Alzheimer and Parkinson Diagnoses in Progranulin Null Mutation Carriers in an Extended Founder Family

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ORIGINAL CONTRIBUTION Alzheimer and Parkinson Diagnoses in Progranulin Null Mutation Carriers in an Extended Founder Family

Nathalie Brouwers, MSc; Karen Nuytemans, MSc; Julie van der Zee, MSc; Ilse Gijselinck, MSc; Sebastiaan Engelborghs, MD, PhD; Jessie Theuns, PhD; Samir Kumar-Singh, MD, PhD; Barbara A. Pickut, MD; Philippe Pals, MD, PhD; Bart Dermaut, MD, PhD; Veerle Bogaerts, MPharm; Tim De Pooter, BS; Sally Serneels, BS; Marleen Van den Broeck, BS; Ivy Cuijt, BS; Maria Mattheijssens, BSN; Karin Peeters, BSN; Raf Sciot, MD, PhD; Jean-Jacques Martin, MD, PhD; Patrick Cras, MD, PhD; Patrick Santens, MD; Rik Vandenberghe, MD; Peter P. De Deyn, MD, PhD; Marc Cruts, PhD; Christine Van Broeckhoven, PhD, DSc; Kristel Sleegers, MD, PhD

Background: Progranulin gene (PGRN) haploinsuffi- Results: We identified 2 patients with AD and 1 pa- ciency was recently associated with ubiquitin-positive tient with PD who carried the null mutation frontotemporal lobar degeneration linked to chromo- IVS0 ϩ 5GϾC, which we reported earlier in an exten- some 17q21 (FTLDU-17). sively characterized Belgian founder family, DR8, segregating FTLDU. Postmortem pathologic diagnosis of the Objective: To assess whether PGRN genetic variability patient with PD revealed both FTLDU and Lewy body contributed to other common neurodegenerative brain pathologic features. In addition, we identified in PGRN diseases, such as Alzheimer disease (AD) or Parkinson only 1 other null mutation, the nonsense mutation disease (PD). p.Arg535X, in 1 patient with probable AD. However, in vitro analysis predicted a PGRN C-truncated protein, al- Design: Mutation analysis of PGRN. though it remains to be elucidated if this shortened transcript leads to haploinsufficiency. Setting: Memory Clinic of the Middelheim General Hospital. Conclusions: Our mutation data indicated that null mutations are rare in patients with AD (3/666=0.45%) and Patients: We analyzed 666 Belgian patients with AD and PD (1/255=0.39%). Also, AD and PD clinical diagnoses 255 with PD. in patients who carry PGRN null mutations likely result from etiologic heterogeneity rather than PGRN haploin- Main Outcome Measures: Results of PGRN sequenc- sufficiency. ing, PGRN transcript analysis, short tandem repeat genotyping, and neuropathologic analysis. Arch Neurol. 2007;64(10):1436-1446

N FAMILIES SEGREGATING UBIQUI- tion and subsequent degradation.4 Also, re- tin-positive frontotemporal lo- duced levels of the respective mutant tran- bar degeneration linked to chro- script have been observed that might mosome 17q21 (FTLDU-17), contribute to haploinsufficiency.4,5 We re- null mutations have been iden- cently provided in silico evidence that at tified in the progranulin gene (PGRN), re- least some of the missense mutations ob- I 1,2 sulting in haploinsufficiency. Most PGRN served in patients with FTLD affect PGRN mutations result in loss of the PGRN tran- structure and/or stability, again resulting script after nonsense-mediated decay in loss of functional protein by protein deg- (NMD) or nuclear retention and degrada- radation.6 tion of messenger RNA (mRNA).1-3 Two Frontotemporal lobar degeneration PGRN mutations affect the translation ini- marks a subtype of neurodegenerative de- tiation codon, preventing translation.3 mentia in which the presenting clinical fea- Apart from null mutations, missense mu- tures consist of behavioral dysfunction and tations scattered throughout the protein personality changes, often with language have been identified in patients with fron- impairments. Despite the fact that these totemporal lobar degeneration (FTLD) and symptoms are noticeable early in disease control individuals. The signal peptide mu- in most patients with FTLD, a differen- Author Affiliations are listed at tation, p.Ala9Asp, is expected to lead to tial diagnosis of FTLD or Alzheimer dis- the end of this article. loss of protein due to protein mislocaliza- ease (AD) often remains difficult to estab-

