Introduction

Frontotemporal dementia, or frontotemporal lobar dementia (FTLD), is a degenerative brain disease with variable clinical phenotype, pathology and genetic background. FTLD is clinically characterized by behavioural changes and/or language impairment, defined as primary progressive aphasia or semantic dementia. Disease onset occurs mostly at presenile age.

The most frequent histopathological subtype of FTLD shows ubiquinated, tau-negative inclusions in the neurites and cytoplasm of neocortical and hippocampal neurons (FTLD-U). The main constituent of the neuronal inclusions was found to be TAR-DNA binding protein 43 (TDP-43), a highly conserved 43 kDa nuclear transcription factor [10, 29]. Four major subtypes of FTLD-U have been delineated based on the distribution of neuronal cytoplasmic inclusions (NCI), dystrophic neurites and the presence of neuronal intranuclear inclusions (NIIs), [9, 25]. In the same year TDP-43 was identified, mutations of the progranulin (PGRN) gene were described in FTLD-U [2, 11]. Neuropathologically, all cases with PGRN mutations share a common FTLD-U subtype, characterized by NCIs and irregular dystrophic neurites in the neocortex and subcortical nuclei [3, 14, 18, 24, 26, 38, 39]. This subtype is referred to as type I by Mackenzie or type 3 by Sampathu and co-workers [26, 35].

Human PGRN contains 13 exons and is located on chromosome 17q21 in close vicinity to MAPT (Microtubulus-associated protein tau), another gene identified as being involved in FTLD. PGRN encodes for the 88 kDa glycoprotein progranulin. Progranulin is a pluripotent growth factor that has been shown to be involved in cell differentiation, inflammation, tissue repair, tumorigenesis and brain development in mice [13, 16, 43]. The pathophysiological pathway by which progranulin mutations are associated with the deposition of TDP-43 protein and with neuronal degeneration is still unclear.

At present, more than 60 PGRN mutations have been identified in patients with FTLD (http://www.molgen.ua.ac.be/FTDMutations/). Pathogenic mutations include missense and nonsense mutations, or small insertions or deletions in the exons or introns of the gene [14]. Most of the mutations lead to frameshift and premature stop codons. Pathogenic mutations in PGRN invariably lead to mutant mRNA transcripts, which undergo nonsense-mediated decay, thereby resulting in haploinsufficiency [2, 11].

Prevalence of mutations in PGRN is suggested to account for 1–15% of all cases with FTLD [8, 14, 22], but up to 26% of familial cases [7, 12, 30]. In a large series from the USA, mutations were found in 10% of all patients with FTLD and 23% in cases of familial FTD [14]. Several other studies from France, Italy, the Netherlands, the UK, Belgium, Finland, and the USA have reported lower frequencies of an average 5% in unselected FTD groups and 4–10% in groups of cases of familial FTD [6–8, 11, 14, 15, 17, 20–22, 31]. The differences in the reported frequencies may be due to differences in the mode of ascertainment of patients, in ethnic variations as well as to founder effects. Overall, the frequency of PGRN mutations is similar to that of mutations in MAPT [33].

Mean age at onset of FTLD patients with mutations in PGRN is around 60 years. The majority of patients with PGRN mutations show the behavioural-variant phenotype with apathy and social withdrawal as prominent characteristics [42]. PGRN mutations have also been found in patients who present with language impairment early in the course of the disease, diagnosed as primary non-fluent progressive aphasia (PPA) [14, 17, 18, 27, 37]. Patients from different families with the same mutation do not necessarily show the same clinical phenotype or age at onset [17].

The aim of our study was to determine the frequency and type of progranulin mutations in a series of 79 unselected FTLD patients from an ethnically largely homogeneous south-western German population. To date, no mutation screening for PGRN has been published for Germany. In order to ascertain all types of mutations, we performed—in addition to the mutation analysis—quantitative PCR analyses, which additionally allows for detection of whole gene and exon deletions, a technique that has only been applied as an exception in earlier PGRN mutation screening studies.

Materials and methods

Patients

79 consecutive outpatients, who attended our university hospital memory clinic between 2001 and 2007 and received a clinical diagnosis of FTLD, were included. Routine neurological and psychiatric exams, neuropsychological testing and cerebral imaging (CT- or MRI-scan) were conducted in all cases. In addition, HMPAO-SPECT was performed in a subset of cases. The diagnosis of FTLD was made in a case conference, based on established criteria [1]. Screening for other known dementia-related mutations, including tau protein mutations, was not performed. Several of our patients have died, but none have been subjected to autopsy.

PGRN mutation analysis

PGRN mutation analysis was performed in all 79 patients. All 13 exons including exon-intron boundaries were sequenced. DNA of EDTA blood samples of patients were extracted with the BACC-2 Nucleon Extraction Kit (Amersham Bioscience, Freiburg, Germany) according to the manufacturer’s instructions. All PGRN exons were first amplified by Fast-PCR using primers listed in Table 1. Amplification products were purified with the QIAquick® Gel Extraction Kit (Qiagene, Hilden, Germany), followed by sequencing using BigDYE®Ready Terminator Sequencing Kit (Applied Biosystems, Foster City, CA, USA). For sequencing, previously-described primers were used [2] (Table 2). Sequencing was performed on the ABI 3130 XL Genetic Analyzer (16 capillary DNA sequencer, Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using SeqPilot software (JSI medical systems, Kippenheim, Germany) (Fig. 1).

