Mutations in Progranulin Gene: Clinical, Pathological, and Ribonucleic Acid Expression Findings

Mutations in Progranulin Gene: Clinical, Pathological, and Ribonucleic Acid Expression Findings Adolfo López de Munain, Ainhoa Alzualde, Ana Gorostidi, David Otaegui, Javier Ruiz-Martínez, Begoña Indakoetxea, Isidro Ferrer, Jordi Pérez-Tur, Amets Sáenz, Alberto Bergareche, Miriam Barandiarán, Juan José Poza, Ramón Zabalza, Irune Ruiz, Miguel Urtasun, Iñaki Fernández-Manchola, Bixen Olasagasti, Juan Bautista Espinal, Javier Olaskoaga, Marta Ruibal, Fermin Moreno, Nieves Carrera, and José Félix Martí Massó Background: There is an increasing interest in the clinico-pathological correlation of mutations in progranulin (PGRN) and frontotemporal lobar degeneration (FTLD) complex diseases. We aim to study the PGRN expression variability in patients with different clinical features for a better understanding of its roles in FTLD disease. Methods: We sequenced the PGRN gene in 72 patients suffering from FTLD (25 familial and 47 sporadic cases) and in 24 asymptomatic at-risk relatives. We also analyzed PGRN expression in blood by quantitative real-time polymerase chain reaction from 37 patients, 8 asymptomatic mutation carriers, and 10 control subjects as well as in brain tissue from 16 patients and 9 control subjects. Results: Four novel mutations were associated with familial and sporadic FTLD and familial dementia associated with amyotrophic lateral sclerosis. We identified a close association between the IVS6-1G⬎A mutation in PGRN and corticobasal syndrome. Brain tissue was available for carriers of two of the four mutations (IVS6-1 G⬎A and P357HfsX3). Immunohistochemical analysis revealed ubiquitin- and TDP43positive and ␶/␣-synuclein negative immunoreactive neuronal intranuclear inclusions. The relative expression of PGRN in the clinical sample was significantly lower in carriers of the IVS6-1 G⬎A than in control subjects. Conclusions: Progranulopathies are a major cause of the main phenotypes included in the FTLD complex. According to our results, the level of expression of PGRN in blood could be a useful marker both for diagnostics of part of the spectrum of FTLD conditions and for monitoring future treatments that might boost the level of PGRN in this disorder. Key Words: Expression, FTLD, progranulin

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rontotemporal lobar degeneration (FTLD) is actually a complex group of non-Alzheimer dementias, whose nosology remains controversial. Up to 20 different nosological features have been used to describe these conditions, including clinical, pathological, and genetic phenomena (1). The main clinical subtypes of FTLD are frontotemporal dementia (FTD), in which behavioral and personality changes predominate, semantic dementia, and progressive primary non-fluent aphasia (PPA), with prominent early language disturbances. In addition, two pathological entities with prominent motor and gait disturbances, corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are also classified as FTLD. Corticodentatonigral degeneration described by Rebeiz et al. (2) and subsequently renamed corticobasal degeneration (CBD) (3) was firstly recognized to have a pathological picture close to Pick disease with distinctive ␶-positive pathology. Nevertheless, cases of

From the Servicio de Neurología (ALdM, JR-M, BI, MB, JJP, RZ, MU, IF-M, BO, JBE, JO, NC, JFMM); Unidad Experimental (ALdM, AA, AG, DO, AS); Servicio de Anatomía Patológica (IR), Hospital Donostia, San Sebastián; Servicio de Neurología (JR-M), Hospital de Mendaro, Mendaro; Servicio de Neurología (AB), Hospital Bidasoa, Hondarribia; Servicio de Neurología (MR, FM), Hospital Ntra. Sra. de la Antigua, Zumarraga, Gipuzkoa; Institut de Neuropatología (IF), Hospital Universitario de Bellvitge, Hospitalet de Llobregat, Barcelona; and the Unitat de Genètica Molecular (JP-T), Institut de Biomedicina de València-CSIC, València, Spain. Address reprint requests to A. López de Munain, M.D., Ph.D., Neurology Department, Hospital Donostia, Paseo del Dr. Beguiristain 105-116, San Sebastián 20014 Spain; E-mail: [email protected]. Received April 6, 2007; revised July 26, 2007; accepted August 29, 2007.

