Early-Onset Familial Lewy Body Dementia With Extensive Tauopathy: A Clinical, Genetic, and Neuropathological Study

J Neuropathol Exp Neurol Copyright Ó 2008 by the American Association of Neuropathologists, Inc. Vol. 68, No. 1 January 2009 pp. 73Y82 ORIGINAL ARTICLE Early-Onset Familial Lewy Body Dementia With Extensive Tauopathy: A Clinical, Genetic, and Neuropathological Study Jordi Clarimón, PhD, Laura Molina-Porcel, MD, Teresa Gómez-Isla, MD, PhD, Rafael Blesa, MD, PhD, Cristina Guardia-Laguarta, BSc, Anna González-Neira, PhD, Montserrat Estorch, MD, PhD, Josep M a Grau, MD, PhD, Lluís Barraquer, MD, PhD, Carles Roig, MD, PhD, Isidre Ferrer, MD, PhD, and Alberto Lleó, MD, PhD Abstract We describe a Spanish family in which 3 of 4 siblings had dementia with Lewy bodies, 2 of them starting at age 26 years and the other at 29 years. The father has recently been diagnosed with Lewy body disease, with onset at 77 years. Neuropathological examination of the brain of the index patient disclosed unusual features characterized by diffuse Lewy body disease and generalized neurofibrillary tangle pathology but with no amyloid deposits in any region. Moreover, Lewy body pathology colocalized with neurofibrillary tangles in most affected neurons. Mutation screening that included all coding exons of presenilin 1 (PSEN1), presenilin 2 (PSEN2), >-synuclein (SNCA), A-synuclein (SNCB), microtubule-associated protein tau (MAPT), leucine-rich repeat kinase 2 (LRRK2), glucocerebrosidase (GBA), and exons 16 and 17 of the amyloid precursor protein (APP) genes did not identify any mutation. Genome-wide single nucleotide polymorphism was performed in 4 family members and ruled out any pathogenic duplication or deletion in the entire genome. In summary, we report a unique family with pathologically confirmed early-onset dementia with Lewy bodies with widespread tau and >-synuclein deposition. The absence of mutations in genes known to cause Lewy body disease suggests that a novel locus or loci are implicated in this neurodegenerative disease. Key Words: >-synuclein, familial Lewy body dementia, generalized tauopathy, synucleinopathy, tau. From the Neurology Department, (JC, LM-P, TG-I, RB, CG-L, JMG, LB, CR, AL), and the Nuclear Medicine Department (ME), Hospital de la Santa Creu i Sant Pau; Genotyping Unit, CeGen, Human Cancer Genetics Program, Spanish National Cancer Center (CNIO), Madrid (AG-N); Institut de Neuropatologia, Servei Anatomia Patològica, IDIBELL- Hospital Universitari de Bellvitge, Facultat de Medicina, Universitat de Barcelona (IF); and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) (JC, TG-I, RB, CG-L, AL, IF), Barcelona, Spain. Send correspondence and reprint requests to: Alberto Lleó, MD, PhD, Memory Unit, Alzheimer s Laboratory, Neurology Department, Hospital de la Santa Creu i Sant Pau, Sant Antoni M a Claret 167, Barcelona 08025, Spain; E-mail: alleo@santpau.cat JC and LM-P contributed equally to this work. This work was supported in part by CIBERNED, FIS grant PI05/1570, and by the European Commission support to IF under the Sixth Framework Programme (BrainNet Europe II, LSHM-CT-2004-503039). J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 INTRODUCTION Lewy body dementia (LBD) is the second leading cause of degenerative dementia in older people, accounting for 10% to 15% of cases at autopsy (1). Clinically, it is characterized by fluctuating cognitive impairment, recurrent visual hallucinations, and parkinsonism (1, 2). Pathologically, LBD is characterized by widespread occurrence of Lewy bodies (LBs), mainly composed of aggregated >- synuclein. Coexistent Alzheimer disease (AD)-type pathology (i.e. cortical amyloid plaques and neurofibrillary tangles [NFTs]) can also be found in many patients. Interestingly, NFTs are typically observed in the hippocampal region, even in patients with minimal AD pathology (3). Lewy body dementia usually occurs as a sporadic