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There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39-76), and disease duration 7.6 ± 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation. [ABSTRACT FROM AUTHOR]

Objective: To develop a visual rating scale for posterior atrophy (PA) assessment and to analyse whether this scale aids in the discrimination between Alzheimer's disease (AD) and other dementias.Methods: Magnetic resonance imaging of 118 memory clinic patients were analysed for PA (range 0-3), medial temporal lobe atrophy (MTA) (range 0-4) and global cortical atrophy (range 0-3) by different raters. Weighted-kappas were calculated for inter- and intra-rater agreement. Relationships between PA and MTA with the MMSE and age were estimated with linear-regression analysis.Results: Intra-rater agreement ranged between 0.93 and 0.95 and inter-rater agreement between 0.65 and 0.84. Mean PA scores were higher in AD compared to controls (1.6  ±  0.9 and 0.6  ±  0.7, p  <  0.01), and other dementias (0.8 ± 0.8, p  <  0.01). PA was not associated with age compared to MTA (B = 1.1 (0.8) versus B = 3.1 (0.7), p  <  0.01)). PA and MTA were independently negatively associated with the MMSE (B = -1.6 (0.5), p  <  0.01 versus B = -1.4 (0.5), p  <  0.01).Conclusion: This robust and reproducible scale for PA assessment conveys independent information in a clinical setting and may be useful in the discrimination of AD from other dementias. [ABSTRACT FROM AUTHOR]

The objective of the study was to illustrate the utility of positron emission tomography (PET) imaging using [C]PIB and [F]FDDNP together with cerebrospinal fluid (CSF) measures of amyloid-beta1 to 42 (Abeta42), total tau (t-tau) and tau phosphorylated at threonine 181 (p-tau) in the in vivo diagnosis of specific dementia syndromes. Two siblings fulfilling diagnostic criteria for familial Alzheimer's disease (AD) were investigated using [C]PIB and [F]FDDNP PET in combination with CSF measures of Abeta42, t-tau and p-tau. PET data were compared with paired [C]PIB and [F]FDDNP data from age-matched sporadic AD patients (n=9) and healthy controls (n=6). [C]PIB retention and CSF levels of Abeta42 in both patients resembled those of controls suggesting the presence of nonamyloid pathology. Genetic testing confirmed the absence of mutations in the presenilin 1 gene in 1 patient; subsequent testing revealed the R406W tau mutation in both individuals leading to a diagnosis of frontotemporal dementia [F]FDDNP retention broadly correlated with CSF levels of t-tau and p-tau. Despite both individuals harbouring the same mutation, [F]FDDNP retention and CSF t-tau and p-tau were elevated in 1 patient, but not in the other. [C]PIB imaging and CSF measures of Abeta42 are useful in refuting the presence of underlying amyloid pathology. This, in combination with elevated levels of CSF t-tau and p-tau, has potential value in differential diagnosis of frontotemporal dementia from AD. [ABSTRACT FROM AUTHOR]

Aim: We examined whether impairment in specific cognitive domains in Alzheimer's disease (AD) differed according to APOE genotype and age at onset. Methods: Cognitive functions of 229 consecutive AD patients were assessed using Visual Association Test (VAT), Memory Impairment Screen+ (MIS+), VAT object naming, fluency test and Trail Making Test (TMT). Dementia severity was assessed using MMSE. ANOVAs were performed with APOE genotype and age at onset as independent variables and sex, education and MMSE as covariates. Results: 28% of patients were APOE epsilon4-negative, 58% heterozygous and 14% homozygous. A significant association between APOE genotype and VAT and MIS+ was found when correcting for sex and education. An interaction effect between APOE genotype and age at onset on VAT and VAT object naming was found, with young carriers performing worse than young noncarriers. By contrast, when additionally correcting for MMSE, a significant association between APOE genotype and VAT object naming, TMT-A and TMT-B was found, with noncarriers performing worse than carriers. Conclusion: Memory was more impaired among APOE epsilon4 carriers than among noncarriers. By contrast, naming, executive functions and mental speed were more impaired among APOE epsilon4 noncarriers. This suggests that the APOE genotype modifies the clinical phenotype in terms of cognitive impairment in AD. Copyright (c) 2007 S. Karger AG, Basel. [ABSTRACT FROM AUTHOR]