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corticobasal syndrome (CBDS) were reported in association with other pathological hallmarks as ␶-negative inclusions of the motor neuron disease or lacking distinctive pathology (4,5). Each of these clinical forms of FTLD probably reflects different topographical distributions of underlying pathologies (6). Nevertheless, clinical and pathological overlap is very common and, accordingly, clinical misdiagnosis is frequent (7,8). The pathology of the FTLD is diverse and includes cases characterized by the abnormal deposition of hyperphosphorylated intracytoplasmatic ␶ protein (MAPT) in neurons and glial cells as well as cases without ␶ inclusions. The latter are a heterogeneous group of disorders with ubiquitin-positive but ␶and ␣-synuclein-negative neuronal inclusions (FTLD-U), motor neuron disease, and other rare disorders with no distinctive histopathology or neuronal inclusions composed of neuronal intermediate filaments (9 –15). In tauopathies, genetic studies identified mutations in MAPT in a small proportion of cases (15). In FTLD-U, other genes such as CHMP2B (16), valosin-containing protein (VCP) (17), and, more recently, progranulin (PGRN) (18 –25) have been considered as causative factors. The variety of clinical diagnoses and anatomopathological pictures, together with the relative complexity of the genetic etiology of the FTLD complex, points toward the existence of common mechanisms playing a role in the development of the disease in all these clinical conditions that differ, inter alia, in either the localization of the insult or in its nature. In any case, the lack of a reliable biological marker that could help in the diagnosis of these disorders adds an additional layer of complexity to their study. If such a marker became available, all areas of study, including diagnosis, research, and therapeutic intervention, would be greatly enhanced by it. BIOL PSYCHIATRY 2008;63:946 –952 © 2008 Society of Biological Psychiatry

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A. López de Munain et al. In this work, we present the clinical, molecular, and pathological analysis of a series of patients with familial and sporadic diseases within the confines of the FTLD complex, and we analyze the level of expression of PGRN in peripheral cells to assess its potential value as a biomarker for this disease.

Methods and Materials Patients In this study we have included 72 patients: a clinical series of 57 patients from the hospitals of Gipuzkoa whose biological samples were obtained between 1995 and 2006, and a retrospective series of 15 neuropathological samples from the Brain Bank at the Institute of Neuropathology and University of BarcelonaHospital Clinic obtained between 1998 and 2006. All patients included were diagnosed by applying consensus criteria as published elsewhere (26 –30). The clinical diagnosis was validated in a meeting of five expert neurologists taking into account clinical features, magnetic resonance imaging, single photon emission computed tomography, and complementary tests. The condition was considered as familial when: 1) other living relatives were diagnosed with any clinical syndrome within the FTLD spectrum, 2) clinical reports or pathological material of dead relatives confirmed the diagnosis of FTLD, or 3) enough clinical information from previously affected relatives was available to determine a clinical diagnosis. The clinical diagnosis of patients was considered as primary or secondary depending on the order in which specific clinical traits appeared, according to Kertesz et al. (30). A brief questionnaire was completed by each patient or survivor providing information on gender, ethnic origin, current age or age at death, age at onset, and primary or secondary diagnosis. This was used in conjunction with radiological, neuropathological, and molecular data. Where possible, neuropsychological evaluation was made by an experienced neuropsychologist (Supplement 1). After receiving approval from the Donostia Hospital Ethics Board and obtaining informed consent, we collected blood samples from a population of 130 healthy individuals (74 men and 56 women, mean age 72.8 ⫾ 7.3 years) to determine the potential pathogenicity of the mutations observed. These control samples, whose neuropsychological evaluation did not show any cognitive decline, are further described in an Alzheimer study by Blázquez et al. (31). In addition, brain tissues from 9 healthy individuals (8 men and 1 woman, 63.8 ⫾ 11.9 years) were obtained from the Institute of Neuropathology Brain Bank to have a reference for expression studies in brain. Molecular Procedures For genomic studies, DNA was extracted from leukocytes by standard procedures, and total RNA was extracted with VersaGene RNA Purification System (Gentra, Minneapolis, Minnesota). The DNA was obtained from frozen brain necropsy tissue with the QIAamp DNA Micro Kit (Qiagen, Valencia, California), and total RNA was obtained with RNeasy Lipid Tissue Mini Kit (Qiagen), according to the manufacturer’s instructions. MAPT Analysis. Relevant exons (exons 1 and 9 –13) of the gene MAPT were analyzed by direct sequencing of the coding region plus flanking intronic sequences (32). PGRN Gene Sequencing and Expression Analysis. The 12 coding exons of PGRN were amplified by polymerase chain reaction (PCR) with previously published primers (18,19) and others that we designed (primers available upon request). The PCR products were sequenced in both directions on an ABI 3130