disease, but small numbers of familial cases have been reported (4Y11). Genetic studies of some of these families have been invaluable for elucidating some of the molecular bases of LBD. A genomic triplication containing the >-synuclein gene (SNCA) segregated with a phenotype ranging clinically and pathologically from Parkinson disease (PD) to LBD, in a large family known as the Iowa kindred (12). A p.e46k mutation in the same gene was found in a Spanish family with autosomal dominant PD, PD with dementia, and LBD (10). The p.p123h mutation in the A-synuclein gene (SNCB) has also been found in a family with LBD (13). Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been associated with a range of clinical phenotypes including PD, PD with dementia, LBD, and amyotrophy (14). Interestingly, striking neuropathological heterogeneity has been related to mutations in this locus and includes pure nigral degenerative disease without >-synuclein pathology, PD with brainstem LBs, diffuse Lewy body pathology, and tau pathology mainly in the substantia nigra (14). Mutation analysis in the glucocerebrosidase gene (GBA), responsible for Gaucher disease, has suggested that 1 quarter of LBD patients may carry mutations in this locus (15). A recent screening of 2 common mutations in GBA disclosed, however, that only 3.5% of LBD cases carried these pathogenic variants (16). Finally, early-onset parkinsonism with dementia has been linked to a mutation in the presenilin-1 gene (PSEN1) (17). Together, these data clearly indicate genetic heterogeneity in LBD. 73

Clarimón et al J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 The present study describes the clinical characteristics of a family comprising 3 siblings who died from very early-onset LBD. Neuropathological examination of the index patient revealed an unusual neuropathological phenotype consisting of widespread LB and NFT pathology, but no amyloid deposits in any brain region. Sequencing of genes related to AD, LBD, and PD, as well as whole genome genotyping, did not reveal any point mutation or genomic copy number alteration. MATERIALS AND METHODS Patients The family was identified at the Hospital Sant Pau, Barcelona. The patients were recruited between 1983 and 2006, and all of them were examined by neurologists with expertise in neurodegenerative diseases. All medical records were retrospectively reviewed to confirm the diagnosis, applying current consensus diagnostic criteria for LBD (18). Blood samples were obtained from all members after written informed consent was given. 123 I metaiodobenzylguanidine myocardial scintigraphy is reduced in dementia with Lewy bodies (DLB); it has been shown to be useful in distinguishing patients with AD and LBD and is a supportive feature in the current clinical criteria for DLB (18Y20). Cardiac sympathetic dysfunction was assessed by metaiodobenzylguanidine myocardial scintigraphy in patient I-2 with informed consent. After 30-minute resting period, an intravenous FIGURE 1. Clinical phenotype of the Spanish family with early-onset Lewy body disease. (A) Family pedigree. Squares represent men; circles represent women. Black symbols indicate affected individuals, and diagonal bands indicate deceased family members. Ages of onset for individuals with disease or current ages of healthy individuals are depicted underneath. Ages at death are shown in italics below the ages of onset. (B) Magnetic resonance imaging (MRI) of patient II-1 shows atrophy in frontotemporal regions. (C) MRI of patient I-2 shows atrophy in parietal and temporal regions. (D) Dopamine transporter scan in patient I-2 demonstrates bilateral symmetrical reduction of striatal tracer uptake. (E) A 123 I metaiodobenzylguanidine myocardial scintigraphy study in patient I-2 shows a marked cardiac sympathetic denervation (left) compared with a healthy individual (right). 74 Ó 2008 American Association of Neuropathologists, Inc.