Table 1 PGRN fragment amplification primers
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Table 2 PGRN sequencing primers
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Fig. 1
figure 1

PGRN mutation analysis

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PGRN deletion analysis

Copy number changes of PGRN were tested by quantitative real-time PCR (qPCR) using SYBR® GREEN l (Qiagene, Hilden, Germany) as described previously [4]. PCR amplicons listed in Table 3 were designed using PRIME program (Genetic Computer Group, Wisconsin, USA) and quantified against two reference amplicons in human subtelomer region 3p26.3 and 4p15.2 [4, 5]. qPCR was performed on ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Data was analyzed with the help of Sequence Detection Software (SDS version 2.2.1, Applied Biosystems, Foster City, CA, USA).

Table 3 PGRN real-time PCR amplification primers
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Results

Clinical characterization of the FTD patients

The series was comprised of 43 men and 36 women. Mean age at onset was 62 years, ranging between 41 and 83 years. 19 patients (24%) had a positive family history of dementia. Among these, 11 patients (14%) had a positive family history for FTD with instances of early-onset in at least one first-degree relative (n = 8), or with age at onset >65 years (n = 3). In the other 8 cases with FTD, at least one parent had suffered dementia at a much higher age, presumably Alzheimer’s disease. In three cases, sufficient information on family history was not available.

The clinical FTD subtype was “frontal” with leading behavioural and executive cognitive disorder in 44, primary progressive aphasia (PPA) in 24, semantic dementia (SD) in 8, mixed behavioural variant and PPA in 1, mixed FTD and CBD in 1, and FTD with Parkinsonism in 1. Mean age of disease onset of patients with the “frontal type” was 61.2 years, and 64.9 and 57.0 years in patients with PNA and SD, respectively (Table 4).

Table 4 Demographic data, grouped by FTLD subtypes
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DNA sequence analysis of PGRN

Sequence analysis revealed two pathogenic PGRN mutations (3%). Both mutations were nonsense mutations (p.R418X, p.K259X) leading to the generation of a premature stop codon (Fig. 1A).

PGRN deletion Analysis: qPCR analysis revealed no deletions within or including the PGRN gene in our cohort.

Clinical phenotype of the two patients with mutations within the PGRN gene

Case 1

This woman presented with progressive symptoms of language impairment at age 58. One year later, she had become quiet, withdrawn, and inefficient in all her activities. Upon examination, she appeared friendly, cooperative, not depressed. Spontaneous speech was markedly reduced. There were few semantic, and no phonematic paraphasias. She showed difficulties in comprehending instructions, utilisation behaviour, rule breaks, and marked symptoms of perseveration in fluency, clock reading and drawing tasks. The Mini Mental score was 18. Further testing showed dysnomia (9 of 15 in the short Boston naming test), and impaired fluency (five animals, no S-words in one minute). CERAD figure copying was borderline (8). Clock drawing (Shulman score 3–4) and clock reading (score 8) [36], as well as trail making “A” performance (118 s) were pathological. Verbal memory performance was also impaired (15 of 30 in three trials of CERAD word list learning, 4 at delayed recall, 95% at delayed recognition).

An MRI was described as showing external and internal brain atrophy, mostly temporo-parietal. A brain HMPAO perfusion SPECT performed at our clinic showed extensive bilateral frontotemporal hypoperfusion, mostly on the left side, extending to the parietal cortex and also involving the thalami and basal ganglia.

A diagnosis of frontotemporal dementia was made. The presence of early and pronounced general cognitive and behavioural symptoms and the pattern of imaging findings suggest classification as a case of the predominantly “frontal” type FTD (Fig. 1B).

Family history

Both parents of this patient had reached an age >75 years. Her mother was chronically ill and bedridden before she died. More precise information on her medical diagnosis was not available. This case of FTD was therefore considered to be sporadic.

PGRN mutation analysis

Sequence analysis showed a nonsense mutation in exon 10 in a heterozygous state, leading to the generation of a premature stop codon.

Case 2

This man presented with a change of behaviour and with cognitive deficits that became evident at age 58: increased talking, irritability, neglect of former activities, emotional indifference, word finding difficulty, forgetfulness and adherence to irrelevant stimuli. Upon examination, he was cooperative, not depressed, but highly stimulus-bound, making irrelevant remarks and reading aloud whatever was in sight. His sentences tended to be long, complicated, and were sometimes not finished. His overall speech production was reduced. There was no impairment of language comprehension. He showed symptoms of perseveration. The Mini Mental score was 26. Further testing showed normal Token Test, CERAD figure drawing and recall performance. Boston naming (13 of 15) was borderline, and performance at another naming test was pathological (Aachen Aphasia Test, 98/120). Verbal fluency was borderline (16 animals in one minute). Verbal memory performance was pathological (11 of 30, delayed recall 2, delayed recognition 90%).