system with Big Dye v3.1 following manufacturer’s protocol (Applied Biosystems, Foster City, California). The real-time (RT)PCR was performed on samples with the IVS6-1 G⬎A mutation from exon 6 to exon 11 and the resulting PCR products were sequenced to detect aberrant transcripts and determine whether premature termination of the PGRN coding sequence resulted in nonsense-mediated decay (NMD) of the mutant messenger RNAs (mRNAs). In addition, through quantitative PCR (qRT-PCR: 7300 Real Time system, Applied Biosystems), we measured the relative expression of the PGRN gene in blood and in brain tissues. In each sample the levels of GAPDH and PGRN-mRNA were measured in triplicate with predesigned Taqman assays (Applied Biosystems). The relative quantity of RNA was measured by the ⌬⌬CT method (33) relative to the data from neurologically healthy control subjects (mean age ⬎ 60 years) (see Supplement 1 for primer sequences). Neuropathological Studies. A postmortem neuropathological study was available for 20 patients. Detailed descriptions will be restricted to the cases with mutations in PGRN. The brain weight was 900 g in case 03-013 and 1000 g in case 06-420, with a postmortem delay of 14 hours and 30 hours, respectively. Cortical atrophy was present in the frontal and temporal lobes, particularly in their medial and lateral surfaces, as well as in the cingulate cortex. The hippocampi were mildly atrophic, and there was depigmentation of the substantia nigra. Similar lesions were observed in both cases. In case 03-013, one-half of the brain was immediately cut into coronal sections (1-cm-thick), frozen on dry ice, and stored at ⫺80°C until use. For morphological examinations, the brains were fixed by immersion in 4% buffered formalin for 3 weeks at 4°C. Neuropathological studies were carried out on paraffin sections (4-␮m-thick) of different brain structures (Supplement 1). The sections were stained with hematoxylin and eosin, Klüver Barrera, and a series of specific antibodies (Supplement 1) following the EnVision ⫹ system peroxidase procedure (Dako, Glostrup, Denmark). The peroxidase reaction was visualized with diaminobenzidine and hydrogen peroxide.

Results Patients We studied 72 unrelated patients, 20 of whom had undergone a postmortem neuropathological examination. Of these 72 patients, 57 suffered from different phenotypes of the FTLD complex (29 primary or secondary CBDS, 6 PPA, 1 semantic dementia, 15 FTD, 1 amyotrophic lateral sclerosis [ALS]-dementia, 1 ALS-dementia-parkinsonism, and 4 ALS), 3 suffered from Pick disease, and the remaining 12 suffered from PSP. Moreover, 25 cases were familial (9 CBDS, 3 PPA, 10 FTD, 3 ALS), and 47 were sporadic (Table 1). Of the 25 familial cases, 19 were of Basque origin. Molecular Studies MAPT Analysis. The unique mutation detected in MAPT gene was P301L in exon 10 in all three cases of familial FTD presenting ␶ inclusions. PGRN Sequencing Analysis. We found four different mutations in the PGRN gene among our clinical series of patients (Supplement 1). Two mutations caused truncated proteins by either altering splicing mRNA (IVS6-1 G⬎A) or by changing the reading frame of the messenger (c.1068-1070delC; p.P357HfsX3). The IVS6-1 G⬎A mutation appeared in 11 patients (7 with CBDS, as primary or secondary diagnosis, 2 with PPA, and 2 with FTD), www.sobp.org/journal

