Received 12 November 2006; accepted revised manuscript 19 January 2007.
Mutations Other Than Null Mutations Producing a Pathogenic Loss of Progranulin in Frontotemporal Dementia Julie van der Zee*1,2,5, Isabelle Le Ber*9,10,11, Sebastian Maurer-Stroh19, Sebastiaan Engelborghs3,5,6, Ilse Gijselinck1,2,5, Agnès Camuzat14, Nathalie Brouwers1,2,5, Rik Vandenberghe7, Kristel Sleegers1,2,5, Didier Hannequin15, Bart Dermaut8, Joost Schymkowitz19, Dominique Campion16, Patrick Santens8, Jean-Jacques Martin4,5, Lucette Lacomblez10,12,18, Tim De Pooter1,2,5, Karin Peeters1,2,5, Maria Mattheijssens1,2,5, Martine Vercelletto17, Marleen Van den Broeck1,2,5, Marc Cruts1,2,5, Peter P. De Deyn3,5,6, Frederic Rousseau19, Alexis Brice9,10,12,13, and Christine Van Broeckhoven1,2,5†
1Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB; 2Laboratory of Neurogenetics; 3Laboratory of Neurochemistry and Behavior; 4Laboratory of Neuropathology, Institute Born-Bunge; 5University of Antwerp, Antwerpen, Belgium; 6Memory Clinic, Division of Neurology, Middelheim General Hospital, Antwerpen, Belgium; 7Departments of Neurology and Neuropathology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium; 8Department of Neurology, Ghent University Hospital, University of Ghent, Gent, Belgium; 9INSERM U679, Neurology and Experimental Therapeutics, Paris, France ; 10Fédération des Maladies du Système Nerveux, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France; 11Centre de Neuropsychologie et du Langage, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France ; 12Université Pierre et Marie Curie, Faculté de Médecine, Paris, France; 13Département de Génétique, Cytogénétique et Embryologie, Hôpital Pitié-Salpêtrière, AP-HP, Paris, France; 14INSERM U679, Hôpital de la Salpêtrière, Paris, France; 15INSERM U614, Rouen University Hospital, France; 16Département de Neurologie, Rouen University Hospital, France; 17Service de Neurologie, CHU Guillaume et René Laënnec, Nantes, France; 18Service de Pharmacologie, Hôpital Pitié-Salpêtrière, Paris, France; 19SWITCH Laboratory, VIB, Free University of Brussels, Brussels, Belgium
*These authors contributed equally to this work. †Correspondence to: Prof. Dr. Christine Van Broeckhoven Ph.D., D.Sc., VIB - Department of Molecular Genetics, Neurodegenerative Brain Diseases Group, University of Antwerp - CDE, Universiteitsplein 1 BE-2610 Antwerpen, Belgium; Tel.: +32 3 2651001; Fax: +32 3 2651012; E-mail: [email protected] Communicated by Jean-Louis Mandel
Frontotemporal dementia (FTD) [MIM# 600274] is a genetically complex disorder with multiple genes associated to disease etiology [Hutton et al., 1998; Baker et al., 2006; Cruts et al., 2006a; Skibinski et al., 2005; Watts et al., 2004]. Up to 50% of FTD patients have a positive family history of dementia, mainly with autosomal dominant inheritance [Chow et al., 1999; Poorkaj et al., 2001; Rosso et al., 2003]. The majority of FTD families have been linked to chromosome 17q21. In 1998, mutations in the microtubule associated protein tau gene (MAPT) [MIM# 157140] were identified in a set of families with FTD and parkinsonism linked to chromosome 17q21 (FTDP-17) [Hutton et al., 1998] and so far 39 MAPT mutations have been identified in 115 families worldwide (AD&FTD Mutation database: http://www.molgen.ua.ac.be/FTDmutations) [Rademakers et al., 2004]. At autopsy, MAPT mutation carriers consistently showed extensive tau pathology [Rademakers et al., 2004]. Over the years however, evidence accumulated for the presence of a second gene at 17q21 involved in FTD [van der Zee et al., 2007; Cruts et al., 2006b]. Recently heterogeneity at the 17q21 locus was explained by the identification of mutations in the progranulin gene (GRN, PGRN) [MIM# 138945], located 1.7 Mb centromeric of MAPT. Subsequent mutation data showed that PGRN mutations are a major cause of FTD, and are associated with tau-negative neuropathology characterized by ubiquitin-immunoreactive (ub-ir) neuronal inclusions (FTDU-17, FTD with ub-ir inclusions linked to chromosome 17) [MIM# 607485] [Baker et al., 2006; Cruts et al., 2006a; Gass et al., 2006]. These cytoplasmic and pathognomic lentiform intranuclear inclusions were observed in layer II of the frontal and temporal neocortex and in the dentate fascia of the hippocampus [Lipton et al., 2004; Mackenzie and Feldman, 2005].
PGRN consists of one non-coding exon 0 and 12 coding exons covering 8 kb of genomic sequence. It encodes progranulin (PGRN), a 593 amino acid secreted precursor glycoprotein of 68.5 kDa, composed of a signal peptide followed by 7.5 tandem repeats of a 12-cysteinyl granulin motif, that can be proteolytically cleaved to form a family of 6 kDa granulin peptides [He and Bateman, 2003]. PGRN is a pluripotent widely expressed growth factor with constitutive roles in development, cell cycle progression, cell motility, wound repair and inflammation. In tumor tissue PGRN was found to be overexpressed [Daniel et al., 2000; He et al., 2003; He and Bateman, 2003]. In the central nervous system PRGN is highly expressed in neurons of the cerebral cortex, particularly in the granule cells of the hippocampus, and in the Purkinje cells of the cerebellum [Daniel et al., 2000]. In other neurodegenerative diseases upregulation of PGRN was observed in microglia of Creutzfeldt-Jacob patients and in spinal cord of patients suffering from amyotrophic lateral sclerosis (ALS) [MIM# 105400] [Baker and Manuelidis, 2003; Malaspina et al., 2001].
