 0

M 53 4 + 0 + 0 0 0 0 0

Numbering of individuals in pedigree and table is different to protect patient confidentiality. 0 no changes observed, + observed changes 1 Still living 2 Dysphagia.

Neuropathological features The neuropathological features of three patients (IV-14, IV16 and IV-17) from this Swedish family have been previously reported [27]. In this paper, we describe the neuropathology of an additional affected family member (IV-4). The brain weight was 1,048 g. The brain was generally atrophic, most prominently in the frontal and temporal lobes, but also to a lesser extent in the parietal lobes. The cerebral gyri were thinned and the sulci widened. The lateral ventricles were slightly dilated. The hippocampi displayed mild bilateral atrophy and the pigmented nuclei in the brain stem were somewhat pale. Microscopic examination revealed mild neuronal loss in hippocampal CA1 region. No argyrophilic inclusions or senile plaques were seen with Bielschowsky silver staining and extensive screening for tau and amyloid-β immunoreactivity was found to be negative. Staining with ubiquitin Fig. 2 a Two TDP-43 positive lentiform intranuclear inclusions (arrows) and b small dystrophic neurites (arrows) as well as a granular glial cytoplasmic inclusion (arrowhead) were detected in the superficial temporal cortex of patient IV-4. In the hippocampal dentate gyrus of patient IV-4, the ubiquitin (c) and TDP-43 (d) positive neuronal cytoplasmic inclusions were relatively compact and round, but also granular cytoplasmic inclusions were present. Scale bars=15 μm (a, b) and 10 μm (c, d)

and TDP-43 antibodies revealed discrete but definite immunostaining with a pattern corresponding to type 3, according to the system of Sampathu et al. [29] or type 1 according to Mackenzie et al. [30]. In specimens from cerebral cortices, there were neuronal inclusions, both lentiform intranuclear and granular cytoplasmic (NCI), as well as small dystrophic neurites and glial cytoplasmic inclusions in superficial cortical layers (Fig. 2a,b). In hippocampal dentate gyrus, compact round NCIs were detected mainly with ubiquitin staining, whereas in hippocampal pyramidal neurons, TDP-43-positive NCIs usually had a granular appearance (Fig 2c,d). No TDP-43 immunoreactive cytoplasmic inclusions were found in the brain stem and spinal cord, including their motor nerve cells. Immunostaining for α-synuclein (and ubiquitin) disclosed several immunoreactive inclusions in the substantia nigra, and a few in locus ceruleus and gyrus cinguli, in addition to some immunoreactive neurites in the substantia nigra

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and locus ceruleus (Fig. 1, Electronic supplementary material). Mutation screening of the PGRN gene Sequence analysis of affected members of the family revealed a single nucleotide deletion in PGRN exon 1 (g.102delc, Fig. 3a), resulting in a frameshift of the coding sequence and the introduction of a premature termination codon in exon 2 (Gly35GlufsX19). The Gly35fs mutation was identified in all affected members in generation 4 (except in two affected patients for which DNA samples were not available) as well as in three unaffected relatives from the same generation (Fig. 1). Individuals IV-2 and IV19 are unaffected by disease at the current ages of 62 and 65 years, and individual IV-11 was unaffected at the latest contact at 60 years of age. In addition, individual III-2 (DNA sample not available) who died of a cerebral haemorrhage at the age of 65 years displayed no symptoms of dementia, although this individual was most likely a mutation carrier since four of the children were affected by disease. We did not find this mutation in any other individuals from this family, and it was not identified in 165 healthy control subjects investigated. Analysis of PGRN wild-type and mutant alleles Size separation analysis of PCR fragments spanning PGRN exon 1 obtained from genomic DNA showed only the wildtype allele in healthy relatives, whilst affected family members displayed one wild-type and one mutant PGRN allele, which confirmed the 1-bp deletion mutation (Fig. 3b). Analysis of PCR fragments extending from PGRN exons 1 to 2 obtained from lymphoblast cDNA from mutation carriers predominantly displayed the wild-type PGRN allele, consistent with the expected degradation of the mutated mRNA (Fig. 4a). Sequence analysis of lymphoblast cDNA showed only the wild-type allele (Fig. 4b). Fig. 3 a Genomic DNA sequence of PGRN exon 1 from one unaffected (upper panel) and one affected (lower panel) individual demonstrating the one base pair deletion which causes the PGRN Gly35fs mutation. b PCR fragment size analysis from genomic DNA shows the wild-type allele in an unaffected individual (upper panel) and the mutant and wildtype alleles in an affected individual (lower panel)

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Analysis of PGRN mRNA levels Levels of PGRN mRNA were compared in mutation carriers and healthy individuals by synthesising cDNA from lymphoblast mRNA from two mutation carriers and two healthy relatives and performing qPCR analysis. This revealed an approximate 60% decrease of PGRN mRNA in the mutation carriers compared to healthy relatives (Fig. 4c). Inhibition of PGRN nonsense-mediated decay in lymphoblast cells To confirm that the near absence of the mutated allele is a consequence of NMD, we treated lymphoblasts from one unaffected and one affected family member with CHX, an inhibitor of NMD. After 8 h of incubation, lymphoblast cells from the affected individual showed an increased level of mutant mRNA as demonstrated by PCR fragment size analysis (Fig. 4a).