Frontotemporal dementia (FTD) is the second most common cause of early-onset dementia after Alzheimer disease (AD). Efforts in the field mainly focus on familial forms of disease (fFTDs), while studies of the genetic etiology of sporadic FTD (sFTD) have been less common. In the current work, we analyzed 4,685 sFTD cases and 15,308 controls looking for common genetic determinants for sFTD. We found a cluster of variants at the MAPT (rs199443; p = 2.5 × 10 -12 , OR = 1.27) and APOE (rs6857; p = 1.31 × 10 -12 , OR = 1.27) loci and a candidate locus on chromosome 3 (rs1009966; p = 2.41 × 10 -8 , OR = 1.16) in the intergenic region between RPSA and MOBP, contributing to increased risk for sFTD through effects on expression and/or splicing in brain cortex of functionally relevant in-cis genes at the MAPT and RPSA-MOBP loci. The association with the MAPT (H1c clade) and RPSA-MOBP loci may suggest common genetic pleiotropy across FTD and progressive supranuclear palsy (PSP) (MAPT and RPSA-MOBP loci) and across FTD, AD, Parkinson disease (PD), and cortico-basal degeneration (CBD) (MAPT locus). Our data also suggest population specificity of the risk signals, with MAPT and APOE loci associations mainly driven by Central/Nordic and Mediterranean Europeans, respectively. This study lays the foundations for future work aimed at further characterizing population-specific features of potential FTD-discriminant APOE haplotype(s) and the functional involvement and contribution of the MAPT H1c haplotype and RPSA-MOBP loci to pathogenesis of sporadic forms of FTD in brain cortex., Competing Interests: Declaration of interests O.A.A. has received speakers’ honoraria from Janssen, Lundbeck, and Sunovion and is a consultant to Cortechs.ai. C.C. received research support from GSK and EISAI. The funders of the study had no role in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. C.C. is a member of the advisory board of Vivid Genomics and Circular Genomics. M.A.N. and H.L.L. hold part of a competitive contract awarded to Data Tecnica International LLC by the National Institutes of Health to support open science research. M.A.N. currently serves on the scientific advisory board for Character Bio Inc. and Neuron23 Inc. I.R.M. receives license royalties for patent related to PGRN therapy and is a member of the scientific advisory committee for Prevail Therapeutics. H.R.M. is employed by UCL. In the last 12 months he reports paid consultancy from Roche, Aprinoia, AI Therapeutics, and Amylyx; lecture fees/honoraria from BMJ, Kyowa Kirin, and Movement Disorders Society; and research grants from Parkinson’s UK, Cure Parkinson’s Trust, PSP Association, Medical Research Council, and the Michael J. Fox Foundation. H.R.M. is a co-applicant on a patent application related to C9ORF72—Method for diagnosing a neurodegenerative disease (PCT/GB2012/052140). R.P. has received honoraria for advisory boards and speaker engagements from Roche, EISAI, Eli Lilly, Biogen, Janssen-Cilag, Astra Zeneca, Schwabe, Grifols, Novo Nordisk, and Tabuk. R.S.-V. served in advisory board meetings for Wave Life Sciences, Ionis, and Novo Nordisk; has received personal fees for participating in educational activities from Janssen, Roche Diagnostics, and Neuraxpharm; and has received funding to her institution for research projects from Biogen and Sage Pharmaceuticals. S.W.S. received research support from Cerevel Therapeutics and is a member of the scientific advisory board of the Lewy Body Dementia Association and the Multiple System Atrophy Coalition. J.S.Y. serves on the scientific advisory board for the Epstein Family Alzheimer’s Research Collaboration. J.H. does consulting and gives talks for Eli-Lilly, Roche, and Eisai and is on the Ceracuity advisory board., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Hearing loss in older adults is associated with increased dementia risk. Underlying mechanisms that connect hearing loss with dementia remain largely unclear., Methods: We studied the association of hearing loss and biomarkers for dementia risk in two age groups with normal cognition: 65 participants from the European Medical Information Framework (EMIF)-Alzheimer's disease (AD) 90+ study (oldest-old; mean age 92.7 years, 56.9% female) and 60 participants from the EMIF-AD PreclinAD study (younger-old; mean age 74.4, 43.3% female). Hearing function was tested by the 'digits-in-noise test' and cognition by repeated neuropsychological evaluation. Regressions and generalised estimating equations were used to test the association of hearing function and PET-derived amyloid burden, and linear mixed models were used to test the association of hearing function and cognitive decline. In the oldest-old group, mediation analyses were performed to study whether cognitive decline is mediated through regional brain atrophy., Results: In oldest-old individuals, hearing function was not associated with amyloid pathology (p=0.7), whereas in the younger-old individuals hearing loss was associated with higher amyloid burden (p=0.0034). In oldest-old individuals, poorer hearing was associated with a steeper decline in memory, global cognition and language, and in the younger-old with steeper decline in language only. The hippocampus and nucleus accumbens mediated the effects of hearing loss on memory and global cognition in the oldest-old individuals., Conclusions: Hearing loss was associated with amyloid binding in younger-old individuals only, and with cognitive decline in both age groups. These results suggest that mechanisms linking hearing loss with risk for dementia depends on age., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Alzheimer's disease is characterized by regional reductions in cerebral blood flow (CBF). Although the gold standard for measuring CBF is [ 15 O]H 2 O PET, proxies of relative CBF, derived from the early distribution phase of amyloid and tau tracers, have gained attention. The present study assessed precision of [ 15 O]H 2 O derived relative and absolute CBF, and compared precision of these measures with that of (relative) CBF proxies. Dynamic [ 15 O]H 2 O, [ 18 F]florbetapir and [ 18 F]flortaucipir PET test-retest (TrT) datasets with eleven, nine and fourteen subjects, respectively, were included. Analyses were performed using an arterial input model and/or a simplified reference tissue model, depending on the data available. Relative CBF values (i.e. K 1 / K 1 ' and/or R 1 ) were obtained using cerebellar cortex as reference tissue and TrT repeatability (i.e. precision) was calculated and compared between tracers, parameters and clinical groups. Relative CBF had significantly better TrT repeatability than absolute CBF derived from [ 15 O]H 2 O ( r  = -0.53), while best TrT repeatability was observed for [ 18 F]florbetapir and [ 18 F]flortaucipir R 1 ( r  = -0.23, r  = -0.33). Furthermore, only R 1 showed, better TrT repeatability for cognitively normal individuals. High precision of CBF proxies could be due to a compensatory effect of the extraction fraction, although changes in extraction fraction could also bias these proxies, but not the gold standard.