J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 Familial Lewy Body Dementia With Tauopathy TABLE. Case Clinical Features of the Early-Onset Lewy Body Dementia Family Age at Onset, Years Age at Death or Current Age,* Years Dementia Parkinsonism Visual Hallucinations Fluctuations Neuropsychological Profile Visuospatial Memory Deficits Language II-1 26 38 + + + + +++ + II-2 29 44 + + + + + +++ + II-3 26 29 + + + + ++ I-2 77 80* + + + ++ +++ + +, mild deficits; ++, moderate deficits; +++, severe deficits. injection of 11 mbq of 123 I metaiodobenzylguanidine was administered. Average counts per pixel in the heart and mediastinum were used to calculate the heart-to-mediastinum ratio. Normal values at our center are set at 1.56 for individuals older than 60 years (19). Neuropathology and Immunohistochemistry The neuropathological study on patient II-1 was carried out on sections of the frontal (area 8), primary motor, primary sensory, parietal, temporal superior, temporal inferior, anterior cingulate, anterior insular, and primary and associative FIGURE 2. >-Synuclein immunohistochemistry in brain sections of the index patient (II-1) shows abundant Lewy bodies and Lewy neurites in the temporal cortex (T3) (A), CA1 region of hippocampus (B), entorhinal cortex (C), frontal cortex (area 8) (D), vagus nucleus (E), and ventral reticular nucleus (F). Paraffin sections lightly stained with hematoxylin. Original magnification: (AYD) 200; (E, F) 400. Ó 2008 American Association of Neuropathologists, Inc. 75

Clarimón et al J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 visual cortices; entorhinal cortex and hippocampus; caudate, putamen and pallidum; medial and posterior thalamus; subthalamus; nucleus basalis of Meynert; amygdala; midbrain (2 levels), pons and medulla oblongata; and cerebellar cortex and dentate nucleus. Tissue samples were fixed and embedded in paraffin. Dewaxed sections, 5 Km thick, were stained with hematoxylin and eosin and Klüver-Barrera or processed for immunohistochemistry following the EnVision+ system peroxidase procedure (DAKO, Glostrup, Denmark). Antibodies to glial fibrillary acidic protein (DAKO), AA4- amyloid (Roche, Basel, Switzerland), and ubiquitin (DAKO) were used at dilutions of 1:250, 1:50, and 1:200, respectively. Anti-CD68 (DAKO), a marker of microglia, was diluted at 1:100. AT8 antibody (Innogenetics, Alpharetta, GA) was used at a dilution of 1:50. Phosphospecific tau rabbit polyclonal antibodies Thr181, Ser199, Ser202, Ser262, Ser396, and Ser422 (Calbiochem, San Diego, CA) were used at a dilution of 1:100, except for anti-phospho-tauthr181, which was used at a dilution of 1:250. Antibodies to 3R and 4R tau (Upstate, Billerica, MA) were used at dilutions of 1:800 and 1:50, respectively. Rabbit polyclonal anti->-synuclein antibody (Chemicon, Billerica, MA) was used at a dilution of 1:3000, and mouse monoclonal anti-phosphorylated >- synucleinser129 antibody (Wako, Tokyo, Japan) at a dilution of 1:2000. Rabbit polyclonal antibodies to C-terminal TAR- DNA binding protein-43 (TDP-43; Abcam, Cambridge, UK) and monoclonal TDP-43 antibodies (Abnova, Heidelberg, Germany) were used at dilutions of 1:1000 and 1:500, respectively. Mouse anti-polyglutamine clone 1C2 (Millipore, Billerica, MA) was used to detect polyglutamine expansions; Huntington disease tissue was used as a positive control. Sections processed for amyloid and >-synuclein immunohistochemistry were first pretreated with formic acid and incubated with methanol and H 2 0 2 in PBS and normal serum. After incubation with the primary antibody, the sections were incubated with EnVision + system peroxidase for 15 minutes at room temperature. Sections were lightly counterstained with hematoxylin. For double-labeling immunofluorescence and confocal microscopy, dewaxed sections were stained with a saturated solution of Sudan black B (Merck, Whitehouse Station, NJ) for 30 minutes, then rinsed in 70% ethanol and washed in distilled water. The sections were incubated with rabbit polyclonal anti->-synuclein (Chemicon) at a dilution of 1:3000, mouse monoclonal anti-phosphorylated >-synucleinser129 antibody (Wako) diluted at 1:2000, mouse anti-at8 antibody (Innogenetics) at a dilution of 1:50, or rabbit polyclonal FIGURE 3. Double-labeling immunofluorescence of >-synuclein (green) and phosphorylated >-synuclein Ser129 (red) in brain sections of patient II-1 (AYC) demonstrate almost complete colocalization of the 2 antibodies. Control sections without primary antibodies show a negative staining (DYF). Original magnification: 200. 76 Ó 2008 American Association of Neuropathologists, Inc.