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 lish.7 Furthermore, patients with FTLD and AD often The control group existed of 459 unrelated, healthy, Dutch- develop parkinsonian symptoms later in their disease,8,9 speaking Belgian individuals (mean±SD age at inclusion, and in PGRN null mutation carriers, parkinsonism has 58.6±16.0 years; 54.9% female) and included individuals already been reported.3 On the other hand, dementia is (n=275) without neurologic or psychiatric antecedents and in- common in patients with Parkinson disease (PD).10,11 dividuals (n=184) selected among those married into families with data on neurologic diseases collected for genetic linkage The PGRN gene encodes a precursor protein, PGRN, studies. which contains a secretory signal peptide and 7.5 tan- All studies were approved by the medical ethics committee demly repeated granulin domains (GRN A through G and of the ZNA Middelheim General Hospital, the University Hos- paragranulin), which are characterized by a highly con- pital Antwerp, and the University of Antwerp. After informed served motif of 12 cysteine residues that form 6 disul- consent, blood samples from probands were collected for ge- fide bridges.12 The PGRN gene is expressed in a wide va- netic studies. For mutation carriers, samples from additional riety of tissues, including neurons in the cerebral cortex, relatives were collected for haplotype-phase determination and the Purkinje cells in the cerebellum, and pyramidal cells segregation studies. in the hippocampus.12 PGRN and at least some of the GRNs have been shown to have growth modulatory prop- PGRN SEQUENCING erties, and PGRN transcripts have been found up- regulated in neurodegenerative disorders, such as In all patients and control individuals, we performed a mutation analysis of all coding exons, 1 to 12, and noncoding exon Creutzfeldt-Jakob disease and amyotrophic lateral scle- 2 12 0, as previously described. A total of 20 ng of genomic DNA rosis. These characteristics, along with the fact that PGRN (gDNA) was amplified by polymerase chain reaction (PCR), and null mutations cause FTLD, support a role for PGRN in amplification products were sequenced (Applied Biosystems neuronal survival. 3730xl DNA Analyzer using the Big Dye Terminator v3.1 Cycle Because patients with FTLD, AD, and PD display a broad Sequencing kit; Applied Biosystems, Foster City, California). spectrum of overlapping clinical symptoms and PGRN po- Sequences were analyzed using novoSNP.19 tentially exerts a role as a neuronal survival factor, we ar- gued that PGRN mutations might potentially be impli- PGRN TRANSCRIPT ANALYSIS cated in the etiology of AD and PD. Consequently, we performed an extensive mutation analysis of PGRN in large Lymphocytes were isolated from total blood with Ficoll den- groups of well-characterized patients with AD and PD. sity gradient centrifugation (Greiner Bio-One, Wemmel, Bel- gium), and mRNA was isolated from lymphocytes using the Chemagic mRNA Direct Kit (Chemagen, Leiden, the Nether- METHODS lands). First-strand complementary DNA (cDNA) was synthe- sized using the SuperScript III First-Strand Synthesis System STUDY GROUPS for RT (reverse transcription)–PCR kit (Invitrogen, Carlsbad, California) and random hexamer primers. After PCR amplifi- The AD group consisted of 666 patients (mean±SD age at on- cation of part of the transcript (exons 5-6 to 3Ј untranslated set, 74.6±8.8 years; 65.5% female) derived from a prospective region), genotypes at the mutated position were generated by study of neurodegenerative and vascular dementia in Bel- sequencing as described.2 gium.13,14 All patients were examined at the Memory Clinic of the Middelheim General Hospital, Antwerp, Belgium (by P.P.D.D., S.E., and B.A.P.), and underwent diagnostic neuro- SHORT TANDEM REPEAT GENOTYPING psychological examination (including Middelheim Frontality ϩ Ͼ Score [MFS]15) and structural (computed tomography and mag- In patients carrying IVS0 5G C and their family members, netic resonance imaging) and functional neuroimaging (single- we genotyped 14 STR markers located in and flanking the 8-cM 20 photon emission computed tomography).13,14 Consensus diag- ancestral DR8 haplotype, as previously described. Allele fre- nosis of probable (n=627) or possible (n=39) AD was given quencies for each STR marker were estimated in 102 unre- 20 by at least 2 neurologists (P.P.D.D., S.E., and B.A.P.) based on lated Belgian control individuals. the National Institute of Neurological and Communicative Dis- eases and Stroke/Alzheimer’s Disease and Related Disorders As- NEUROPATHOLOGIC ANALYSIS sociation criteria.16 In 48 patients with probable AD, clinical diagnosis was confirmed after autopsy. In 173 patients (26.0%), Brain autopsy was performed on patient DR205.1, who had given the disease was considered familial based on the presence of at prior informed consent, at the University Hospital Gasthuis- least 1 first-degree relative with dementia. In 365 patients, the berg. Brain hemispheres were fixed in buffered formalin and cerebrospinal fluid (CSF) was sampled and levels of amy- tissue was further processed for paraffin, embedding from the ␤ ␤ loid- peptide (A 1-42), total tau, and tau phosphorylated at right and left frontal and parietal cortices, hippocampus, basal 17 threonine 181 (P-tau181P) were determined as described. ganglia, midbrain, pons, medulla oblongata, and cerebellum. Belgian patients with PD (n=255; mean±SD age at onset, Sections 10 µm thick were sliced and stained with hematoxylin- 59.4±10.9 years; 43.1% female) were derived from 2 indepen- eosin, cresyl violet, Bodian, and Gallyas. Sections 5 µm thick dent studies. Eighty-two patients were selected from the same were also sliced from all brain regions, and immunohisto- prospective study13 as the patients with AD. Additionally, a set chemical analysis was performed with the following antibod- of 173 patients with PD was selected from a retrospective epi- ies: 4G8 (anti-A␤; Senetek, Maryland Heights, Missouri), AT8 demiologic study of PD.18 In both studies, a diagnosis of PD (against abnormally phosphorylated paired helical filament– required 3 of 4 of the following features to be present: brady- tau; Innogenetics, Zwijnaarde, Belgium), ubiquitin (UBI) kinesia, rigidity, tremor, and/or asymmetrical onset. In addi- (Dako, Glostrup, Denmark), ␣-synuclein (SNCA) (Dako), tion, a response to levodopa had to be present. Individuals had anti–glial fibrillary acidic protein (GFAP) (Dako), and rabbit a positive family history of PD if at least 1 first-degree relative TAR DNA-binding protein 43 antisera (TDP-43) (Proteintech presented with parkinsonism (3.5%). Group Inc, Chicago, Illinois). Antigen retrieval for A␤ immu-