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Table 1. Clinical and Molecular Characteristics of Patients with CBDS, PPA, SD, FTD, and ALS as Primary or Secondary Diagnosis

Case 03-013 05-396 03-246 04-251 06-140 06-221 06-222 06-225 06-226 06-235 06-236 06-269 06-248 06-280 02-071 06-348 06-296 05-171 03-022 04-492 06-255 06-300 04-550 06-253 06-303 06-252 06-288 06-240 06-249 06-420 06-287 06-263 06-251 95-027 06-405 06-346 06-267 98-279 00-440 06-297 06-461 03-051 00-330 98-083 06-286 05-470 06-144 02-334 06-145 06-247 06-150 06-281 06-282 06-292 06-293 06-304 06-229 1 2

Clinical Secondary Family Gender Presentation Diagnosis Yes Yes Yes Yes No No No No Yes No No No No Yes Yes No No No Yes Yes No No Yes Yes No No No Yes No Yes Yes No No No Yes Yes Yes No No Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No

M F F M F M F M M F M F F F F M M F F M F F F F M F F F M M M F F M F F F M M F M M F M M M M F M F M F M F F F F F F

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CBDS CBDS CBDS CBDS CBDS CBDS CBDS CBDS FTD CBDS CBDS CBDS CBDS/PSP CBDS/PSP FTD/LBD CBDS CBDS FTD FTD FTD PPA FTD PPA PPA PPA PPA PPA PPA SD FTD FTD FTD FTD FTD FTD FTD FTD FTD FTD FTD FTD ALS ALS-FTD ALS ALS ALS ALS PSP PSP PSP PSP PSP PSP PSP PSP PSP PSP PSP AD

Other Familial Diagnosis (n)

FTD FTD FTD FTD — — — — CBDS — — — — — CBDS — — CBDS CBDS CBDS CBDS CBDS FTD FTD

ALS (1), FTD (1) — — FTD (1) FTD (1)

FTD

FTD (1)/AD (2)

— — — — — — — — — — — — — — — — — — — — — — — — — — — — —

FTD (1) FTD (1), CBDS (1) FTD (1) FTD (1)

FTD (1)

FTD (1) PPA (1), FTD (1)

FTD (1)/DS (1) FTD (1) — — — FTD (1) FTD (1) FTD (1) — — — FTD(2) ALS (2)/PD (1)/FTD (1) FTD (1) ALS (3) — — — — — — — — — — — — — — —

Current Age at Age Onset Age at ␶ (yrs) (yrs) Death (yrs) Neuropathology Mutation — 66 71 — 72 57 73 68 73 90 82 73 76 80 — 81 75 64 70 58 75 65 70 66 73 66 82 79 75 — 72 60 70 — 58 77 66 — 65 59 — — — 29 61 74 — — 62 71 75 69 72 80 88 76 — —

53 64 65 60 72 55 69 63 71 84 79 57 71 74 56 78 73 58 65 52 68 61 66 64 72 63 75 76 74 44 70 60 67 70 55 71 63 71 47 64 59 55 63 31 28 59 69 73 70 62 69 71 64 64 77 88 72 — —

57 — — 64 — — — — — — — — — — 67 — — — — — — — — — — — — — — 51 — — — 78 — — — — 53 — — 57 65 32 — — — 81 73