J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 Familial Lewy Body Dementia With Tauopathy anti<a-amyloid (DAKO) at a dilution of 1:50. After washing in PBS, sections were incubated with secondary antibodies Alexa488 anti-rabbit and Alexa546 anti-mouse (Molecular Probes, Eugene, OR) at a dilution of 1:400. Nuclei were stained with TO-PRO-3-iodide (Molecular Probes) diluted at 1:1000. Sections were examined with a Leica TCS-SL confocal microscope. Mutation Analysis and Genotyping Patient II-2 was screened for mutations in the coding exons and exon-intron boundaries 3Y12 of the PSEN1 and presenilin-2 (PSEN2) genes, the coding exons 16 and 17 of APP gene, the coding exons 2 to 13 of the microtubuleassociated protein tau (MAPT) gene, the coding exons 2 to 6 of the SNCA gene, the coding exons 3 to 7 of the SNCB gene, the 1 to 51 exons of the LRRK2 gene, the coding exons 2 to 13 of the progranulin (PGRN) gene, the coding exons 2 to 6 of the TAR-DNA binding protein-43 (TDP-43) gene, and the exons 1 to 11 of the GBA gene. All exons and their intronic flanking regions were amplified by means of polymerase chain reaction, using FastStart Taq DNA Polymerase (Roche Molecular Biochemicals, Mannheim, Germany) and respective primers (oligonucleotide sequences provided upon request). DNA fragments purified from polymerase chain reactions were used as templates in sequencing reactions using Big Dye Terminator (version 3.1) Cycle Sequencing Ready Reaction DNA Sequencing Kit (Applied Biosystems, Foster City, CA), and the product was subsequently run in ABI PRISM 3730 Genetic Analyzer (Applied Biosystems). Sequence analysis was carried out using Sequencher 4.1.4. (Gene Codes Corporation, Inc., Ann Arbor, MI). The extended MAPT H1 haplotype was determined by the presence of the 238-bp deletion in intron 9, which defines the H2 haplotype (21). Whole Genome Genotyping Genome-wide single nucleotide polymorphism (SNP) typing using the Illumina HumanHap550 Genotyping Bead- Chip was performed in individuals I-1, I-2, II-1, and II-4. A total of 561,466 SNP markers were genotyped for each sample. The experiments were carried out according to the manufacturer s specifications (Illumina, Inc, San Diego, CA). DNA samples with GenCall scores G0.15 at any locus were considered Bno calls.[ Genotype success rates greater than 99.7% were obtained for all assays. Visualization of gene copy variation was performed through Genome Viewer tool within Illumina s BeadStudio v2.2.22 software. Two parameters were visualized using this tool: log R ratio and B allele frequency, plotted along the entire genome for all SNPs on the array. Briefly, the value of the log R ratio is the log 2 ratio of the observed normalized R value for the individual SNP divided by the expected normalized R value for the SNPs theta value. An R above 1 is indicative of an increase in copy number (duplication or triplication), and values below 1 suggest a deletion. FIGURE 4. Phospho-tau immunohistochemistry (AT8 antibody) performed in brain sections of the index patient (II-1) shows multiple neurofibrillary tangles and dystrophic neurites in the entorhinal cortex (A), CA1 region of the hippocampus (B), dentate gyrus (C), and substantia nigra (D). Paraffin sections lightly stained with hematoxylin. Original magnification: (AYD) 400. Ó 2008 American Association of Neuropathologists, Inc. 77

Clarimón et al J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 78 Ó 2008 American Association of Neuropathologists, Inc.