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Mutation Age at Family Patient Presentation Onset, y Historya Locationb Alias Genomicc Predicted RNAd Predicted Proteine DR142.1 AD 66 ? IVS 0 IVS0 ϩ 5GϾC g.96241GϾC ... p.0 DR25.14 AD 76 ϩ IVS 0 IVS0 ϩ 5GϾC g.96241GϾC ... p.0 DR205.1 PD 54 ϩ IVS 0 IVS0 ϩ 5GϾC g.96241GϾC ... p.0 DR181.1 AD 72 ? EX 11 EX11 ϩ 190CϾT g.103432CϾT c.1603CϾT p.Arg535X

Abbreviations: AD, Alzheimer disease; EX, exon; IVS, intron; PD, Parkinson disease. a Family history of dementia: plus sign indicates that patient had first-degree family members who were affected; question mark, unknown. b Exon numbering starts with noncoding first exon EX 0. c Numbering relative to the reverse complement of GenBank Accession No. AC003043.1 and starting at nucleotide 1. d Numbering according to the largest PRGN transcript (GenBank Accession No. NM_002087.2) and starting at the translation initiation codon. e Numbering according to the largest PGRN isoform (GenPept Accession No. NP_002078.1).

gDNA cDNA DR181.1

C C T G C T G C TC G A G A C A A C C C T G C T G C TC G A G A C A A C

Control

C C T G C T G C C G A G A C A A C C C T G C T G C C G A G A C A A C

Figure 1. Nonsense-mediated decay analysis of p.Arg535X in patient DR181.1 compared with a control individual. Sequence traces are shown of genomic DNA (gDNA) and complementary DNA (cDNA) prepared from blood lymphocytes.

nohistochemical analysis was performed by treating sections The g.96241GϾC mutation identified in 2 patients with 98% formic acid for 5 minutes at room temperature for with AD and 1 with PD is located close to the splice- 4G8, SNCA, and TDP-43 and by boiling in citrate buffer donor site of intron 0 (IVS0 ϩ 5GϾC) (Table 1) and is (pH=6) for GFAP and UBI. All dilutions were made in 0.1M known to prevent splicing of intron 0, leading to loss of phosphate-buffered saline with 0.1% bovine serum albumin. the mutant transcript.2 Haplotype sharing analyses using Staining was performed with appropriate secondary antibodies and streptavidin–biotin–horseradish peroxidase with 14 STR markers flanking PGRN indicated that all 3 car- chromogen 3Ј,3Ј-diaminobenzidine (Roche, Nutley, New Jer- riers shared 1.61 cM (4.37 Mb) of the 8.0-cM disease hap- 2,20 sey), as previously described.21 lotype of founder family DR8 (Table 2). Pedigree analysis could not identify other living affected relatives of AD patient DR142.1 and PD patient DR205.1, pre- RESULTS venting cosegregation studies of IVS0 ϩ 5GϾC with disease. The other patient with AD was the first cousin of PGRN MUTATION ANALYSES the index patient DR25.1 of 1 known branch of the Bel- gian DR8 founder family and was subsequently labeled Direct exonic sequencing of PGRN identified 1 patient DR25.14. with AD who carried a novel nonsense mutation and 2 patients with AD and 1 with PD with a known splice- CLINICAL CHARACTERISTICS donor site mutation in intron 0 (Table 1). The g.103432CϾT mutation is located in exon 11 of PGRN Patient DR181.1, who carried p.Arg535X, was clinically (EX11 ϩ 190CϾT) (Table 1) and predicts a premature diagnosed as having probable AD 1 year after disease on- termination codon (PTC) at position 535 and a C- set. She presented with impairment of recent memory and truncated protein, p.Arg535X. Comparison of se- attention deficits (Table 3). Neuropsychological exami- quences obtained from the patient’s cDNA and from gDNA nation revealed a Mini-Mental State Examination score of (Figure 1) showed equal amounts of wild-type and mu- 19/30 with severe recent memory deficits and moderate tant alleles, indicating that the mutant transcript es- impairment of praxis, concentration, long-term memory, caped NMD. and orientation in time. Three years later, her cognitive

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Genetic Physical Linked Frequency Linked Marker Location, cM Location, Mb Alleleb Allele, %c DR142.1 DR25.14 DR205.1 D17S1818 60.40 34.42 ...... 446 430 430 D17S1814 61.48 35.37 465 19 465 465 465 D17S800 62.01 36.31 367 10 361 367 367 D17S1787 62.01 36.98 181 35 181 181 181 D17S1793 63.09 37.61 392 81 392 392 392 D17S951 63.62 39.18 143 23 143 143 143 PGRN ... 39.78 ...... D17S1861 63.62 40.16 278 6 278 278 278 D17S934 63.62 40.41 359 27 359 359 359 Chr17-16 . . . 40.68 401 22 401 401 399 D17S810 63.62 40.84 186 30 186 186 186 MAPT . . . 41.33 ...... D17S920 64.16 42.17 326 64 326 326 326-332 D17S931 66.85 42.36 277 9 277 277 265 Chr17-43 . . . 42.68 233 47 233 233 239 D17S1795 68.44 45.28 ...... 399 397 391

Abbreviations: MAPT, microtubule-associated protein tau; STR, short tandem repeat. a Bold indicates minimal founder haplotype (1.61 cM). b Ancestral haplotype identified in the DR8 founder family.20 c Allele frequencies were calculated in 92 control chromosomes. Genetic locations of the STR markers were obtained from the Marshfield gender-averaged map. Physical locations relative to National Center for Biotechnology Information genome build 35.