To date, 37 different PGRN mutations have been identified in 67 FTD patients worldwide (AD&FTD Mutation database: http://www.molgen.ua.ac.be/FTDmutations). These include 18 frameshift mutations, 7 splice site mutations, 9 nonsense mutations, 1 missense mutation in the signal peptide and 2 mutations destroying the Met1 translation initiation codon [Baker et al., 2006; Cruts et al., 2006a; Gass et al., 2006; Mukherjee et al., 2006; Huey et al., 2006; Bronner et al., 2006; Le Ber et al., 2007]. Also a splice site mutation that leads to exon 1 skipping, removed the Met1 codon and associated Kozac sequence [Gass et al., 2006]. Two splice site mutations in the splice donor site of exon 0 lead to read-trough of intron 0 and subsequent nuclear mRNA degradation [Cruts et al., 2006a; Le Ber et al., 2007], while the remaining other mutant transcripts are degraded by nonsense mediated mRNA decay [Baker et al., 2006; Cruts et al., 2006a]. Therefore, all these mutations create PGRN null alleles leading to a 50% loss of functional PGRN, and are expected to exert their pathogenic nature through haploinsufficiency. Although the exact biological function of PGRN in the central nervous system remains elusive, the loss of functional protein observed in FTDU-17 supports a role for PGRN in maintaining neuronal survival.
In the present study we investigated the pathogenic nature of PGRN missense mutations and sequence variations in the 5’ regulatory region identified in two studies, a Belgian (N = 136) and a French (N = 196) FTD patient series [Cruts et al., 2006a; Le Ber et al., 2007], using in silico conservation and structural analyses to assess their effect on PGRN expression levels and the biological function of PGRN.
MATERIALS AND METHODS
Subjects The Belgian patient sample consisted of 136 FTD patients who were diagnosed using a standard protocol and
established clinical criteria as described [Engelborghs et al., 2003; Neary et al., 1998; Cruts et al., 2006a]. For the French series, DNA samples from 196 index patients with FTD were collected through a French research network of neurologists [Le Ber et al., 2006]. The diagnosis of FTD was based on the Lund and Manchester criteria as
described [The Lund and Manchester Groups, 1994; Le Ber et al., 2007]. Previous mutation analyses identified a MAPT mutation in three Belgian and six French FTD patients, and a presenilin 1 (PSEN1) [MIM# 104311] mutation in one Belgian patient [van der Zee et al., 2006; Dermaut et al., 2004; Le Ber et al., 2007]. In addition to patients, 459 unrelated neurologically healthy Belgian and 187 French control individuals were analyzed for PGRN variations. The research protocols for this study were approved by the Medical Ethical Committee of the University of Antwerp, the Medical Research Ethics Committee of “Assistance Publique-Hôpitaux de Paris”, and the ethics committee of the Salpêtrière Hospital. Descriptives of the Belgian and French study populations are summarized in Table 1.
Table 1. Descriptives of Belgian and French Study Samples
1AAO: age at onset in years ± the standard deviation. 2Positive family history was defined as having at least one first degree relative with dementia or FTD. 3AAI: age at inclusion in years ± the standard deviation.
PGRN Sequencing Analysis
PGRN mutation analysis was performed in 136 Belgian patients and 190 French patients without MAPT mutations as well as in the French and Belgian control individuals as described [Cruts et al., 2006a; Le Ber et al., 2007]. All PGRN exons and intron-exon boundaries were sequenced, including the non-coding exon 0 and a conserved region in intron 0 (g.96237 – g.96983; numbering is relative to the reverse complement of GenBank Accession Number AC003043 and starting at nt 1.). Total genomic DNA was prepared from peripheral blood according to standard procedures. The exons and part of intron 0 were PCR amplified on genomic DNA (20ng) using previously described primers [Cruts et al., 2006a] and an additional primer set for intron 0 (IVS0-F 5’-GGCCATGTGAGCTTGAGGTT-3’, IVS0-R 5’-GAGGGAGTATAGTGTATGCTTCTACTGAATA-3’). Amplification products were purified with 1 U antarctic phosphatase (New England Biolabs, Ipswich, MA USA) and 1 U exonuclease I (New England Biolabs) and sequenced in both directions using the BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) on an ABI3730 automated sequencer (Applied Biosystems). Sequences were analyzed using the Software Package novoSNP [Weckx et al., 2005].
Mutation Nomenclature Genomic DNA (gDNA) mutation numbering is relative to the reverse complement of GenBank Accession
Number AC003043.2 and starting at nt 1. Complementary DNA (cDNA) mutation numbering is relative to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation site +1. The protein mutation numbering is according to the largest PGRN isoform (GenPept Accession Number NP_002078.1).