77 76

Ubiquitin ⫹ np np np np np np np np np np np np np np np np np np np np np np np np np np np np Ubiquitin ⫹ np np np np np np np np DLDH np np Ubiquitin ⫹ np np np np np PSP np np np np np np np np np Mixed PSP/CBD CBD

— — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —

PGRN Mutation IVS6-1 G⬎A IVS6-1 G⬎A IVS6-1 G⬎A IVS6-1 G⬎A — — — — — — — — — — IVS6-1 G⬎A — — — IVS6-1 G⬎A IVS6-1 G⬎A — — IVS6-1 G⬎A IVS6-1 G⬎A — — — — — P357HfsX3 — — — R177H IVS6-1 G⬎A — — — — — IVS6-1 G⬎A — V5L — — — — — — — — — — — — — — —

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A. López de Munain et al. Table 1. (continued)

Case 3 4 5 6 7 8 9 10 11 12 13 14 15

Family

Gender

Clinical Presentation

No No No No No No No Yes Yes Yes No No No

M F M F M M F M M M M M M

PiD CBD MSA CBD FTD PSP PSP FTD FTD FTD PiD FTD ⫹ AD PiD

Secondary Diagnosis

Other Familial Diagnosis (n)

Current Age (yrs)

Age at Onset (yrs)

Age at Death (yrs)

— — — — — — — FTD FTD FTD — — —

— — — — — — — — — — — — —

— — — — — — — — — — — — —

— — — — — — — — — — — — —

68 72 82 75 67 65 71 52 58 58 74 56 68

Neuropathology CBD CBD CBD ⫹ AGD CBD CBD PSP PSP FTD-T ⫹ FTD-T ⫹ FTD-T ⫹ PiD PiD PiD

␶ Mutation

PGRN Mutation

— — — — — — — P301L P301L P301L — — —

— — — — — — — — — — — — —

AD, Alzheimer disease; AGD, argyrophilic grain dementia; ALS, amyotrophic lateral sclerosis; CBDS, corticobasal syndrome; DLDH, dementia lacking distinctive histology; FTD, frontotemporal dementia; LBD, Lewy Body Disease; MSA, multiple system atrophy; np, not present; PD, Parkinson disease; PiD, pick disease; PPA, primary progressive aphasia; PSP, progressive supranuclear palsy; SD, semantic dementia.

all of them of Basque origin, and in 4 affected living relatives from three different kindreds. The P357HfsX3 mutation was identified in one patient with familial FTD. The other two mutations identified are p.R177H (c.530C⬎A), which appeared in one patient with sporadic FTD, and p.V5L (c.13G⬎C), identified in a patient with familial ALS-FTD. None of these four mutations were found in 130 neurologically healthy control subjects. In addition, we identified 14 carriers of the mutations amongst the 24 asymptomatic at-risk relatives available for the study; 12 healthy individuals carried the IVS6-1 G⬎A mutation, and 2 healthy individuals were found to carry the R177H mutation. Neuropsychological examinations were performed on 7 PGRN⫹ and 14 PGRN⫺ patients (Supplement 1). The CBDS patients carrying the mutation IVS6-1 G⬎A displayed an earlier and more extended global cognitive disorder. Furthermore, these patients presented an earlier age of onset (59.3 ⫾ 5.6 years) when compared with the CBDS patients without PGRN mutations (68.9 ⫾ 8.6) (Student t test p ⬍ .05) (Table 1). PGRN Expression Analysis. The RNA was obtained from blood from 37 patients, 9 of whom were symptomatic IVS6-1 G⬎A carriers (all women, mean age 68.4 ⫾ 3.3 years); the remaining 28 PGRN⫺ patients were 18 women and 10 men, mean age 71.5 ⫾ 11.1 years. Additionally, we analyzed blood from 8 asymptomatic PGRN⫹ carriers (4 women and 4 men, mean age 49.25 ⫾ 12.8 years) and 10 control subjects (6 women and 4 men, mean age 62.2 ⫾ 13.1 years). The carriers of the IVS6-1 G⬎A mutation did not show an aberrant transcript when we sequenced the complementary DNA. This suggests an NMD process in accordance with previous expression studies of PGRN (14,15). The relative expression of PGRN in the clinical sample was significantly lower in carriers of the IVS6-1 G⬎A mutation when compared with control subjects (Kruskal-Wallis and MannWhitney U, p ⬍ .01). No significant differences were seen between symptomatic and asymptomatic carriers of the mutation (Figure 1). The level of PGRN mRNA in brain of a patient carrier of the IVS6-1 G⬎A mutation was similar to that found in control subjects (n ⫽ 9) (data not shown). Analysis of Penetrance We analyzed the penetrance in each kin, taking into account only the sib relationships where the current age of the youngest