Discussion In this paper, we report the clinical, neuropathological and genetic features of a Swedish family with FTD. This family was one of the first FTD families described with linkage to chromosome 17q21, and we have now identified a Gly35fs mutation in the PGRN gene shared by affected family members. Significant heterogeneity in clinical symptoms has been previously described for patients carrying PGRN mutations, but common diagnoses are FTD, PNFA or corticobasal syndrome [31]. Individuals in this Swedish family displayed a large spectrum of initial symptoms, but they all shared a rapid disease progression with non-fluent aphasia, loss of spontaneous speech, personality and behavioural changes. Neuropathologically, affected members of the family available for examination shared mainly the same features of severe atrophy, especially of the frontal

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Fig. 4 a PCR fragment size analysis of lymphoblast cDNA shows that the mutant mRNA allele (at 168 bp) is barely detectable in the affected individual (upper panel). Treatment with cycloheximide (CHX, 500 μM) for 8 h results in a selective increase of the mutant mRNA allele (lower panel). b Sequencing of lymphoblast cDNA of PGRN exon 1 from an affected individual reveals only the wild-type allele (lower panel). The genomic DNA sequence from the same individual is included for comparison (upper panel). c Quantitative PCR analysis shows an approximate 60% reduction in PGRN mRNA levels in lymphoblast cells from individuals carrying the Gly35fs mutation (n=2) compared to unaffected family members (n=2). Data are shown as the average of triplicate samples and error bars denote SEM

lobes, microvacuolation and gliosis. One affected individual (IV-4) available for additional immunohistochemical staining displayed intraneuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites positive for ubiquitin and TDP-43 in superficial cortical layers, consistent with the pathology reported for other patients carrying PGRN mutations [31]. Interestingly, α-synuclein pathology was also detected in the substantia nigra and locus ceruleus in this patient. One earlier report from an additional three individuals from the same family describes occasional Lewy bodies in the substantia nigra in one individual [27]. Another report from a family with a different PGRN mutation describes two individuals out of five with α-synuclein pathology [32]. The significance of α-synuclein pathology in cases with PGRN mutations is at the moment not known. The PGRN Gly35fs mutation identified in this family introduces a premature termination codon, expected to activate a mechanism of NMD of mutant mRNA and resulting haploinsufficiency, which has previously been described for other PGRN mutations [22, 23]. Analysis of lymphoblast PGRN mRNA from two mutation carriers revealed an approximate 60% decrease compared to healthy relatives. PCR fragment analysis as well as sequence analysis of lymphoblast cDNA from mutation carriers mainly showed the wild-type allele, further supporting the near absence of mutant PGRN mRNA. However, we could

detect minor amounts of the mutant mRNA allele in the PCR fragment size analysis. This is consistent with reports of premature termination codon containing mRNA being reduced to 5–25% of the normal level as a result of NMD [33]. The effects of NMD on the mutant PGRN mRNA allele were further confirmed by inhibiting NMD with CHX which resulted in increased levels of the mutant PGRN mRNA allele, in accordance with earlier reports [22, 23]. We identified the PGRN Gly35fs mutation in all affected members investigated as well as in three unaffected members of this family. The mean age at onset in the family is 54 years, with a variation from 46 to 59 years. The unaffected mutation carriers of this family have passed the age at onset with 1 to 6 years. The Gly35fs mutation has been previously described in two unrelated patients with FTLD; however, in this report, only information of the clinical or pathological diagnosis, onset age and age at death was provided [34]. One of these Gly35fs carriers had an onset age of 83 years, far above the mean age at onset in our family. Previous studies have shown evidence of highly variable age at onset among PGRN mutation carriers and reduced penetrance in some PGRN mutation families [34]. The APOEɛ4 allele, known to be a risk factor for Alzheimer’s disease, has in previous studies been suggested to correlate to a later age at onset in PGRN mutation carriers [34, 35]. However, genotyping of APOE in our family did not reveal a correlation between APOE genotype and age at onset (data not shown).

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In conclusion, the PGRN Gly35fs mutation causes FTD with a variable clinical presentation in a large Swedish family, most likely through NMD of mutant PGRN mRNA and resulting haploinsufficiency. The family also includes three mutation carriers unaffected by disease, supporting previous observations of a variable age at onset or reduced penetrance among PGRN mutation carriers. Acknowledgement We thank Ms. Maud Salomonsson and Ms. Liisa Lempiäinen for skilled histopathological and immunohistochemical assistance. This work was supported by EU contract LSHM-CT2003-503330 (APOPIS), the Swedish Research Council, the Swedish Brain Foundation, the Swedish Alzheimer Foundation, the Swedish Dementia Association, Stohne’s Foundation, the Swedish Lion Research Foundation and the EVO Research Fund of Helsinki University Hospital. Experiments comply with the current laws of Sweden.

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