J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 Familial Lewy Body Dementia With Tauopathy RESULTS Clinical Characteristics In this family, there were 4 affected individuals across 2 generations (Fig. 1A). A brief description of family members I-2, II-1, II-2, II-3, and II-4 follows: Patient II-1 (Index Patient) This was a 28-year-old man without any relevant medical history who presented with a 2-year history of progressive walking difficulties and falls. He complained of general slowness and difficulties in performing fine movements that interfered with his work as a plumber. In the last year, he had also developed problems in remembering recent events and pronouncing words. At the initial visit, he showed hypomimia, bilateral cogwheel rigidity, and bradykinesia with positive palmomental and grasping reflexes. Gait was slow with reduced arm swing. Neuropsychological examination showed poor verbal memory with spared delayed recall and constructive apraxia (Table). Brain magnetic resonance imaging showed marked atrophy in both frontotemporal regions with enlargement of frontal horns of the ventricular system (Fig. 1B). The clinical picture progressed over the following years, and he developed fluctuations. The patient would stare into space for long periods repeating the same word and yet be alert at other times. He showed behavioral changes, with mood swings and episodes of bizarre behavior. Over time, the patient showed a generalized cognitive impairment, marked fluctuations, dysphagia, myoclonus, and severe parkinsonism without response to L-Dopa or dopamine agonists. L-Dopa induced bilateral dyskinesias. The patient died of aspiration pneumonia at the age of 38 years. Complete autopsy with neuropathological examination was performed. Patient II-2 The proband s brother was a 31-year-old man with a 2-year history of headache, dizziness, and concentration difficulties. He had episodes of syncope. As a result of the symptoms, he had stopped working as a mechanic. At the first visit, the patient showed hypomimia, rigidity, and bradykinesia with parkinsonian gait and reduced arm swing. There was no response to L-Dopa or dopamine agonists. Two years later, he developed fluctuations with frequent confusion and delusional ideas about his work. Neuropsychological evaluation at the age of 33 years showed poor visual and verbal memory with perseverations and deficits in visuospatial tests (Table). Brain computed tomographic scan at the same time showed mild cortical atrophy. Parkinsonism and cognitive deficits worsened over the following years. At age 37 years, the patient showed reduced speech and dysarthria, severe rigidity, generalized spontaneous myoclonus, and visual hallucinations. He also developed episodes of reduced levels of consciousness. He died of pneumonia at the age of 44 years. Autopsy was denied. Patient II-3 The proband s sister was a 26-year-old woman who had grown up in a town different from her siblings. She developed progressive word-finding difficulties, apathy, and slowness in walking and performing daily activities. Examination showed anomia, dysarthria, marked bradykinesia, brisk deep tendon reflexes, and parkinsonian gait with positive grasping, palmomental, and sucking reflexes. Neuropsychological examination showed anomia and deficits in orientation and verbal memory (Table). Brain magnetic resonance imaging showed mild atrophy in frontal and perisilvian regions. Motor and cognitive symptoms progressively worsened. The patient died of uncertain cause while sleeping at the age of 29 years. Patient I-2 Ten years after patient II-3 s death, the father developed cognitive symptoms and parkinsonism at the age of 77 years. He complained of forgetfulness, word-finding difficulties, visual hallucinations, and general slowness. Neurological examination showed hypomimia, bilateral bradykinesia without tremor, and positive palmomental and sucking reflexes. Neuropsychological evaluation showed deficits in memory, language, and visuospatial functions (Table). His mini-mental state examination (MMSE) was 18/30 at the first visit. Brain magnetic resonance imaging showed atrophy in parietal and temporal regions