Introduction: Synapse dysfunction is emerging as an early pathological event in frontotemporal dementia (FTD), however biomarkers are lacking. We aimed to investigate the value of cerebrospinal fluid (CSF) neuronal pentraxins (NPTXs), a family of proteins involved in homeostatic synapse plasticity, as novel biomarkers in genetic FTD., Methods: We included 106 presymptomatic and 54 symptomatic carriers of a pathogenic mutation in GRN , C9orf72 or MAPT , and 70 healthy non-carriers participating in the Genetic Frontotemporal dementia Initiative (GENFI), all of whom had at least one CSF sample. We measured CSF concentrations of NPTX2 using an in-house ELISA, and NPTX1 and NPTX receptor (NPTXR) by Western blot. We correlated NPTX2 with corresponding clinical and neuroimaging datasets as well as with CSF neurofilament light chain (NfL) using linear regression analyses., Results: Symptomatic mutation carriers had lower NPTX2 concentrations (median 643 pg/mL, IQR (301-872)) than presymptomatic carriers (1003 pg/mL (624-1358), p<0.001) and non-carriers (990 pg/mL (597-1373), p<0.001) (corrected for age). Similar results were found for NPTX1 and NPTXR. Among mutation carriers, NPTX2 concentration correlated with several clinical disease severity measures, NfL and grey matter volume of the frontal, temporal and parietal lobes, insula and whole brain. NPTX2 predicted subsequent decline in phonemic verbal fluency and Clinical Dementia Rating scale plus FTD modules. In longitudinal CSF samples, available in 13 subjects, NPTX2 decreased around symptom onset and in the symptomatic stage., Discussion: We conclude that NPTX2 is a promising synapse-derived disease progression biomarker in genetic FTD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: Growing evidence suggests a role of occupation in the emergence and manifestation of dementia. Occupations are often defined by complexity level, although working environments and activities differ in several other important ways. We aimed to capture the multi-faceted nature of occupation through its measurement as a qualitative (instead of a quantitative) variable and explored its relationship with different types of dementia., Methods: We collected occupational information of 2121 dementia patients with various suspected etiologies from the Amsterdam Dementia Cohort (age 67 ± 8, 57% male; MMSE 21 ± 5). Our final sample included individuals with Alzheimer's disease (AD) dementia (n = 1467), frontotemporal dementia (n = 281), vascular dementia (n = 98), Lewy body disease (n = 174), and progressive supranuclear palsy/corticobasal degeneration (n = 101). Within the AD group, we used neuropsychological data to further characterize patients by clinical phenotypes. All participants were categorized into 1 of 11 occupational classes, across which we evaluated the distribution of dementia (sub)types with χ 2 analyses. We gained further insight into occupation-dementia relationships through post hoc logistic regressions that included various demographic and health characteristics as explanatory variables., Results: There were significant differences in the distribution of dementia types across occupation groups (χ 2  = 85.87, p < .001). Vascular dementia was relatively common in the Transportation/Logistics sector, and higher vascular risk factors partly explained this relationship. AD occurred less in Transportation/Logistics and more in Health Care/Welfare occupations, which related to a higher/lower percentage of males. We found no relationships between occupational classes and clinical phenotypes of AD (χ 2  = 53.65, n.s.)., Conclusions: Relationships between occupation and dementia seem to exist beyond the complexity level, which offers new opportunities for disease prevention and improvement of occupational health policy.