Microsatellite Genotyping
In the Belgian patient DR121.1 and the French patient F98/001, who both carried the PGRN c. 1294C>T, p.Arg432Cys mutation, 14 microsatellite (STR) markers spanning an 8 cM region around PGRN were genotyped
for allele sharing analysis, as described [van der Zee et al., 2006]. Twenty ng genomic DNA was amplified in multiplex PCRs, at annealing temperature of 58°C, with fluorescently labeled primers. PCR products were sized on an ABI 3730 automated sequencer (Applied Biosystems), and genotypes were assigned using in-house developed genotyping software.
In Silico Analyses Evolutionary conservation analysis was performed using the Sorting Intolerant From Tolerant (SIFT v.2)
program [Ng and Henikoff, 2003] to estimate the severity of amino acid mutations caused by single nucleotide polymorphisms (SNPs) by comparison to the evolutionary available pool and variability of amino acids at the mutated positions in an alignment of homologous sequences. Different inputs of selected homologues and their alignment were used: 61 unaligned sequences from BLink [Wheeler et al., 2004], SIFT aligns, remove 100% identical; ClustalX [Jeanmougin et al., 1998] alignment of 61 sequences from BLink, remove 100% identical; Query sequence, SIFT finds homologues and aligns, remove 100% identical. Scores <0.05 are predicted to affect protein function, scores ≥ 0.05 are predicted to be tolerated (Table 2).
Table 2. PGRN Missense Mutations in FTD Patients
Variation Patients SIFT6
Alias1 Genome2 Predicted RNA3
Predicted protein4 Origin Family
history5Onset (years) A B C
EX7+35C>T g.101973C>T c.743C>T p.Pro248Leu French - 71 0.02 0 0 EX7+65G>A g.102003G>A c.773G>A p.Ser258Asn French - 53 0.14 0.24 0.03
EX10+115C>T g.103031C>T c.1294C>T p.Arg432Cys French +* 65 0.19 0.02 0.19 Belgian - 66
Missense mutations were absent in 646 control individuals.1EX:exon, exon numbering starts with noncoding first exon EX0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3Numbering according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation codon. 4Numbering according to the largest PGRN isoform (GenPept Accession Number NP_002078.1). 5A negative family history indicates that no first degree relatives were reported with dementia or FTD. 6SIFT consensus predictions [Ng and Henikoff, 2003]: A) 61 unaligned sequences from BLink [Wheeler et al., 2004], SIFT aligns, remove 100% identical. B) ClustalX [Jeanmougin et al., 1998] alignment of 61 sequences from BLink, remove 100% identical. C) Query sequence, SIFT finds homologues and aligns, remove 100% identical. Scores <0.05 are predicted to affect protein function (in bold), scores ≥ 0.05 are predicted to be tolerated. *PGRN c.1294C>T, p.Arg432Cys was also detected in an affected cousin of the index patient.
To assess effects of mutations on structure and stability of granulin domains, we modeled the full structures of
individual granulin domains based on the repetitive occurrence of the disulfide connected beta-hairpin stack motif (crystal structure PDB 1g26 [Tolkatchev et al., 2000b]) using SwissPDB-Viewer [Guex and Peitsch, 1997], Modeller [Fiser and Sali, 2003], ProQ [Wallner and Elofsson, 2003] and FoldX [Schymkowitz et al., 2005] (Fig. 2). Differences in free energy resulting from the mutations were estimated using FoldX analogous to the SNPeffect method [Reumers et al., 2006], with the exception of an additional penalty for forming or breaking disulfide bonds [Czaplewski et al., 2004].
To estimate the effect of 5’ regulatory region variations on putative transcription factor binding sites we performed a MatInspector analysis (http://www.genomatix.de) [Cartharius et al., 2005]. A core similarity cut-off value of 1 and an optimized matrix similarity -0.05 were used (Table 3).
Progranulin Mutations in FTD 5
Table 3. PGRN 5’ Regulatory Variations in FTD Patients
Promoter mutations were absent in 646 control individuals. 1EX: exon, IVS: intron, exon numbering starts with noncoding first exon EX 0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3A negative family history indicates that no first degree relatives were reported with dementia or FTD. 4MatInspector analysis [Cartharius et al., 2005], a core similarity cut-off value of 1 and an optimized matrix similarity -0.05 were used. *Neuropathological diagnosis at autopsy was conforming to FTDU.
RESULTS
Genetic Variability of PGRN Apart from 13 previously reported null mutations [Cruts et al., 2006a; Le Ber et al., 2007] (Fig. 1), extensive
mutation analysis of PGRN in 332 FTD patients identified 11 exonic and five intronic variants, as well as ten variants in the 5’ and two in the 3’ regulatory regions of PGRN. Three missense mutations (Table 2, Figs. 1,2) and three sequence variations in the 5’ regulatory region (Table 3, Fig. 1) were detected only in patients and were absent in 1292 control chromosomes. Also three silent mutations were unique to patients (Table S1). The remaining 19 variants were present in patients as well as control individuals and consisted of 11 rare (Table S2) and eight frequent polymorphisms (Table S3).
Figure 1. Genetic variability of PGRN observed in Belgian and French FTD patients. Schematic presentation of PGRN illustrating the mutations identified in 136 Belgian and 196 French FTD patients. Coding mutations are given by their cDNA position numbered according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at the translation initiation site +1. Mutations in the 5’ regulatory region are given by their gDNA position relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. Previously identified null mutations are depicted in the upper half, promoter and missense mutations observed in this study in the lower half. Mutations identified in Belgian patients are in red, those identified in French patients are in green. c.1294C>T was identified in one Belgian and one French FTD patient.