state had severely deteriorated (Mini-Mental State Exami- The PD patient DR205.1 had his condition diag- nation score of 3/30). The patient also displayed severe fron- nosed at the age of 56 years, 1 year after the onset of symp- tal lobe signs (MFS of 7/10). A CSF biomarker analysis toms. Symptoms included global and cogwheel rigidity, ␤ showed decreased A 1-42 and increased total tau levels, a hypomimia, bradykinesia, shuffling gait, postural insta- 22 finding compatible with AD. However, CSF P-tau181P lev- bility, and a discrete resting tremor. The patient re- els were within normal limits. Although patient DR181.1 sponded well to levodopa treatment. The patient re- had no relatives with dementia, a sibling of the patient was ported loss of concentration, but a neuropsychological diagnosed as having PD. examination revealed no abnormalities. Two years later, In Table 3 we summarize the medical information of however, progressive memory problems were recorded, 16 patients of the DR8 founder family who carried the accompanied by apathy, hypophonia, and reduced ver- IVS0 ϩ 5GϾC null mutation. Both AD patients DR25.14 bal expression. Behavioral observation and neuropsy- and DR142.1 presented with symptoms of forgetful- chological testing revealed loss of insight and judg- ness. In addition to severe impairment of recent memory, ment, changes in sexual behavior, impaired control of which first became apparent at the age of 76 years, pa- emotions, mutism, and echolalia, with comparatively tient DR25.14 had deficits of long-term memory. She be- spared memory and spatial abilities. The cognitive prob- came apathetic, displaying lack of initiative. Further- lems were not typical for Lewy body disease or PD with more, reduced verbal fluency was noted, but naming was dementia but were compatible with pronounced frontal unaffected. The patient was disoriented in both time and dysfunction in light of PD or with FTLD. Patient DR205.1 space and displayed impaired problem solving. The CSF had an MFS of 6/10. The mother and 2 maternal aunts biomarker profile was typical for AD, with decreased lev- of patient DR205.1 had late-onset dementia, but there were ␤ els of A 1-42 and increased levels of total tau and P-tau181P. no relatives with PD. Patient DR25.14 had a family history of dementia, with Since the identification of the DR8 founder mutation a mother, a maternal aunt, and a grandmother who had and subsequent PGRN screening in a series of patients late-onset dementia and 2 first cousins who were diag- with FTLD, we have identified 1 other patient with FTLD nosed as having FTLD (Figure 2). For patient DR142.1, (DR119.1) who had the IVS0 ϩ 5GϾC mutation, defin- early symptoms included repetitive telephone calls and ing a novel branch of founder family DR8.2 Patient repetition of stories and questions at the age of 66 years. DR119.1 presented with word-finding difficulties at the Because of disorientation in space, she got lost on sev- age of 45 years and social withdrawal.23 Memory was pre- eral occasions. Toward later stages of disease, a loss of served. Her speech was characterized by shortening and decorum became obvious. A year before her death, spon- simplification of sentences and phonemic paraphasia, taneous speech was impaired, and she displayed verbal which led to a diagnosis of progressive nonfluent apha- and motor stereotypies. Age at onset varied consider- sia. The patient’s father died at the age of 65 years after a ably between these patients with AD (ie, 66 and 76 years), 5-year period of progressive language impairment and which is in accordance with the wide onset range noted behavioral changes. Furthermore, patient DR8.15, a first in patients with FTLDU in family DR82,20 (Figure 3). cousin of the index patient DR8.1, developed primary pro- Both patients had an MFS score of 4/10. gressive aphasia at the age of 63 years. With unimpaired

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DR181.1/F AD 72 77 ↓Memory; CSF A␤1-42 and total Cortical and subcortical Relative parietotemporal NA ↓concentration tau typical for atrophy; cerebellar HP with involvement

AD, P-tau181P atrophy; discrete of both frontal lobes; normal for age PWML diastasis of frontal cortical activity DR2.1 (DR2 FTLD 66 71† ↓Memory, Disinhibition, Global mainly NA NA III-8)/M ↓concentration ↓verbal fluency subcortical atrophy (CT) DR2.3 (DR2 PNFA 63 72† PNFA, apathy Global Relative bilateral frontal, Frontotemporal atrophy III-6)/F corticosubcortical parietal and temporal with FTLDU; atrophy RϾL; PWML HP, RϾL (SPECT) concurrent AD (MRI) (Braak A-II) DR2.17 (DR2 FTLD 69 71 ↓Spontaneous Global moderate cortical Relative frontal and NA III-10)/M speech, and subcortical frontoparietal HP, perseveration, atrophy (MRI) LϾR; relative HP of dysarthria, apathy left thalamus; diastasis of frontal cortical activity (SPECT) DR8.1 (DR8 FTLD 62 69† ↓Word finding, Frontotemporoparietal Relative bilateral frontal FTLDU; frontal III-28)/F apathy, cortical and HP, LϾR (SPECT) atrophy Ͼ temporal; disinhibition, subcortical atrophy marked neuronal ↓Memory LϾR (MRI) loss in ZC of the SN; rare brainstem Lewy bodies DR8 III-18 FTLD 51 55† ↓Spontaneous ↓Memory, control Global cortical and Severe relative bifrontal NA (DR8 speech, echolalia, of emotions, loss subcortical atrophy HP, LϾR (SPECT) III-18)/F apathy, ⌬behavior of decorum (CT) DR8.15/F PPA 63 64 Aphasia, verbal Postcontusional bilateral NA apraxia frontal and R temporal (MRI) DR25.1 FTLD 69 75† ↓Spontaneous Repetitive Cortical and subcortical Severe relative bilateral FTLDU; frontal (DR25 speech, ↓Initiative, movement of the frontal atrophy; frontal, parietal, and atrophy Ͼ temporal; III-17)/F hyperorality hands periventricular white temporal HP; marked neuronal matter lesions (CT) scintigraphic loss in ZC of the SN; indications of NFT in hippocampus subcortical loss (maximum, 10/mm2) (SPECT). DR25.5 FTLD 70 74† Aphasia, apathy, Cortical and subcortical Bilateral frontal, parietal, Frontotemporal atrophy (DR25 ⌬behavior, atrophy, maximal and temporal HP with FTLDU III-18)/M ⌬personality frontally, LϾR; LϾR; right cerebellar PWML (MRI) HP (PET) DR25.14/F AD 76 78† ↓Memory, ↓verbal CSF biomarker Corticosubcortical Relative frontoparietal NA expression, apathy profile typical for atrophy, PWML, HP extending into AD lacunar infarctions in both anterior the basal ganglia temporal lobes; bilaterally (CT) preserved sensorimotor cortex activity (SPECT)