Objective: Frontotemporal lobar degeneration (FTLD) is the second most prevalent dementia in young patients and is characterized by the presence of two main protein aggregates in the brain, tau (FTLD-Tau) or TDP43 (FTLD-TDP), which likely require distinct pharmacological therapy. However, specific diagnosis of FTLD and its subtypes remains challenging due to largely overlapping clinical phenotypes. Here, we aimed to assess the clinical performance of novel cerebrospinal fluid (CSF) biomarkers for discrimination of FTLD and its pathological subtypes., Methods: YKL40, FABP4, MFG-E8, and the activities of catalase and specific lysosomal enzymes were analyzed in patients with FTLD-TDP ( n  = 30), FTLD-Tau ( n  = 20), AD ( n  = 30), DLB ( n  = 29), and nondemented controls ( n  = 29) obtained from two different centers. Models were validated in an independent CSF cohort ( n  = 188)., Results: YKL40 and catalase activity were increased in FTLD-TDP cases compared to controls. YKL40 levels were also higher in FTLD-TDP compared to FTLD-Tau. We identified biomarker models able to discriminate FTLD from nondemented controls (MFG-E8, tTau, and A β 42 ; 78% sensitivity and 83% specificity) and non-FTLD dementia (YKL40, pTau, p/tTau ratio, and age; 90% sensitivity, 78% specificity), which were validated in an independent cohort. In addition, we identified a biomarker model differentiating FTLD-TDP from FTLD-Tau (YKL40, MFGE-8, β HexA together with β HexA/tHex and p/tTau ratios and age) with 80% sensitivity and 82% specificity., Interpretation: This study identifies CSF protein signatures distinguishing FTLD and the two main pathological subtypes with optimal accuracy (specificity/sensitivity > 80%). Validation of these models may allow appropriate selection of cases for clinical trials targeting the accumulation of Tau or TDP43, thereby increasing their efficiency and facilitating the development of successful therapies.