PGRN Missense Mutations We investigated in silico the effect of c.743C>T, p.Pro248Leu; c.773G>A, p.Ser258Asn and c.1294C>T,
p.Arg432Cys on PGRN protein sequence conservation and structure (Fig. 2, Table 2). Two missense mutations p.Pro248Leu and p.Arg432Cys were predicted to be pathogenic. SIFT analysis predicted that Pro248Leu would dramatically perturb protein function (p=0.00), which was in accordance with the structural modeling that revealed
6 van der Zee et al.
a significant destabilizing effect of 6.22 ± 0.54 kcal/mol on the granulin domain. In the granulin domain structure Pro248 is located in a loop connecting two β-hairpins where it is most likely essential to constrain a sharp and rigid turn (Fig. 2B). Moreover, Pro248 is adjacent to two Cys residues of the granulin B domain at a position which is 100% conserved between the seven granulin domains. Arg432Cys is located between granulin domains C and D (Fig. 2A). Depending on the parameters used, SIFT analysis predicted that this mutation perturbed the biological function of PGRN (p=0.02) (Table 2). In addition, no Cys residues are normally observed between the granulin domains. Arg432Cys was detected in one Belgian (DR121.1, onset age 66 years) and one French FTD patient (F98/001, onset age 65 years). Allele sharing analysis using markers located in and around PGRN demonstrated that six consecutive STR markers in a region of 5.36 Mb centromeric of PGRN were shared as well as all intragenic PGRN SNPs (Fig. 3). In 102 control individuals, EM estimation could not reveal this shared haplotype, neither was this allele combination observed.
Ser258Asn, located in granulin domain B, affects an amino acid residue that is conserved across orthologues but not between the granulin domains (Fig. 2A). SIFT analysis predicted a moderately significant effect for this mutation on protein function (p=0.03) (Table 2), however structural modeling failed to show a destabilizing effect on the granulin domain.
Figure 2. PGRN missense mutations relative to sequence conservation and structure. A: PGRN sequence alignment of individual granulin domains. Conserved Cys residues are highlighted in dark blue, other conserved amino acids in light blue. PGRN missense mutations are in red. B: Molecular modeling of granulin domains. The complete structure of a granulin domain was reconstructed based on the crystal structure of the N-terminal module of Granulin A (PDB 1g26 [Tolkatchev et al., 2000a]) and the inherent symmetry of the disulfide-dominated structure. It comprises six disulfide bonds and can be split into three self-similar overlapping modules. The two missense mutations located in a granulin domain were mapped on the reconstructed model.
Progranulin Mutations in FTD 7
Figure 3. The FTDU-17 genomic region. The red vertical bar represents the minimal candidate region delineated by FTD families 1083 [Rademakers et al., 2002] and UBC17 [Mackenzie et al., 2006] combined with FTD patients DR121.1 and F98/001, carrying the PGRN p.Arg432Cys mutation and sharing a common haplotype at 17q21. The candidate region of the Belgian founder family DR8 is also given [van der Zee et al., 2006]. Positions of PGRN and MAPT are indicated. Genetic sizes are according to the Marshfield gender-averaged linkage map; physical sizes according to the human reference sequence NCBI build 35.
PGRN Sequence Variations in 5’ Regulatory Region The three promoter variants were analyzed using MatInspector to assess whether they potentially altered
transcription factor binding (TFB) specificities (Table 3). MatInspector analysis predicted gain and/or loss of TFB sites for all three variations. One promoter mutation, g.96172G>T (EX0+148G>T) was predicted to create a CDP site and loss of a CDE site. CDP (CCAAT displacement protein) is a transcriptional factor for many diverse cellular genes that are involved in most cellular processes, including differentiation, development, and proliferation [Nishio and Walsh, 2004], and CDE is able to regulate gene transcription in a cell cycle-dependent manner [Lange-zu et al., 2000]. g.96282G>T (IVS0+46G>T) predicted gain of one Sp2 domain. Sp/XKLF proteins are shown to regulate transcription of genes involved in cell cycle control, oncogenesis, and differentiation [Moorefield et al., 2004]. Finally, g.96425C>T (IVS0+189C>T) predicted loss of one EGR1 site and gain of one PAX5 site. EGR1 belongs to the early growth response family of zinc finger transcription factors and is involved in many processes related to growth, differentiation and injury repair [McKee et al., 2006]. The PAX5 transcription factor has an important role in development of both B-lymphocytes and brain [Steinbach et al., 2001].
DISCUSSION
Recent identification of PGRN as the gene responsible for FTD with ub-ir neuronal brain pathology linked to chromosome 17, has contributed significantly to our understanding of the genetic etiology of FTD. Most PGRN mutations reported to date are frameshift, nonsense or splice site mutations that lead to loss of mutant transcript and thus functional protein. In the present study we investigated the pathogenic nature of PGRN missense mutations and sequence variations in the 5’ regulatory region that we identified in a systematic study of PGRN in a Belgian and French FTD series comprising 332 patients.