(continued)

memory and activities of daily living, her language im- atrophied, and the substantia nigra and locus coeruleus pairment was characterized by aphasia with reduced were severely depigmented. Histochemical and immu- fluency, excessive phonologic paraphasia of spoken and nohistochemical data confirmed severe neuronal loss written language, verbal apraxia, and perseverations. and showed gliosis in all neocortical regions analyzed, One year after disease onset, the patient started display- with many surviving neurons containing lipofuscin. ing behavioral changes such as disinhibited laughter Anti-UBI immunoreactivity visualized a huge burden of and stereotypic involuntary movements of the tongue threadlike inclusions in layers II and III, deeper cortical and jaw. layers (Figure 4A), and white matter (Figure 4B). These inclusions stained positive with UBI and SNCA PATHOLOGIC CHARACTERISTICS but not with AT8 antibodies. In the neocortical regions and basal ganglia, UBI-positive and tau- and SNCA- The AD patients DR25.14 and DR142.1 died without negative inclusions were observed. Lenticular, cat-eye autopsy (Table 3). The PD patient DR205.1 died at the types of inclusions were especially abundant in age of 61 years, and gross examination of autopsied the basal ganglia (Figure 4C), overlapping with infre- brain samples showed severe cortical atrophy, particu- quent Lewy body inclusions observed in the brainstem, larly of the frontal lobe. The caudate nucleus was also caudate nucleus, and occasionally cortical regions

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 Table 3. Overview of Clinical and Pathologic Characteristics of Mutation Carriers (cont)

Age at Death†/ Structural Functional Patienta/ Clinical Age at Current Presenting Additional Neuroimaging Neuroimaging Sex Diagnosis Onset, y Age, y Impairments Features (CT/MRI) (SPECT/PET) Findings DR26.1 PNFA 65 68† Progressive apraxia of Global subcortical and Relative frontal, NA (DR26 speech cortical atrophy temporal, and parietal II-1)/M maximal frontally and HP, LϾR; relative HP temporally, LϾR of basal ganglia and (MRI) lentiform nucleus, R cerebellar HP(SPECT) DR27.1 FTLD 58 64† ⌬Behavior (decorum, ↓ Verbal fluency, Corticosubcortical Bilateral frontal, Frontotemporal atrophy (DR27 aggression) extrapyramidal atrophy, maximal temporal. and parietal with FTLDU III-4)/F rigidity frontotemporally HP, RϾL; R HP at RϾL; PWML (MRI) parietooccipital transition; L cerebellar HP (PET) DR28.1 PNFA 57 62† PNFA Parkinsonism NA Relative frontal, Frontotemporal and (DR28 (tremor, rigidity) temporal, and parietal parietal lobe atrophy III-3)/M HP, LϾR (SPECT) with FTLD; mild neuronal loss in ZC of the SN; SP (maximum, 40/mm2) with rare NFT DR31.1 PNFA 66 70† PNFA Global cortical and Marked relative bilateral Frontotemporal atrophy (DR31 minor subcortical frontal, and temporal with FTLDU; II-1)/M temporal atrophy HP, LϾR; diastasis of neuronal loss in ZC LϾR (MRI) frontal cortical activity of the SN; rare (SPECT) perivascular plaque deposits; NFT (maximum, 7/mm2) DR119.1/F PNFA 45 47 ↓Word finding, Anterior temporal NA NA ↓verbal expression atrophy LϾR (MRI) DR142.1/F AD 66 76† ↓Memory ↓Spontaneous Slightly asymmetric Relative frontal, NA speech, loss of (RϾL) subcortical temporal, and parietal decorum and cortical atrophy. HP, RϾL; diastasis of PWML (CT) frontal cortical activity; preserved sensorimotor cortex activity (SPECT) DR205.1/M PD and frontal 56 61† Resting tremor, Mutism, echolalia, NA NA FTLDU; frontal dysfunction rigidity, loss of judgment, atrophy Ͼ temporal; bradykinesia disinhibition concurrent PD; SP (maximum, 120/mm2) with rare NFT

Abbreviations: A␤1-42, amyloid-␤ peptide; AD, Alzheimer disease; CSF, cerebrospinal fluid; CT, computed tomography; FTLD, frontotemporal lobar degeneration; FTLDU, ubiquitin-positive frontotemporal lobar degeneration; HP, hypoperfusion; L, left; MRI, magnetic resonance imaging; NA, not available; NFT, neurofibrillary tangle; PD, Parkinson disease; PET, positron emission tomography; PNFA, progressive nonfluent aphasia; PPA, primary progressive aphasia; PWML, periventricular white matter lesions; R, right; SN, substantia nigra; SP, senile plaques; SPECT, single-photon emission computed tomography; ZN, zona compacta; downward arrow, decreased; upward triangle, change in. aPatients with identifiers between brackets represent individuals for whom detailed clinical and/or pathologic data were described previously.2,20