Behavioural variant frontotemporal dementia (bvFTD) is characterized by behavioural and social cognitive disturbances, while various psychiatric and neurodegenerative disorders may have similar clinical symptoms. Since neurodegenerative disorders are eventually progressive, whereas primary psychiatric disorders are not, this study aimed to investigate whether the change in clinical symptoms over time differed between groups and which biomarkers predicted rate of decline. Disease trajectories (median follow-up = 3 years) of frontal and stereotyped behaviour, general and frontal cognitive functioning, and social cognition were examined in bvFTD (n = 34), other neurodegenerative (n = 28) and primary psychiatric disorders (n = 43), all presenting with late-onset frontal lobe syndrome (45-75 years), using linear mixed models. To gain more insight in underlying pathological processes driving disease progression, we studied the association of baseline cerebrospinal fluid (CSF) (neurofilament light (NfL) and YKL-40 levels, phosphotau 181 to total tau ratio) and neuroimaging markers with disease trajectories. Frontal behavioural symptoms (e.g., disinhibition, apathy) worsened over time in bvFTD, whereas they improved in psychiatric disorders and remained stable in other neurodegenerative disorders. General and frontal cognitive decline was observed in bvFTD and other neurodegenerative disorders, but not in psychiatric disorders. None of the groups showed change in stereotypy and social cognition. For all diagnostic groups, higher CSF NfL levels were associated with faster frontal cognitive decline. A modest association was observed between caudate volume and stereotyped behaviour. Tracking frontal behavioural symptoms and cognition has potential to distinguish bvFTD from other disorders. CSF NfL levels seem to be associated with decline in frontal cognitive functioning., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Dementia is generally characterized by cognitive impairment that can be accompanied by psychotic symptoms; for example, visual hallucinations are a core feature of dementia with Lewy bodies, and delusions are often seen in Alzheimer's disease. However, for behavioral variant of frontotemporal dementia (bvFTD), studies on the broad spectrum of psychotic symptoms are still lacking. The aim of this study was to systematically and prospectively subtype the wide spectrum of psychotic symptoms in probable and definite bvFTD., Methods: In this study, a commonly used and validated clinical scale that quantifies the broad spectrum of psychotic symptoms (Positive and Negative Symptom Scale) was used in patients with probable and definite bvFTD (n=22) and with a primary psychiatric disorder (n=35) in a late-onset frontal lobe cohort. Median symptom duration was 2.8 years, and the patients were prospectively followed for 2 years., Results: In total, 22.7% of bvFTD patients suffered from delusions, hallucinatory behavior, and suspiciousness, although the majority of the patients exhibited negative psychotic symptoms such as social and emotional withdrawal and blunted affect (95.5%) and formal thought disorders (81.8%). "Difficulty in abstract thinking" and "stereotypical thinking" (formal thought disorders) differentiated bvFTD from psychiatric disorders. The combined predictors difficulty in abstract thinking, stereotypical thinking, "anxiety", "guilt feelings," and "tension" explained 75.4% of variance in the diagnosis of bvFTD versus psychiatric diagnoses ( P <0.001)., Conclusion: Delusions, hallucinatory behavior, and suspiciousness were present in one-fifth of bvFTD patients, whereas negative psychotic symptoms such as social and emotional withdrawal, blunted affect, and formal thought disorders were more frequently present. This suggests that negative psychotic symptoms and formal thought disorders have an important role in the psychiatric misdiagnosis in bvFTD; misdiagnosis in bvFTD might be reduced by systematically exploring the broad spectrum of psychiatric symptoms., Competing Interests: Disclosure Welmoed Krudop received funding from the Mooiste Contact Fonds, KPN. Philip Scheltens received grant support (for the institution: the VUmc, Alzheimer center) from GE Healthcare, Danone Research, and MERCK. He has received speaker’s fees (paid to the institution) from Lilly, GE Health-care, Lundbeck, Danone, and Jansen AI-Pfizer. Yolande AL Pijnenburg received funding from the Nederlandse Hersenstichting. The other authors report no conflicts of interest in this work.