with bilateral leukoaraiosis (Fig. 1C). Dopamine transporter scan showed reduced metabolism in both caudate-putaminal regions (Fig. 1D). Metaiodobenzylguanidine myocardial scintigraphy showed reduced sympathetic cardiac innervation supporting the diagnosis of LBD (ratio heart/mediastinum 1.01; Fig. 1E). The patient s symptoms have continued to worsen, and he is now dependent for most activities of daily living. Neuropathology The macroscopic examination of the brain in patient II-1 revealed atrophy of the frontal and temporal lobes, hippocampus and parahippocampal regions, caudate, and putamen. Sections of the brainstem revealed marked loss of pigment in the substantia nigra and locus ceruleus. No gross abnormalities were seen in the blood vessels, but mild arteriosclerosis was noted. The microscopic examination of hematoxylin and eosinystained and Klüver-BarreraYstained sections showed marked neuronal loss in the substantia nigra pars compacta, locus ceruleus, reticular formation of the brainstem, amygdala, hippocampus (areas CA2 and CA3), entorhinal cortex, neocortex, and striatum. This loss was accompanied by microgliosis and astrocytosis, revealed with anti-cd68 and glial fibrillary acidic protein antibodies, respectively. The FIGURE 5. Double-labeling immunofluorescence and confocal microscopy with >-synuclein (green) and phospho-tau (red) antibodies in brain sections of patient II-1 showing colocalization of the 2 proteins in most abnormal protein aggregates in the frontal cortex (AYC) and hippocampus (DYF). In contrast, there is a lack of subcellular colocalization of phospho-tau and >- synuclein in Lewy bodies (arrow) and neurites (arrowhead) in the substantia nigra (GYI). Control sections without primary antibodies show no staining (JYL). Original magnification: 200. Ó 2008 American Association of Neuropathologists, Inc. 79

Clarimón et al J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 entorhinal and perirhinal cortex showed spongiosis in the superficial layers. Granulovacuolar degeneration was observed in pyramidal neurons of the hippocampus on hematoxylin and eosinystained sections. A preliminary study stressing the large number and widespread distribution of ubiquitin-immunoreactive abnormal neurites in this patient was reported previously (22). This aspect, together with the massive presence of >-synuclein-immunoreactive LBs and Lewy neurites (LNs), was herein confirmed. The distribution of LBs and LNs was widespread and included selected nuclei of the medulla oblongata (including dorsal nucleus of the vagus nerve and reticular formation), locus ceruleus, substantia nigra pars compacta, as well as the nucleus basalis of Meynert, amygdala, entorhinal, perirhinal cortex and subiculum, all areas of the hippocampus (with massive aberrant neurites in CA1, CA2, and CA3 subfields), dentate gyrus, caudate, putamen and accumbens, and cerebral cortex (Fig. 2). The involvement of the cerebral cortex affected the gyrus cinguli, insular cortex, frontal lobes, primary motor and sensory areas, and parietal and occipital cortex, sparing the primary visual area. Lewy neurites were stained not only with anti-ubiquitin antibodies, as previously reported, but also with anti->-synuclein antibodies. Lewy bodies and LNs were also stained with antiphospho->-synuclein Ser129 antibodies. Double-labeling immunofluorescence and confocal microscopy revealed that the vast majority of >-synuclein deposited in aberrant aggregates was phosphorylated (Fig. 3). Similar results were obtained using different combinations of monoclonal and polyclonal antiy>-synuclein and antiyphospho-synuclein antibodies. In addition to the widespread synucleinopathy, the AT8 antibody disclosed the presence of a massive tauopathy affecting the same regions affected by the synucleinopathy (Fig. 4). Anti-phospho-tau antibodies (including clone AT8, anti-phospho-tau Thr181, Ser262, Ser202, Ser396, and Ser342) revealed NFT and pretangles in all these areas, together with tau-immunoreactive neuropil threads and large neuritic inclusions, particularly in the entorhinal cortex, neocortex, hippocampus, amygdala, and nucleus basalis of Meynert. The use of specific