(Figure 4D). The UBI-positive inclusions (neuronal (9/34=26%) were still cognitively healthy at an age intranuclear inclusions and neuronal cytoplasmic inclu- older than 60 years. Thirty-five percent of patients are sions) contained TDP-43 (Figure 4E). The patient was male. Of 16 patients, detailed medical information was diagnosed as having mixed pathologic features of dif- available that showed prominent language impairment fuse Lewy body disease and FTLDU. Numerous vessel- (progressive nonfluent aphasia and reduced spontane- related A␤-positive dense-core plaques were also ous speech) and behavioral and personality changes, of observed in the cortical and hippocampal regions which apathy was most frequently noted (n=6; (Figure 4F). Table 3). In 3 of 13 patients with FTLD (Table 3), impairment of memory was an early symptom. Parkinson- DR8 FOUNDER FAMILY ism was observed in 1 patient with a diagnosis of progressive nonfluent aphasia, which was attributed to the The current DR8 founder pedigree consists of 10 use of antipsychotic medication. However, for several branches that extend throughout 7 generations and siblings parkinsonian symptoms were reported by comprise up to 250 relatives, 237 of whom had genea- members of the family. logic data (Figure 2). Mean age at onset in 39 patients Pathologic diagnosis of FTLDU was obtained in 8 pa- was 64.4 years, ranging from 45 to 78 years, with most tients (S.K.-S., et al, unpublished data, 2007), with con- (25/39=64%) showing their first symptoms between current PD in patient DR205.1 and AD (Braak stage A-II) 60 and 75 years (Figure 3). Some mutation carriers in patient DR2.3. In 4 patients, mild to marked neuro-

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∗

44 4

0 ∗

∗

3 2

∗

4 4

∗

DR8.1

∗

∗ ∗ 7

68 ∗ 64 75

∗

68 ∗

∗ ∗

43 ∗

. 55

∗

I-1

∗

0 II

4 4

95 9 8

DR8

4 ∗

∗

83 DR25 5 4

∗

.14

∗

6 4

47 5

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8 43 71

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56 1

∗ 52

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6 DR

25 0

6 6

∗ 9 .1

65 0

R25.

0 ∗

67 5

∗ 6

∗ 9

DR2

∗ 49

∗ 2

∗

6 ∗ 7

6 6

68 ∗

35 ∗

4 ∗

5 41

∗

∗ 34 3

∗

∗ 8

∗

7 32

∗ 60

∗

97 47

∗

78 ∗ 61

∗

∗ 66

DR28.1

8 3 6 ∗ 0 8 ∗ 6

4 ∗

76 61 ∗ 3 3 6 ∗ ∗ 5 8 9 5 3 ∗ 5 7 ∗ 1 5 7 0 69 ∗

99 5 67 ∗ 9 75 ∗

65 ∗ 70

4 7 ∗ 3 69 78 80 ∗ DR2.1 5 7 68 ∗ H-I 3 ∗ 3 73 67

32 99 ∗ 74 H-II ∗ D 66 R 3 38 ∗ 1 68 .1 DR2.1 73 43 51 ∗ ∗ 74 DR 54 III ∗ 20 5 5. 84 80 4 ∗ 50 1 91 74 ∗ 65 64 6 5 ∗ 40∗ 32 IV ∗ 41 63 74 .3 50∗ ∗ 66 79 DR2 ∗ 46 71 39 ∗ ∗ ∗ 23 47 V 67 ∗ DR1 ∗ 42 19.1 66 ∗ 82 63 DR142.1 72 44 ∗ ∗ ∗ 21 46 ∗ ∗ VI 51 ∗ 48 ∗

VII 29 ∗

Figure 2. Composite pedigree of the DR8 founder family illustrating the 10 different branches identified to date. Generations H-I and H-II are hypothetical generations. Circles represent females; squares, males; black symbols, patients; gray symbols, individuals with affection status unclear; slash, deceased. Asterisks denote individuals for whom DNA is available. Patients represented with a DR number are those for whom we have clinical and/or pathologic information. The index patient of each branch is indicated with an arrowhead.

nal loss of the zona compacta of the substantia nigra was tient with AD with a novel nonsense mutation, p.Arg535X, observed, with melanin in the astrocytic cytoplasm in 3 and 2 patients with AD and 1 with PD who carried the patients. Rare Lewy bodies were also present in 1 other null mutation IVS0 ϩ 5GϾC, which we previously de- patient (DR8.1; Table 3). In 3 brains (patients DR25.1, scribed in the extended Belgian FTLDU-17 founder fam- DR28.1 and DR31.1; Table 3), rare amyloid deposits or ily.2 So far, 37 of 44 PGRN mutations (http://www.molgen neurofibrillary tangles were observed, which were lim- .ua.ac.be/FTDMutations) predicted the formation of a PTC ited to hippocampal areas in patients DR31.1 and DR25.1. and subsequent loss of the mutant transcript by NMD. In contrast, our data indicated that p.Arg535X was not COMMENT sensitive to NMD. This finding can be explained by the location of the mutation in the penultimate exon 11 of Extensive mutation analysis of PGRN in patients with well- PGRN at 42 nucleotides from the exon 11/12 boundary. diagnosed AD (n=666) and PD (n=255) identified 1 pa- When a PTC is located within less than 50 to 55 nucleo-

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6 No. of Individuals

0 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79 80-84 Age Category, y

Figure 3. Age distribution of patients and healthy individuals who carry the IVS0 ϩ 5GϾC mutation. For patients, ages represent the age at onset; for at-risk individuals, ages represent age at inclusion.