A brain MRI showed no clear atrophy. A brain HMPAO perfusion SPECT performed at our clinic showed symmetric temporo-polar and fronto-basal hypoperfusion.

As in case 1, the pattern of clinical and imaging findings supports classification of this patient as a case of the predominantly “frontal” type of FTD.

Family history

A brother of this patient suffers a degenerative disease which started at age 51 with word finding difficulties. 21 months later, he showed severe aphasia, but only a minor change of personality was noted. There was atrophy mainly of the left temporal lobe (MRI). When we saw him 4 months later, he was still pursuing his job and said to be unimpaired in all technical and practical matters. His drive, mood and behaviour appeared normal. Speech was reduced to “yes” and “no”. Speech comprehension, reading aloud and writing were markedly impaired. Upon testing, the Mini Mental score was 19, Boston naming score was pathological (5/15), verbal memory borderline (15/30; 5 at delayed recall), figure copying and delayed recall were normal. He was classified as a case of progressive nonfluent aphasia. Genotyping could not be performed since the patient was not able any more to give consent to genetic testing. The parents of case 2 were said to have died of cancer at the age of 70 and 73 years, without known dementia. Further information was not available. This case was classified to be a familial case of FTD.

PGRN mutation analysis

Sequence analysis revealed a nonsense mutation in exon 7 in a heterozygous state not described before, leading to the generation of a premature stop codon (Fig. 1C).

Discussion

This is the first report of a German FTD case series screened for progranulin mutations. Patients live or lived in South Baden, which represents about one-quarter of the territory of the federal state of Baden-Württemberg in the south-west part of Germany.

The overall frequency of positive family history for dementia in our FTD patient cohort was 24%. This proportion is below reported frequencies in several earlier series with a positive family history of up to 40–50% of FTLD cases [28, 40]. The possibility remains that the true proportion of dominantly inherited cases is obscured by instances of early death of mutation carriers in the parental generation, siblings that carry mutations but are yet undiagnosed, or illegitimate descent.

Point mutations were identified in two cases of our cohort of 79 patients. In both cases, a point mutation generated a premature termination codon that causes nonsense-mediated mRNA decay and leads eventually to the absence of mutant PGRN transcript.

The mutation p.R418X as identified in case 1 has been reported before [2, 14, 41]. Mean age at onset in a Canadian family (UBC15) described by Baker and colleagues was 60, comparable to the age at onset of our index patient [2]. Gass and coworkers identified another FTLD family in the USA (01-01) with the p.R418X mutation; here age at onset was 49 [14].

The nonsense mutation of case 2 in exon 7 has not been reported before. The clinical phenotypes of case 2 and his brother, who has not been sequenced for PGRN mutations, differed from each other in regard to age at onset and clinical phenotype (frontal type and PNA type). Pronounced phenotypic variability has previously been described in pedigrees with progranulin mutations with respect to clinical picture [19, 32], age at onset and pattern of histopathological changes. In one family, post-mortem examinations showed atypical tau and alpha-synuclein pathology [23]. Assuming that progranulin mutations are always null mutations and uniformly lead to haploinsufficiency, other as yet unknown genetic, epigenetic and environmental factors must be responsible for this variability within and across families.

Two of our patients had a progranulin gene mutation, corresponding to a low proportion of 3% of the entire sample. In previous FTD series that were unselected for family history, the reported rates of progranulin mutations were 2% [17], 1% [8], 5% [14], and 6% [31]. In apparently familial cases, reported rates were 13% in a French series [22], 22% in a North American series [14], and 4–7% in a Dutch series [7]. In contrast to most other mutation screening studies, we performed quantitative real-time PCR analysis to be sure to identify single exon and larger deletions of PGRN. Previously, two patients with heterozygous genomic deletions in PGRN were reported [15, 34]. Thus, our study provides a reliable frequency for PGRN mutations for south-west Germany.

Age at onset was 58 years in our cases 1 and 2. Information on the status of the mother of case 1 is incomplete. Both parents of case 2 and his affected brother lived to their seventies and were apparently not demented. This situation is, in principle, compatible with illegitimate descent, de novo mutation, incomplete penetrance or with a high variability of age at onset. Previous reports of families with progranulin mutation have shown that age at onset can indeed vary greatly, e.g. from 35 to 75 years within one family [23], from 49 to 88 years across affected families [19], and from 35 to 87 years in one kindred [8]. In some affected families, age at onset is not only variable, but clearly higher than the average presenile age range typical of FTD, e.g. 75.8 ± 5.0 years in one generation of kindred 2 reported by Kelley et al. [19].

We conclude that PGRN mutations in unselected populations may be lower due to a founder effect in selected studies and may explain regional differences in PGRN mutations. In addition, it is important to perform deletion analysis to accurately determine the frequency of pathogenic gene mutations.