antibodies revealed that phospho-tau deposits were composed of 4R and 3R tau (data not shown). Double-labeling immunofluorescence and confocal microscopy disclosed colocalization of >-synuclein and phospho-tau in most abnormal protein aggregates in the amygdala, nucleus basalis of Meynert, hippocampus, and cerebral cortex (Fig. 5). Phospho-tau and LBs were present in individual cells in most brain regions but localized apart in the substantia nigra and locus ceruleus. Amyloid deposits were absent, and no senile plaques were observed in any region. The extensive neuronal abnormalities contrasted with glial cell preservation. Only very rare glial cells, probably astrocytes, in the amygdala and subcortical white matter were stained with anti-synuclein antibodies. Similarly, only very few (if any in some sections) periventricular astrocytes were stained with anti-phospho-tau antibodies. Intranuclear inclusions were not observed in hematoxylin and eosinystained sections or in sections stained with 80 anti-polyglutamine clone 1C2 antibody. TAR-DNA binding protein-43 antibodies did not disclose abnormal TDP-43 intracytoplasmic or intranuclear inclusions, or thick neurites and threads (data not shown). Genetic Studies Mutation screening of the index patient did not reveal any pathogenic changes in PSEN1, PSEN2, APP, SNCA, SNCB, MAPT, PGRN, TDP-43, GBA, or LRRK2. Analysis of the extended MAPT H1 haplotype revealed that all family members were heterozygous (H1/H2), whereas patient II-2 was homozygous for the H1 haplotype (H1/H1). To find genomic copy number variations (such as deletions or duplications), DNA from both parents (individuals I-1 and I-2), one of the affected siblings (individual II-2), and his healthy sister (individual II-4) was used for high-resolution genomic profiling of chromosomal aberrations through whole-genome SNP genotyping of È555,000 tagsnps. This approach allows the detection of deletions or duplications with average lengths of 4.8 kb and 90% of genetic dosage alterations equal or greater than 12 kb. A 27.2-kb genomic duplication in chromosome 1p34.2 containing the CTPsynthase gene (CTPS) was found in the healthy mother (I-1) and patient II-2. This has been reported as a polymorphic copy number variation of the human genome (http://projects. tcag.ca/variation/). Also, a 323.5-kb duplication in chromosome 19q13.31, comprising a gene cluster containing the genes PSG-2, PSG-6, PSG-7, and PSG-11, which are members of the pregnancy-specific A-1-glycoprotein family, was found in the mother. No other gene dosage increase was found. Homozygous and heterozygous genomic deletions ranging from 2 to 209 kb were also detected in all individuals, most of these, however, were polymorphic variations. Only one known polymorphic variation (locus 2018 of the Database of Genetic Variation), consisting of a 29.6-kb heterozygous deletion in an intergenic region of chromosome 6q12, was present in patients I-2 and II-2 but not in the healthy family members I-1 and II-4. DISCUSSION We describe a unique family with 4 apparently affected members presenting clinical features consistent with LBD. Families with parkinsonism and dementia have been reported in the literature, but most of them had a PD phenotype with dementia appearing years after the onset of parkinsonism (6, 8Y10, 23Y25). In contrast, families in which all members presented LBD are more infrequent, and the age of onset is usually between the fifth and sixth decade of life (4Y7, 26, 27). In the family reported here, all members showed early cognitive impairment and parkinsonism with visual hallucinations (II-2, I-2) and fluctuations (II-1, II-2). The most striking feature of this family is the early age of onset and minimal variability in age of onset among siblings. Although some atypical features were observed, such as the lack of L-Dopa response in members II-1 and II-2 or the prominent frontal lobe atrophy in member II-1, all patients met the diagnosis of probable DLB according to current clinical criteria (18). The fact that the father has been diagnosed with lateonset LBD might indicate autosomal dominant inheritance Ó 2008 American Association of Neuropathologists, Inc.