tides from the last exon-exon junction, the NMD sur- Although both patients’ conditions could not be neuro- veillance complex cannot be formed and the mutated tran- pathologically confirmed, the CSF biomarker profile of 24 ␤ script escapes degradation. Thus, in the case of patient DR25.14 was typical for AD, with low CSF A 1-42 p.Arg535X, the formation of a truncated protein miss- levels.25 Also, all autopsy diagnoses in the DR8 founder ing 59 C-terminal amino acids can be expected. Since the family were consistent with FTLDU. Mixed FTLDU and patient is still alive, we were unable to test for the pres- AD pathologic features were described for patients from ence or absence of the C-truncated PGRN in the brain, the FTLDU family HDDD126 and were also observed in leaving it undecided whether this mutation exerts a patho- another patient from the DR8 founder family who un- logic function via haploinsufficiency or an aberrant gain derwent autopsy (patient DR2.3; clinically diagnosed as of function. Of interest is that the mutation is located in having FTLD; age at death, 72 years). Pathologic exami- the last GRN domain (GRN E) and might influence its nation of this patient’s brain revealed early AD patho- highly conserved cysteine fold, potentially leading to deg- logic features (Braak A-II) in addition to typical FTLDU-17 radation. However, although PGRN null mutations are abnormalities. frequently found in patients with FTLD (5%-10%), in AD Patient DR205.1 presented with typical PD, al- and PD PGRN null mutations are clearly not a frequent though frontal lobe features were apparent. Cognitive im- cause of disease (0.45% in AD and 0.39% in PD). Inter- pairment is often observed in patients with PD, with an estingly, we identified 7 patient-specific missense mu- average prevalence of 40% in cross-sectional studies11 and tations that were present in 11 patients with AD or PD. a cumulative prevalence approaching 80%.10 It predomi- We recently provided evidence in favor of a pathogenic nantly involves frontal lobe features, including de- nature for several missense mutations6; however, be- creased verbal fluency, apathy, and difficulties in im- cause missense mutations are also present in control in- pulse control and interpreting social clues. Postmortem dividuals,5 further functional assessment will be necessary. examination of the patient’s brain showed concurrent dif- Both the patients with AD and PD who carried the fuse Lewy body abnormalities and severe FTLDU patho- IVS0 ϩ 5GϾC mutation had their conditions diag- logic features. Interestingly, vessel-related amyloid-␤ nosed with internationally accepted clinical diagnostic pathologic features were present as well. criteria. The 2 patients with AD presented with marked Several explanations exist for the high clinical hetero- deficits of recent memory and modest behavioral distur- geneity observed in IVS0 ϩ 5GϾC carriers in founder bances of frontal lobe origin, as was reflected by a rela- family DR8. Because PGRN seems to be important in the tively low MFS of 4. In hindsight, both patients with AD maintenance of neuronal cells, neuronal degeneration due displayed a reduced spontaneous speech and/or apathy, to loss of PGRN might be fully responsible for the ob- a clinical manifestation reminiscent of the affected rela- served clinical heterogeneity (eg, because of neuronal tives of the DR8 founder family.20 However, these symp- damage in the basal ganglia); however, the presence of toms occurred on a background of typical AD symp- pathologic correlates of PD and AD argues against this. toms, such as loss of episodic memory and disorientation. Alternately, in addition to FTLDU, a separate disease

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UBI

SNCA

UBI

TDP-43

UBI Aβ

Figure 4. Brain immunohistochemical analysis of patient DR205.1. A, Ubiquitin (UBI)-positive neuronal cytoplasmic inclusions, neuronal intranuclear inclusions, and threadlike inclusions in layers II and III and in deeper cortical layers (original magnification ϫ4). B, Neuritic pathologic features in the white matter (original magnification ϫ10). C, A severe degree of abnormality was observed in the basal ganglia. Arrowheads point to 2 UBI-positive neuronal intranuclear inclusions in the same section (original magnification ϫ40). D, Infrequent Lewy bodies were also observed in the cortex and the basal ganglion, as shown in the latter region (arrowhead) (␣-synuclein [SNCA] staining, original magnification ϫ40). E, Rabbit TAR DNA-binding protein 43 antisera (TDP-43) staining was localized in both the neuronal cytoplasmic and neuronal intranuclear inclusions (arrowheads), whereas normal nuclear TDP-43 staining was observed in unaffected neurons (original magnification ϫ40). F, Amyloid-␤ (A␤)–stained dense-core plaques were also observed in cortical and hippocampal regions, as shown in the temporal cortical region (original magnification ϫ20).