member was above 50 years old. We estimated the penetrance assuming that the proportion of carriers among the untested individuals was the same as the one in tested individuals. Similarly, because all of the affected individuals carried the mutation, we assumed this was also the case in the affected but untested subjects. Segregation of the mutations with the disease was analyzed in the pedigrees where samples were available. An autosomal dominant pattern of transmission was evident in all familial cases, and 13 sibs met the criteria for penetrance analysis (Table 1). We subjected 17 of 24 affected relatives and 13 of 45 asymptomatic at-risk sibs to molecular analysis. Among the unaffected family members who were tested, 46% (6 of 13) were carriers (Supplement 1). The theoretical prevalence of asymptomatic carriers would be 14.72 (0.46 ⫻ 32), assuming that the same proportion would apply to those that were not tested. On

Figure 1. Relative quantification of progranulin (PGRN) messenger RNA levels in blood of PGRN⫹ symptomatic and asymptomatic carriers as well as in other frontotemporal dementia–PGRN⫺ cases and control subjects.

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the basis of these calculations, we estimated that roughly 45 of the 69 siblings would carry the mutation, assuming that there were no phenocopies in any family (24 affected, 6 demonstrated carriers and 14.7 estimated carriers among the unaffected individuals that were not analyzed), of whom 24 were clinically affected. Accordingly, we estimated a penetrance of 53.3% with an approximate 95% confidence interval of 41.5%– 65.1%. This estimation is the minimum value of the penetrance due to the age depending on the appearance of symptoms. Neuropathological Studies of Cases with Mutations in the PRGN Gene The two cases of FTD with mutations in PGRN displayed similar microscopic changes, including a visible loss of neurons and astrocytic gliosis in the frontal, temporal, insular, and cingulate cortex. This was accompanied by spongiosis in layer II. Mild loss of neurons and gliosis was also evident in the caudate and putamen, whereas loss of pigmented neurons, extracellular neuromelanin, and gliosis were noted in the substantia nigra. There were no ␶ or ␣-synuclein immunoreactive inclusions in neurons or glia in any region. Likewise, no ␤-amyloid deposits were detected. Nevertheless, abundant ubiquitin-immunoreactive aberrant neurites were found in the cerebral cortex (Figures 2 and 3). UBQ-ir NCI (Neuronal Cytoplasmic Inclusions) was observed in the isocortex and cingulate cortex as well as in granular neurons of the dentate gyrus. Finally, a few round or cat-eye–shaped UBQ-ir NII (Neuronal Intranuclear Inclusions) were also detected (Figure 2). All these inclusions were recognized with anti-TDP-43 antibodies. Interestingly, TDP-43 immunoreactivity, which normally Figure 3. Aberrant neurites and intracytoplasmic inclusions (arrows) stained with anti-ubiquitin antibodies in frontotemporal dementia associated with mutations in progranulin gene (case 1). Paraffin sections counterstained with hematoxylin. Bar ⫽ 25 ␮m.

stains the nucleus, was restricted to intranuclear inclusions in affected cells (Figure 2).

Discussion

Figure 2. Aberrant neurites, intracytoplasmic inclusions, and cat-eye– shaped intranuclear inclusions are stained with anti-ubiquitin (A, B) and anti-TDP-43 antibodies (C, D) in frontotemporal dementia associated with mutations in progranulin gene (case 2). Paraffin sections slightly counterstained with hematoxylin. Bar ⫽ 25 ␮m.