Three missense mutations, c.743C>T, p.Pro248Leu; c.773G>A, p.Ser258Asn; and c.1294C>T, p.Arg432Cys were identified in four patients (4/332 or 1.2 %), and were absent in 1292 control chromosomes. In silico predictions based on evolutionary conservation and structure, indicated that at least two mutations, p.Pro248Leu and p.Arg432Cys, are likely pathogenic since they significantly affect protein structure and stability. Pro248, located in granulin domain B, is evolutionary conserved across PGRN orthologues including rodents and between all granulin domains. Further, molecular modeling indicated that Pro248 is located in a loop of the β-hairpin stack of granulin B. Replacement of a Pro residue at this critical turn by a larger Leu residue would inevitably affect folding kinetics and stability of the granulin domain. Arg432, located between granulin domains C and D, is also a
8 van der Zee et al.
highly conserved residue, and its substitution might potentially disturb proteolytic cleavage between the respective domains. Alternatively p.Arg432Cys might be deleterious by adding an extra Cys-residue to a highly Cys-rich protein competing with existing disulfide bounds, and thereby destabilizing the disulfide-dominated fold of the granulin domains. The latter is supported by the observation that no Cys-residues have been observed between granulin domains indicating that intergranular Cys-residues would not be tolerated. Cys-residue mutations interfering with disulfide bridging are also observed in the EGF-like domains of the NOTCH3 gene involved in CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) [MIM# 125310] [Federico et al., 2005]. Therefore, these two missense mutations are predicted to exert a pathogenic loss of function of PGRN by drastically affecting protein folding and/or stability leading to potential interference with protein-protein interactions or misfolding and early degradation. Although Ser258 is conserved in other species including rodents and p.Ser258Asn was excluded in over 600 control individuals, the pathogenicity of this mutation was less clear from the in silico analyses and at this stage we cannot exclude that it might represent a benign polymorphism (frequency < 1/1292 or < 0.08% in control chromosomes) rather than a pathogenic mutation.
Of interest is that we observed p.Arg432Cys in two independently ascertained FTD patients of Belgian (DR121.1) and French (F98/001) ancestry. These patients shared a common haplotype across the PGRN locus and flanking centromeric region of at least 5.36 Mb at 17q21, indicative of a common founder effect. Combined with the previously reported minimal candidate region for FTDU-17 this reduced the FTDU-17 locus to 3.18 Mb centromeric of PGRN and excluding MAPT (Fig. 3). This confirmed our previous data excluding a role for MAPT in FTDU-17 based on genomic sequencing and fluorescent in situ hybridization [Cruts et al., 2006a; Gijselinck et al., 2006]. F98/001 had a positive family history of dementia and p.Arg432Cys was detected in another affected cousin, further supporting the pathogenic nature of this mutation. No familial anamnesis was reported for patient DR121.1. However, this patient was an only child which is probably the explanation why FTD presented as sporadic in this family.
We also observed three patient-specific sequence variations of highly conserved nucleotides (3/332 or 0.9 %) in the 5’ regulatory region of the gene. MatInspector analysis estimated changes in TFB sites for all three variants. g.96172G>T (EX0+148G>T), was identified in a Belgian patient diagnosed with familial FTD and onset age of 51 years. This patient died at the age of 55 years and brain autopsy confirmed the diagnosis of FTD with ub-ir neuronal inclusions (FTDU). These data suggested that changes in PGRN transcriptional activities could be involved in risk for FTD. In agreement with previous genetic findings of loss of transcript and/or protein, one would expect the promoter variations to significantly reduce PGRN expression, however, reporter gene studies will be needed to confirm and asses the effect of these promoter mutations on PGRN transcriptional activity.
In addition to patients, mutation analysis of PGRN in 646 control individuals revealed 25 sequence variations that were present only in control individuals (Table S4). Rare PGRN variants were detected in 11.3 % (73/646) of control individuals versus 12.4 % (41/332) of patients. This observation indicated that the natural genetic variability of PGRN is high and that it depends on the impact of the variation on protein structure and stability if it is pathogenic in nature or not. Considering this, it was puzzling to find the c.473G>A, p.Cys158Tyr mutation in an 82 year old control person, since gain or loss of a Cys-residue is expected to interfere with the disulfide-bridges constraining the granulin fold. This variation is either insufficient to cause disease or this is an example of non-penetrance. Non-penetrance of PGRN null mutations has been reported in the Belgian founder family DR8 segregating PGRN mutation g.96241G>C, IVS0+5G>C as well as in other American FTD families [Gass et al., 2006].
In conclusion, the present study provides indications that initial reports of PGRN mutation frequency might be underestimated. When considering all PGRN mutations (nonsense, frameshift, missense and promoter mutations) (Fig. 1), they would account for 8.43 % of all FTD patients (28/332) and 18.69 % of patients with a positive family history of FTD (20/107). In the group of patients with pathologically confirmed FTD, the PGRN mutation frequency would be 53.33 % (8/15) and rise to 66.67 % of patients with a FTDU diagnosis (8/12). These PGRN mutations most likely exert their pathogenic effect through reduced PGRN protein levels by loss of transcript or reduced transcription (nonsense en frameshift transcripts and promoter mutations), loss of translation (Met1 mutations) or loss of protein function (missense mutations). Altogether, these observations extend the mutation spectrum of PGRN leading to FTD.
Silent mutations were absent in 646 control individuals. 1EX: exon, exon numbering starts with noncoding first exon EX 0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3Numbering according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation codon. 4Numbering according to the largest PGRN isoform (GenPept Accession Number NP_002078.1).
1EX: exon, IVS: intron, UTR: untranslated region, exon numbering starts with noncoding first exon EX 0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3Numbering according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation codon. 4Numbering according to the largest PGRN isoform (GenPept Accession Number NP_002078.1).