Lobar cerebral microbleeds are most often sporadic and associated with Alzheimer's disease. The aim of our study was to identify the underlying genetic defect in a family with cognitive complaints and multiple lobar microbleeds and a positive family history for early onset Alzheimer's disease. We performed exome sequencing followed by Sanger sequencing for validation purposes on genomic DNA of three siblings with cognitive complaints, reduced amyloid-beta-42 in CSF and multiple cerebral lobar microbleeds. We checked for the occurrence of the variant in a cohort of 363 patients with early onset dementia and/or microbleeds. A novel frameshift variant (c.236&#95;237delAC) generating a premature stop codon in the CCM2 gene shared by all three siblings was identified. Pathogenicity of the variant was supported by the presence of cerebral cavernous malformations in two of the siblings and by the absence of the variant exome variant databases. Two siblings were homozygous for APOE-ϵ4; one heterozygous. The cognitive complaints, reduced amyloid-beta-42 in CSF and microbleeds suggest preclinical Alzheimer's disease, but the stability of the cognitive complaints does not. We hypothesize that the phenotype in this family may be due to a combination of the CCM2 variant and the APOE status. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc., (© 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.)

Introduction: To prospectively determine the diagnostic value of cerebrospinal fluid (CSF) levels total-tau (tau) to amyloid-β 1-42 ratio (Aβ 1-42 ) ratio (tau/Aβ 1-42 ratio), phosphorylated-tau (p-tau) to tau ratio (p-tau/tau ratio), neurofilament light chain (NfL) and YKL40 in the late-onset frontal lobe syndrome, in particular for the differential diagnosis of behavioral variant frontotemporal dementia (bvFTD) versus primary psychiatric disorders (PSY)., Method: We included patients with a multidisciplinary 2-year-follow-up diagnosis of probable/definite bvFTD ( n  = 22) or PSY ( n  = 25), who underwent a detailed neuropsychiatric clinical examination, neuropsychological test battery, and magnetic resonance imaging at baseline. In all cases, CSF was collected through lumbar puncture at baseline. We compared CSF biomarker levels between the two groups and measured the diagnostic accuracy for probable/definite bvFTD, using the follow-up diagnosis as the reference standard., Results: The best discriminators between probable/definite bvFTD and PSY were the levels of CSF NfL (area under the curve [AUC] 0.93, P  < .001, 95% confidence interval [CI] 0.85-1.00), p-tau/tau ratio (AUC 0.87, P  < .001, 95% CI 0.77-0.97), and YKL40 (AUC 0.82, P  = .001, 95% CI 0.68-0.97). The combination of these three biomarkers had a sensitivity of 91% (95% CI 66%-100%) at a specificity of 83% (95% CI 65%-95%) with an AUC of 0.94 ( P  < .001, 95% CI 0.87-1.00) for bvFTD. CSF tau/Aβ 1-42 ratio was less accurate in differentiating between bvFTD and PSY., Discussion: We found a good diagnostic accuracy for higher levels of CSF NfL and YKL40 and reduced p-tau/tau ratio in distinguishing bvFTD from PSY. We advocate the use of these CSF biomarkers as potential additional tools to neuroimaging in the diagnosis of bvFTD versus PSY.