J Neuropathol Exp Neurol Volume 68, Number 1, January 2009 Familial Lewy Body Dementia With Tauopathy with genetic anticipation. This hypothesis would be supported by the fact that other authors have also described families in whom probands present LBD at much earlier ages than their affected parents (6). Neither the clinical picture nor the immunohistochemical study supported a disease caused by expansion of triplet repeats encoding polyglutamine tracts. In view of the small size of the present kindred and the prevalence of LBD in older ages, the possibility of a phenocopy phenomenon in the father cannot be ruled out. In this case, a recessive mode of inheritance or a disease triggered by an environmental factor should be also considered. The former seems unlikely, however, because 3 of 4 offspring developed the disease, and our genome-wide SNP study did not indicate features of homozygosity. The latter is also unlikely because the offspring had grown up far apart from one another. Our extensive genetic analysis ruled out any pathogenic mutation, thus suggesting that unknown locus/loci contribute to the genetic heterogeneity that characterizes LBD. We also performed the first high-density SNP genotyping through the whole genome in a family with LBD. Our results suggest that genomic alterations, such as deletions or duplications, are not related with the disease in this particular family. Furthermore, the absence of large homozygosity tracts in any of the siblings argues against a recessive mode of inheritance due to a possible inbreeding between the parents. Unfortunately, due to the small size of the kindred, linkage analysis was not feasible. The neuropathology of the index patient showed abundant LBs and LNs in most brain regions, a feature consistent with diffuse Lewy body disease, LBD, and stage 6 of Braak and Braak (18, 28, 29). This was accompanied by a generalized tauopathy in which phospho-tau AT8 colocalized with abnormal >-synuclein in most protein aggregates. Because brain tissue was only available in 1 affected member, we cannot be completely certain that this pathology segregates with the disease. The low frequency of incidental lesions at the age of the proband_s death and the wide colocalization of >-synuclein and tau strongly suggest that both types of abnormalities play a role in the disease process in this family. >-Synuclein deposition has been identified in a subset of patients with disorders characterized by prominent tau pathology, such as familial and sporadic AD (30, 31), Down syndrome (32), progressive supranuclear palsy (33), Parkinsonism dementia complex of Guam (34), and frontotemporal dementia (35, 36). Typically, colocalization of tau and >-synuclein aggregates in these cases is restricted to the amygdala and other limbic areas (34). The convergence of tau and >-synuclein pathology has also been described in disorders with primary deposition of >-synuclein, such as familial PD, sporadic LBD, and multisystem atrophy (24, 37Y40). In LBD, tau-positive LBs are typically confined to limbic areas and in most of cases associated with AA peptide (37, 41). Only 1 family diagnosed with PD with dementia has been reported to have a few cortical tau-positive LBs without amyloid deposits (42). Our case showed a generalized abnormal >-synuclein and tau pathology with widespread colocalization and without amyloid deposits in any brain region. Because this unusual neuropathological feature was present in multiple brain regions, it is unlikely that the coexistence of >-synuclein and aggregated tau is simply coincidental. Rather, it suggests the existence of a specific common metabolic pathway involved in this particular disorder. Some studies have investigated the existence of a synergistic effect on the abnormal aggregation of tau and >- synuclein (39). Although the exact mechanism remains unknown, in vitro and in vivo studies suggest that >-synuclein induces the formation of tau fibrils and that the 2 proteins synergistically affect the polymerization of each other into fibrillar amyloid lesions (39). A possible link is also supported by the observation that phosphorylated tau and >-synuclein are found in synaptic-enriched fractions in several >-synucleinopathies (43). Taken together, we hypothesize that unknown genetic causes carried by this family may have enhanced the 2 pathologies, first inducing >-synuclein fibrillization and then promoting tau aggregation in most brain regions. In summary, we describe a unique family in which 3 siblings presented with LBD at very early ages and in which extensive genetic studies revealed no mutations or gene dose effects. 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