Table S3. Frequent PGRN Polymorphisms
Variation
Alias1 Genome2 Predicted RNA3 Predicted protein4
rs number
Controls
N = 646 (%) 5'-111delC g.95914delC - - rs17523519 25.93
3'UTR+78C>T g.103778C>T c.1860C>T - rs5848 27.19 1EX: exon, IVS: intron, UTR: untranslated region, exon numbering starts with noncoding first exon EX 0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3Numbering according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation codon. 4Numbering according to the largest PGRN isoform (GenPept Accession Number NP_002078.1).
10 van der Zee et al.
Table S4. Rare PGRN Variants in Control Individuals
3'UTR+268G>T g.103968G>T c.2050G>T - 0.15 1EX: exon, IVS: intron, UTR: untranslated region, exon numbering starts with noncoding first exon EX 0. 2Numbering relative to the reverse complement of GenBank Accession Number AC003043.2 and starting at nt 1. 3Numbering according to the largest PRGN transcript (GenBank Accession Number NM_002087.2) and starting at translation initiation codon. 4Numbering according to the largest PGRN isoform (GenPept Accession Number NP_002078.1).
ACKNOWLEDGMENTS
The authors are grateful to the patients and family members for their kind cooperation in this study, to the personnel of the VIB - Genetic Service Facility (http://www.vibgeneticservicefacility.be) and of the central Biobank of the Institute Born-Bunge, to Evelyn De Leenheir and Bart Van Everbroeck for support with the neuropathology studies, and to the members of the French Research Network on FTD/FTD-MND for the recruitment of patients and control samples including: Alexis Brice (Hôpital Pitié- Salpêtrière, Paris), Françoise Clerget-Darpoux (Hôpital Paul-Brousse, Villejuif), Gilles Defer (CHU Cote de Nacre, Caen), Mira Didic (CHU La Timone, Marseille), Claude Desnuelle (CHU, Nice), Bruno Dubois (Hôpital Pitié-Salpêtrière, Paris), Charles Duyckaerts (Hôpital Pitié-Salpêtrière, Paris), Véronique Golfier (CH, Saint-Brieuc), Didier Hannequin (Rouen University Hospital), Lucette Lacomblez (Hôpital Pitié-Salpêtrière, Paris), Isabelle Le Ber (Hôpital Pitié-Salpêtrière, Paris), Bernard-François Michel (CH Sainte-Marguerite, Marseille), Florence Pasquier (CHU Roger Salengro, Lille), Catherine Thomas-Anterion (CHU Bellevue, Saint-Etienne), Michèle Puel (CHU Purpan, Toulouse), François Salachas (Pitié-Salpêtrière, Paris), François Sellal (Hôpitaux Civils and INSERM U692, Strasbourg), Martine Vercelletto (CHU Laennec, Nantes), Patrice Verpillat (Hôpital Pitié-Salpêtrière, Paris), William Camu (CHU Gui de Chauliac, Montpellier).
Progranulin Mutations in FTD 11
The research described in this paper was partly supported by a Zenith Award of the Alzheimer Association USA, the International Alzheimer Research Foundation (IARF) – Belgium, the EU contract LSHMCT-2003-503330 (APOPIS) , the InterUniversity Attraction Poles (IAP) program P5/19 of the Belgian Federal Science Policy office (BELSPO), the Fund for Scientific Research – Flanders (FWO-F), the Institute for Science and Technology – Flanders (IWT-F), the Medical Research Foundation Antwerp and Neurosearch Antwerp, and the Special Research Fund (BOF) of the University of Antwerp. A.B. was funded by CRIC AP-HP 01107 (to A.B.), GIS-Institut des Maladies Rares and A03081DS/APS03002DSA (to A.B.), J.v.d.Z. is holder of a PhD fellowship of the IWT-F. I.L.B is holder of a fellowship from the France-Alzheimer association. S.M.S. is recipient of a Marie Curie Intra-European Fellowship. The FWO-F provided postdoctoral fellowships to K.S. and S.E., and PhD fellowships to I.G. and N.B.
REFERENCES
Baker CA, Manuelidis L. 2003. Unique inflammatory RNA profiles of microglia in Creutzfeldt-Jakob disease. Proc Natl Acad Sci U S A 100:675-679.
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. 2006. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 442:916-919.
Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T. 2005. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 21:2933-2942.
Chow TW, Miller BL, Hayashi VN, Geschwind DH. 1999. Inheritance of frontotemporal dementia. Arch Neurol 56:817-822.
Cruts M, Gijselinck I, van der ZJ, Engelborghs S, Wils H, Pirici D, Rademakers R, Vandenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den BM, Cuijt I, Vennekens K, De Deyn PP, Kumar-Singh S, Van Broeckhoven C. 2006a. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 442:920-924.
Cruts M, Kumar-Singh S, Van Broeckhoven C. 2006b. Progranulin mutations in ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Curr Alzheimer Res 3:485-491.
Czaplewski C, Oldziej S, Liwo A, Scheraga HA. 2004. Prediction of the structures of proteins with the UNRES force field, including dynamic formation and breaking of disulfide bonds. Protein Eng Des Sel 17:29-36.
Daniel R, He Z, Carmichael KP, Halper J, Bateman A. 2000. Cellular localization of gene expression for progranulin. J Histochem Cytochem 48:999-1009.
Dermaut B, Kumar-Singh S, Engelborghs S, Theuns J, Rademakers R, Saerens J, Pickut BA, Peeters K, Van den Broeck M, Vennekens K, Claes S, Cruts M, Cras P, Martin JJ, Van Broeckhoven C, De Deyn PP. 2004. A novel presenilin 1 mutation associated with Pick's disease but not beta-amyloid plaques. Ann Neurol 55:617-626.