Background: Frontotemporal dementia (FTD) is a heterogeneous disease both at the clinical, genetic, and pathobiological level. The underlying pathological spectrum (termed FTLD, frontotemporal lobar degeneration) is in most cases defined by accumulation of either tau (FTLD-tau) or TDP-43 proteins (FTLD-TDP). Biomarkers to differentiate these subtypes are not yet available, whereas these are essential requirements to study the natural course of disease and for homogeneous inclusion of patients in clinical studies., Objective: To study if a combination of total (t-) and phosphorylated (p-)tau, and t-TDP-43 and p-TDP-43 proteins in cerebrospinal fluid (CSF) is suitable to discriminate FTLD-tau and FTLD-TDP subtypes., Methods: We developed immunoassays for the quantification of t-TDP-43 and p-TDP-43 proteins and used commercially available assays for the quantification of t-tau and p-tau proteins. We quantified these proteins in ventricular CSF samples from neuropathologically defined FTLD-tau and FTLD-TDP cases to study the reflection of underlying brain pathology in CSF composition, and in lumbar CSF samples from FTLD-tau and FTLD-TDP patients to study the diagnostic potential of CSF biomarkers., Results: In ventricular CSF, t-TDP-43 and t-tau levels, when combined into one model, were significantly different between neuropathologically-defined FTLD-tau and FTLD-TDP cases. In a pilot study using lumbar CSF, the p-tau/t-tau ratio, but not t-TDP-43 levels, were significantly different between FTLD-TDP and FTLD-tau patients., Conclusion: We conclude that with current available methods, CSF tau, rather than TDP-43 proteins, may have diagnostic value in the differentiation of FTLD patients with either tau or TDP-43 pathology.

- Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease caused by repetitive head injuries like those seen in sports such as boxing, American football and soccer.- The clinical features of CTE are a range of cognitive, psychiatric and motor symptoms, and histopathology involves deposits of hyperphosphorylated tau protein and the presence of TAR DNA-binding protein (TDP-43) with relatively little beta-amyloid.- CTE is difficult to differentiate clinically from Alzheimer's disease, frontotemporal dementia and psychiatric disorders because of the major symptom overlap between these conditions.- The most important risk factors for developing CTE are the cumulative effect of repetitive head injuries, with or without clinical symptoms, and the duration of exposure to the repetitive injuries (the sporting career).- There is no treatment for CTE at present and the strategy must be primarily geared to prevention.- In view of the large number of people, including those in the Netherlands, who take part in sports in which head injuries may occur, research into CTE is of major societal importance.

Background/objective: Alzheimer's disease (AD) and behavioral variant frontotemporal dementia (bvFTD) are the most common types of early-onset dementia. We applied longitudinal resting state functional magnetic resonance imaging (fMRI) to delineate functional brain connections relevant for disease progression and diagnostic accuracy., Methods: We used two-center resting state fMRI data of 20 AD patients (65.1±8.0 years), 12 bvFTD patients (64.7±5.4 years), and 22 control subjects (63.8±5.0 years) at baseline and 1.8-year follow-up. We used whole-network and voxel-based network-to-region analyses to study group differences in functional connectivity at baseline and follow-up, and longitudinal changes in connectivity within and between groups., Results: At baseline, connectivity between paracingulate gyrus and executive control network, between cuneal cortex and medial visual network, and between paracingulate gyrus and salience network was higher in AD compared with controls. These differences were also present after 1.8 years. At follow-up, connectivity between angular gyrus and right frontoparietal network, and between paracingulate gyrus and default mode network was lower in bvFTD compared with controls, and lower compared with AD between anterior cingulate gyrus and executive control network, and between lateral occipital cortex and medial visual network. Over time, connectivity decreased in AD between precuneus and right frontoparietal network and in bvFTD between inferior frontal gyrus and left frontoparietal network. Longitudinal changes in connectivity between supramarginal gyrus and right frontoparietal network differ between both patient groups and controls., Conclusion: We found disease-specific brain regions with longitudinal connectivity changes. This suggests the potential of longitudinal resting state fMRI to delineate regions relevant for disease progression and for diagnostic accuracy, although no group differences in longitudinal changes in the direct comparison of AD and bvFTD were found.