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In the studied series we found 14 apparently unrelated cases with four different mutations in PGRN and only 3 cases with mutations in MAPT. This corroborates the relatively rare involvement of MAPT among FTD patients, with ⬍ 50 published mutations to date (http://www.molgen.ua.ac.be/FTDMutations). In contrast, mutations in PGRN have appeared frequently in recent months with 40 mutations now associated with the etiology of this disease (14 –21, present work) (Supplement 1). Nevertheless, the observed mutation carrier proportion (14 of 72 cases) could be biased by the presence of a prevalent ancestral IVS6-1G⬎A mutation related to Basque population. Hence, this high frequency must be confirmed in additional, larger prospective clinical studies. We identified a close association between the IVS6-1G⬎A mutation in PGRN and CBDS as primary or secondary diagnosis, lending weight to recent reports of cases carrying similar mutations in PGRN gene with clinical features of CBDS (20,21). In our patient series, all familial cases where the initial clinical characteristics were of CBDS presented IVS6-1 G⬎A mutation. Historically, CBDS was first described as an extrapyramidal motor disorder,and thus cognitive and language problems have been underdiagnosed (34). However, further review of patients has highlighted the cognitive decline, and in particular, language

A. López de Munain et al. disturbances were recognized as a late feature in the CBDS evolution (3) or as an initial concomitant undifferentiated clinical trait in pure PPA (35). In our patients with the IVS6-1 G⬎A mutation, the language disorder is described as aphemic stuttering that evolved into non-fluent aphasia and finally mutism. When compared with PGRN⫺ patients, carriers of PGRN mutations displayed an earlier age of onset and earlier and more extended global cognitive impairment. These features were coupled to more pronounced language involvement that rapidly evolved toward an anarthric condition. In summary, in the context of CBDS, these three clinical characteristics—familial condition, earlier onset, and more widespread clinical features involving more severe language and cognition defects— could help in predicting progranulopathies, although other authors did not support this idea (36). According to our data, IVS6-1 G⬎A mutation carriers displayed a predominant phenotype; this phenomenon also occurs in other diseases such as PPA (24). However, in most of our families, clinical heterogeneity existed in the intrafamilial evolution, similarly to what has been referred by others (25). Another important clinical finding is the discovery of a case of ALS associated with the IVS6-1 G⬎A mutation. Several authors initially proposed a similar pattern of ubiquitin immunoreactivity in FTD with ubiquitin-positive inclusions and motor neuron disease-dementia (37). However, a recent comparative work on the neuropathology of FTLD showed that the distribution of ubiquitin immunoreactivity differed depending on the presence of mutations in the PGRN gene (38). Our findings open the possibility that there is more widespread diffusion of NII and NCI to lower motor neurons than previously suspected (38). All three cases with mutations in MAPT displayed ␶ inclusions, and none of the neuropathologically confirmed tauopathies presented mutations in PGRN. Furthermore, both cases carrying mutations in PGRN that were subjected to neuropathological studies displayed ␶ -, ubiquitin ⫹, and TDP-43 ⫹ NII. Both findings suggest that each genetic form displays its pathological presentation. It is worth stressing that TDP-43 inclusions are not restricted to cases with mutations in the PGRN gene, because PGRN mutations have been encountered in only a proportion of cases with similar neuropathological characteristics. Furthermore, TDP-43 immunoreactivity is a facet of ubiquitin ⫹ ␶ - inclusions in FTD and ALS (39,40). Owing to the young age of most of the asymptomatic carriers analyzed in this work, our estimation of incomplete penetrance must be interpreted cautiously because of the wide range of the onset of symptomatology and the young age of asymptomatic carriers with respect to the mean age at onset of this mutation. Further analysis including more aged sib-ships will be required to elucidate this issue. The finding of carriers of the IVS6-1 A⬎G mutation that remain asymptomatic until advanced age or do not develop the clinical disease until late 80s implies the existence of other modifier factors (genetic or environmental) that determine the age of onset or the initial clinical picture. We tried to correlate these findings in blood with a study in brain tissue from a patient with the mutation. Nevertheless, we do not find differences between mutated-brain and normal-control subjects. This lack of association could be explained by the study of a unique mutatedbrain and/or by a problem derived by the time elapsed between death and necropsy. Our results of RNA expression at the peripheral level confirm that FTLD linked to mutations in PGRN seems due to haploinsufficiency for this protein. It remains undetermined which other mechanisms are playing to justify the outliers= cases.