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/23/2021 mechanism or a modifying factor might be present be- Antwerpen; Division of Neurology, Ghent University Hos- cause of age-related or genetic factors, giving rise to dif- pital, Ghent (Drs Dermaut and Santens); and Divisions of ferent diseases and, in the case of the patients described Pathology (Dr Sciot) and Neurology (Dr Vandenberghe), herein, to different clinical phenotypes. Incomplete pen- University Hospital Gasthuisberg, University of Leuven, etrance and the wide range of ages at onset observed in Leuven, Belgium. the DR8 founder family2 argue in favor of the existence Correspondence: Christine Van Broeckhoven, PhD, DSc, of modifying factors. Our observations suggest that etio- VIB Department of Molecular Genetics, Neurodegenera- logic heterogeneity exists in these patients and that the tive Brain Diseases Group, University of Antwerp CDE, different pathomechanisms drive each other in an exac- Universiteitsplein 1, BE-2610 Antwerpen, Belgium erbated phenotype. Loss of neuroprotection through par- ([email protected]). tial loss of PGRN might lower a threshold for PD or AD Author Contributions: Study concept and design: Brouwers, pathologic features to arise, and in turn, incipient PD or Kumar-Singh, Pickut, De Deyn, Cruts, Van Broeckhoven, AD pathologic features might exacerbate those of FTLDU. and Sleegers. Acquisition of data: Brouwers, Nuytemans, Relatively severe FTLDU abnormalities in the basal gan- van der Zee, Engelborghs, Kumar-Singh, Pickut, Pals, glia and the vessel-related amyloid A␤ pathologic fea- Dermaut, Bogaerts, De Pooter, Serneels, Van den Broeck, tures in patient DR205.1 point in this direction. More- Cuijt, Mattheijssens, Peeters, Sciot, Martin, Cras, Santens, over, we previously observed signs of clinical Vandenberghe, De Deyn, and Van Broeckhoven. Analy- heterogeneity in the Dutch FTLDU-17 family 1083 with sis and interpretation of data: Brouwers, Nuytemans, patients with both FTLD and AD segregating a PGRN null Gijselinck, Theuns, Kumar-Singh, Pickut, De Pooter, mutation, p.Gln125X.1,2 Family 1083 was originally iden- Serneels, Van den Broeck, Martin, Vandenberghe, De tified as an early-onset AD family through its AD pro- Deyn, Van Broeckhoven, and Sleegers. Drafting of the band.27 Extensive clinical follow-up studies of family 1083 manuscript: Brouwers, Van Broeckhoven, and Sleegers. showed that overall clinical diagnoses were more con- Critical revision of the manuscript for important intellec- sistent with clinical FTLD, although in some patients a tual content: Nuytemans, van der Zee, Gijselinck, diagnosis of AD could not be excluded, and in 1 patient, Engelborghs, Theuns, Kumar-Singh, Pickut, Pals, parkinsonism coexisted.28 Dermaut, Bogaerts, De Pooter, Serneels, Van den Broeck, In conclusion, null mutations in PGRN are not a ma- Cuijt, Mattheijssens, Peeters, Sciot, Martin, Cras, Santens, jor cause of AD or PD. Furthermore, we provided evi- Vandenberghe, De Deyn, Cruts, Van Broeckhoven, and dence of intrafamilial clinical heterogeneity of PGRN null Sleegers. Obtained funding: Van Broeckhoven. Adminis- mutations, since carriers of the same mutation (eg, trative, technical, and material support: Engelborghs, Pickut, IVS0 ϩ 5GϾC) presented clinically with AD, PD, or Bogaerts, Serneels, Van den Broeck, Mattheijssens, Peeters, FTLD. Whether the clinical manifestation of AD or PD Sciot, and De Deyn. Study supervision: Theuns, Martin, can be explained by a wide phenotypic spectrum of PGRN De Deyn, Van Broeckhoven, and Sleegers. Clinical ex- mutations or the co-occurrence of FTLD and AD or PD pertise: Pals. due to other, age-related factors remains to be eluci- Financial Disclosure: None reported. dated. Diagnostic testing of PGRN should, however, be Funding/Support: This work was supported by the Fund considered in patients with familial clinical AD or PD for for Scientific Research–Flanders (FWO-F), the Institute whom mutations in other relevant genes have been ex- for Science and Technology–Flanders (IWT-F), the Spe- cluded. cial Research Fund of the University of Antwerp, the Stich- ting voor Alzheimer Onderzoek, and the Interuniver- Accepted for Publication: June 27, 2007. sity Attraction Poles (IUAP) program P6/43 of the Belgian Author Affiliations: Neurodegenerative Brain Diseases Federal Science Policy Office, Belgium; and by a Zenith Group, Department of Molecular Genetics, VIB (Mss Award from the Alzheimer’s Association to Dr Van Brouwers, Nuytemans, van der Zee, Gijselinck, Bogaerts, Broeckhoven. Dr De Deyn was funded by the Medical Re- Serneels, Van den Broeck, Cuijt, Mattheijssens, and Peeters, search Foundation and Neurosearch, Antwerp. Drs Drs Theuns, Kumar-Singh, Cruts, Van Broeckhoven, and Sleegers, Cruts, Theuns, and Engelborghs are postdoc- Sleegers, and Mr De Pooter); Laboratory of Neurogenetics toral fellows of the FWO-F, and Mss Brouwers, Gijselinck, (Mss Brouwers, Nuytemans, van der Zee, Gijselinck, and Bogaerts are doctoral fellows of the FWO-F. Mss Bogaerts, Serneels, Van den Broeck, Cuijt, Mattheijssens, Nuytemans and van der Zee are holders of a doctoral fel- and Peeters, Drs Theuns, Kumar-Singh, Cruts, Van lowship from the IWT-F. Broeckhoven, and Sleegers, and Mr De Pooter), Labora- Additional Contributions: We are grateful to the par- tory of Neurochemistry and Behaviour (Drs Engelborghs ticipants in this study for their cooperation and the per- and De Deyn), and Laboratory of Neuropathology (Dr sonnel of the Genetic Service Facility (http://www Martin), Institute Born-Bunge; University of Antwerp (Mss .vibgeneticservicefacility.be/) and the Biobank of the Brouwers, Nuytemans, van der Zee, Gijselinck, Engelborghs, Institute Born-Bunge. Bogaerts, Serneels, Van den Broeck, Cuijt, Mattheijssens, and Peeters, Drs Engelborghs, Theuns, Kumar-Singh, Martin, De Deyn, Cruts, Van Broeckhoven, and Sleegers, REFERENCES and Mr De Pooter); Memory Clinic and Division of Neu- 1. Baker M, Mackenzie IR, Pickering-Brown SM, et al. Mutations in progranulin cause rology, ZNA Middelheim General Hospital (Drs tau-negative frontotemporal dementia linked to chromosome 17. 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