Engelborghs S, Dermaut B, Goeman J, Saerens J, Marien P, Pickut BA, Van den Broeck M, Serneels S, Cruts M, Van Broeckhoven C, De Deyn PP. 2003. Prospective Belgian study of neurodegenerative and vascular dementia: APOE genotype effects. J Neurol Neurosurg Psychiatry 74:1148-1151.
Federico A, Bianchi S, Dotti MT. 2005. The spectrum of mutations for CADASIL diagnosis. Neurol Sci 26:117-124.
Fiser A, Sali A. 2003. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol 374:461-491.
Gass J, Cannon A, Mackenzie IR, Boeve B, Baker M, Adamson J, Crook R, Melquist S, Kuntz K, Petersen R, Josephs K, Brown SP, Graff-Radford N, Uitti R, Dickson D, Wzsolek Z, Gonzalez J, Beach TG, Bigio E, Johnson N, Weintraub S, Mesulam M, White CL, III, Woodruff B, Caselli R, Hsiung GY, Feldman H, Knopman D, Hutton M, Rademakers R. 2006. Mutations in progranulin are a major cause of ubiquitin-positive frontotemporal lobar degeneration. Hum Mol Genet 15:2988-3001.
Gijselinck I, Bogaerts V, Rademakers R, van der ZJ, Van Broeckhoven C, Cruts M. 2006. Visualization of MAPT inversion on stretched chromosomes of tau-negative frontotemporal dementia patients. Hum Mutat 27:1057-1059.
Guex N, Peitsch MC. 1997. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714-2723.
He Z, Bateman A. 2003. Progranulin (granulin-epithelin precursor, PC-cell-derived growth factor, acrogranin) mediates tissue repair and tumorigenesis. J Mol Med 81:600-612.
He ZH, Ong CHP, Halper J, Bateman A. 2003. Progranulin is a mediator of the wound response. Nature Medicine 9:225-229.
12 van der Zee et al.
Huey ED, Grafman J, Wassermann EM, Pietrini P, Tierney MC, Ghetti B, Spina S, Baker M, Hutton M, Elder JW, Berger SL, Heflin KA, Hardy J, Momeni P. 2006. Characteristics of frontotemporal dementia patients with a Progranulin mutation. Ann Neurol 60:374-380.
Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, Houlden H, Pickering-Brown S, Chakraverty S, Isaacs A, Grover A, Hackett J, Adamson J, Lincoln S, Dickson D, Davies P, Petersen RC, Stevens M, de Graaff E, Wauters E, van Baren J, Hillebrand M, Joosse M, Kwon JM, Nowotny P, Che LK, Norton J, Morris JC, Reed LA, Trojanowski J, Basun H, Lannfelt L, Neystat M, Fahn S, Dark F, Tannenberg T, Dodd PR, Hayward N, Kwok JB, Schofield PR, Andreadis A, Snowden J, Craufurd D, Neary D, Owen F, Oostra BA, Hardy J, Goate A, van Swieten J, Mann D, Lynch T, Heutink P. 1998. Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393:702-705.
Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ. 1998. Multiple sequence alignment with Clustal X. Trends Biochem Sci 23:403-405.
Lange-zu DC, Brandeis M, Berr F, Mossner J, Engeland K. 2000. A CDE/CHR tandem element regulates cell cycle-dependent repression of cyclin B2 transcription. FEBS Lett 484:77-81.
Le Ber I, Guedj E, Gabelle A, Verpillat P, Volteau M, Thomas-Anterion C, Decousus M, Hannequin D, Vera P, Lacomblez L, Camuzat A, Didic M, Puel M, Lotterie JA, Golfier V, Bernard AM, Vercelletto M, Magne C, Sellal F, Namer I, Michel BF, Pasquier J, Salachas F, Bochet J, Brice A, Habert MO, Dubois B. 2006. Demographic, neurological and behavioural characteristics and brain perfusion SPECT in frontal variant of frontotemporal dementia. Brain 129:3051-3065.
Le Ber I, van der Zee J, Hannequin D, Gijselinck I, Campion D, Puel M, Laquerriere A, De Pooter T, Camuzat A, Van den Broeck M, Dubois B, Sellal F, Lacomblez L, Vercelletto M, Duyckaerts C, Cruts M, Verpillat P, Van Broeckhoven C, Brice A, and the French Research Network on FTD/FTD-MND. 2007. PGRN gene mutations in both sporadic and familial frontotemporal dementia. Human Mutat: Accepted.
Lipton AM, White CL, III, Bigio EH. 2004. Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration. Acta Neuropathol (Berl) 108:379-385.
Mackenzie IR, Baker M, West G, Woulfe J, Qadi N, Gass J, Cannon A, Adamson J, Feldman H, Lindholm C, Melquist S, Pettman R, Sadovnick AD, Dwosh E, Whiteheart SW, Hutton M, Pickering-Brown SM. 2006. A family with tau-negative frontotemporal dementia and neuronal intranuclear inclusions linked to chromosome 17. Brain 129:853-867.
Mackenzie IRA, Feldman HH. 2005. Ubiquitin immunohistochemistry suggests classic motor neuron disease, motor neuron disease with dementia, and frontotemporal dementia of the motor neuron disease type represent a clinicopathologic spectrum. Journal of Neuropathology and Experimental Neurology 64:730-739.
Malaspina A, Kaushik N, de Belleroche J. 2001. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem 77:132-145.