BIOL PSYCHIATRY 2008;63:946 –952 951 Little knowledge about the normal function of PGRN is known in the central nervous system apart from its role in neurodevelopment. Interestingly, PGRN is expressed by microglia and neurons. Moreover, several reports have shown upregulated PGRN levels by microarrays in several neuropathological conditions (41). In fact, several studies reported increased PGRN levels upon injury in the central nervous system but not in acute damage models (42). Moreover, PGRN expression has been reported in activated microglial cells (13), suggesting a possible role of this trophic factor in neuroprotection and inflammatory responses associated with neurodegeneration in the central nervous system (43). Interestingly, PGRN is detected in neuritic plaques from Alzheimer postmortem brains (44). Microglia and certain populations of neurons are the major cell types that express PGRN in the central nervous system (18,38). As in any neurodegenerative disorder, neuronal loss in FTLD-U is accompanied by microglial activation. Ahmed et al. (41) suggest that, because PGRN is related to microglial proliferation and activation, this gene could be implicated in neuroinflammation and brain repair. A closer look at the microglial functional properties and the inflammatory response in PGRN ⫹ and ⫺ cases would be useful in understanding the biological role of PGRN in FTLD-U. Although the discovery of several asymptomatic carriers raises several problems related to ethical issues, it also opens new research opportunities. First of all, the factors that regulate the penetrance of these mutations are poorly understood, confounding the development of routine genetic counseling and presymptomatic diagnosis. Second, setting aside the evident ethical problems that this question raises, asymptomatic carriers constitute an attractive group for monitoring the appearance of the early signs or symptoms of the disease, making it possible to check the sensitivity of other diagnostic tools and the efficacy of neuroprotective agents. ALdM and AA contributed equally to this work. This study was funded by ILUNDAIN FUNDAZIOA, Diputación Foral de Gipuzkoa (GBR project) and Mutua Madrileña Automovilista. Work at JP-T laboratory was funded by a grant from the Ministerio de Educación y Ciencia (SAF2006-00724). All authors declare no conflicts of interest. We wish to thank the patients and families who took part in the study and Sabin Urcelay and Maite Monasterio from the Gipuzkoa Blood Bank for their help in obtaining control samples. We also wish to thank Jose Ignacio Emparanza and Iratxe Urreta from the Clinical Epidemiology Unit for statistical and design advice, Asunción Iribarren and Olaia Zuriarrain for their invaluable technical assistance, and Marisa Martínez and Navi Coll for their help in blood extractions. We also thank Mark Sefton and Catherine Gerhardt for invaluable help in the final preparation of the manuscript. David Otaegui is a predoctoral fellow with a grant from the Basque Government. Supplementary material cited in this article is available online. 1. Kertesz A (2003): Pick’s complex and FTDP-17. Mov Disord 18(suppl 6):S57–S62. 2. Rebeiz JJ, Kolodny EH, Richardson EP (1968): Corticodentanonigral degeneration with neuronal chromasia. Arch Neurol 18:20 –33. 3. Gibb WRG, Luthert PJ, Marsden CD (1989): Corticobasal degeneration. Brain 112: 1171–92. 4. Grimes DA, Bergeron CB, Lang AE (1999): Motor neuron disease-inclusion dementia presenting as cortical-basal ganglionic degeneration. Mov Disord 14:674 – 680.

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