McKee SC, Thompson CS, Sabourin LA, Hakim AM. 2006. Regulation of expression of early growth response transcription factors in rat primary cortical neurons by extracellular ATP. Brain Res 1088:1-11.
Moorefield KS, Fry SJ, Horowitz JM. 2004. Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells. J Biol Chem 279:13911-13924.
Mukherjee O, Pastor P, Cairns NJ, Chakraverty S, Kauwe JS, Shears S, Behrens MI, Budde J, Hinrichs AL, Norton J, Levitch D, Taylor-Reinwald L, Gitcho M, Tu PH, Tenenholz GL, Liscic RM, Armendariz J, Morris JC, Goate AM. 2006. HDDD2 is a familial frontotemporal lobar degeneration with ubiquitin-positive, tau-negative inclusions caused by a missense mutation in the signal peptide of progranulin. Ann Neurol 60:314-322.
Neary D, Snowden JS, Gustafson L, Passant U, Stuss D, Black S, Freedman M, Kertesz A, Robert PH, Albert M, Boone K, Miller BL, Cummings J, Benson DF. 1998. Frontotemporal lobar degeneration - A consensus on clinical diagnostic criteria. Neurology 51:1546-1554.
Ng PC, Henikoff S. 2003. SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res 31:3812-3814.
Nishio H, Walsh MJ. 2004. CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription. Proc Natl Acad Sci U S A 101:11257-11262.
Poorkaj P, Grossman M, Steinbart E, Payami H, Sadovnick A, Nochlin D, Tabira T, Trojanowski JQ, Borson S, Galasko D, Reich S, Quinn B, Schellenberg G, Bird TD. 2001. Frequency of tau gene mutations in familial and sporadic cases of non-Alzheimer dementia. Arch Neurol 58:383-387.
Rademakers R, Cruts M, Dermaut B, Sleegers K, Rosso SM, Van den Broeck M, Backhovens H, van Swieten J, van Duijn CM, Van Broeckhoven C. 2002. Tau negative frontal lobe dementia at 17q21: significant finemapping of the candidate region to a 4.8 cM interval. Mol Psychiatry 7:1064-1074.
Rademakers R, Cruts M, Van Broeckhoven C. 2004. The role of tau (MAPT) in frontotemporal dementia and related tauopathies. Hum Mutat 24:277-295.
Reumers J, Maurer-Stroh S, Schymkowitz J, Rousseau F. 2006. SNPeffect v2.0: a new step in investigating the molecular phenotypic effects of human non-synonymous SNPs. Bioinformatics 22:2183-2185.
Progranulin Mutations in FTD 13
Rosso SM, Donker KL, Baks T, Joosse M, De Koning I, Pijnenburg Y, de Jong D, Dooijes D, Kamphorst W, Ravid R, Niermeijer MF, Verheij F, Kremer HP, Scheltens P, van Duijn CM, Heutink P, Van Swieten JC. 2003. Frontotemporal dementia in The Netherlands: patient characteristics and prevalence estimates from a population-based study. Brain 126:2016-2022.
Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. 2005. The FoldX web server: an online force field. Nucleic Acids Res 33:W382-W388.
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, Sorensen SA, Gydesen S, Fisher EM, Collinge J. 2005. Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia. Nat Genet 37:806-808.
Steinbach JP, Kozmik Z, Pfeffer P, Aguzzi A. 2001. Overexpression of Pax5 is not sufficient for neoplastic transformation of mouse neuroectoderm. Int J Cancer 93:459-467.
The Lund and Manchester Groups. 1994. Clinical and neuropathological criteria for frontotemporal dementia. J Neurol Neurosurg Psychiatry 57:416-418.
Tolkatchev D, Ng A, Vranken W, Ni F. 2000b. Design and solution structure of a well-folded stack of two beta-hairpins based on the amino-terminal fragment of human granulin A. Biochemistry 39:2878-2886.
Tolkatchev D, Ng A, Vranken W, Ni F. 2000a. Design and solution structure of a well-folded stack of two beta-hairpins based on the amino-terminal fragment of human granulin A. Biochemistry 39:2878-2886.
van der Zee J, Gijselinck I, Pirici D, Kumar-Singh S, Cruts M, Van Broeckhoven C. 2007. Frontotemporal lobar degeneration with ubiquitin-positive inclusions: a molecular genetic update. Neurodegenerative Dis: In Press.
van der Zee J, Rademakers R, Engelborghs S, Gijselinck I, Bogaerts V, Vandenberghe R, Santens P, Caekebeke J, De Pooter T, Peeters K, Lubke U, Van den Broeck M, Martin JJ, Cruts M, De Deyn PP, Van Broeckhoven C, Dermaut B. 2006. A Belgian ancestral haplotype harbours a highly prevalent mutation for 17q21-linked tau-negative FTLD. Brain 129:841-852.
Wallner B, Elofsson A. 2003. Can correct protein models be identified? Protein Sci 12:1073-1086.
Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP, Kimonis VE. 2004. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 36:377-381.
Weckx S, Del Favero J, Rademakers R, Claes L, Cruts M, De Jonghe P, Van Broeckhoven C, De Rijk P. 2005. novoSNP, a novel computational tool for sequence variation discovery. Genome Res 15:436-442.
Wheeler DL, Church DM, Edgar R, Federhen S, Helmberg W, Madden TL, Pontius JU, Schuler GD, Schriml LM, Sequeira E, Suzek TO, Tatusova TA, Wagner L. 2004. Database resources of the National Center for Biotechnology Information: update. Nucleic Acids Res 32 Database issue:D35-D40.