Few data are available concerning the role of risk markers for Alzheimer's disease (AD) in progression to AD dementia among subjects with mild cognitive impairment (MCI). We therefore investigated the role of well-known AD-associated single-nucleotide polymorphism (SNP) in the progression from MCI to AD dementia. Four independent MCI data sets were included in the analysis: (a) the German study on Aging, Cognition and Dementia in primary care patients (n=853); (b) the German Dementia Competence Network (n=812); (c) the Fundació ACE from Barcelona, Spain (n=1245); and (d) the MCI data set of the Amsterdam Dementia Cohort (n=306). The effects of single markers and combined polygenic scores were measured using Cox proportional hazards models and meta-analyses. The clusterin (CLU) locus was an independent genetic risk factor for MCI to AD progression (CLU rs9331888: hazard ratio (HR)=1.187 (1.054-1.32); P=0.0035). A polygenic score (PGS1) comprising nine established genome-wide AD risk loci predicted a small effect on the risk of MCI to AD progression in APOE-ɛ4 (apolipoprotein E-ɛ4) carriers (HR=1.746 (1.029-2.965); P=0.038). The novel AD loci reported by the International Genomics of Alzheimer's Project were not implicated in MCI to AD dementia progression. SNP-based polygenic risk scores comprising currently available AD genetic markers did not predict MCI to AD progression. We conclude that SNPs in CLU are potential markers for MCI to AD progression.

Background: The major genetic risk factor for late onset Alzheimer's disease (AD) is the APOE-ɛ4 allele. However, APOE-ɛ4 homozygosity is not fully penetrant, suggesting co-occurrence of additional genetic variants., Objective: To identify genetic factors that, next to APOE-ɛ4 homozygosity, contribute to the development of AD., Methods: We identified a family with nine AD patients spanning four generations, with an inheritance pattern suggestive of autosomal dominant AD, with no variants in PSEN1, PSEN2, or APP. We collected DNA from four affected and seven unaffected family members and performed exome sequencing on DNA from three affected and one unaffected family members., Results: All affected family members were homozygous for the APOE-ɛ4 allele. Statistical analysis revealed that AD onset in this family was significantly earlier than could be expected based on APOE genotype and gender. Next to APOE-ɛ4 homozygosity, we found that all four affected family members carried a rare variant in the VPS10 domain of the SORL1 gene, associated with AβPP processing and AD risk. Furthermore, three of four affected family members carried a rare variant in the TSHZ3 gene, also associated with AβPP processing. Affected family members presented between 61 and 74 years, with variable presence of microbleeds/cerebral amyloid angiopathy and electroencephalographic abnormalities., Conclusion: We hypothesize that next to APOE-ɛ4 homozygosity, impaired SORL1 protein function, and possibly impaired TSHZ3 function, further disturbed Aβ processing. The convergence of these genetic factors over several generations might clarify the increased AD penetrance and the autosomal dominant-like inheritance pattern of AD as observed in this family.

Background: Heterogeneous disease trajectories of mild cognitive impairment (MCI) and dementia are frequently encountered in clinical practice, but there is still insufficient knowledge to understand the reasons and mechanisms causing this heterogeneity. In addition to correlates of the disorder, patient characteristics such as their health status, social environment, comorbidities and frailty may contribute to variability in trajectories over time. The current paper outlines the study design and the study population of and provides an overview of the data collected in the Clinical Course of Cognition and Comorbidity in Mild Cognitive Impairment (4C-MCI cohort, n = 315) and Dementia (4C-Dementia cohort, n = 331) Study., Methods: The two complementary longitudinal cohorts part of the 4C study began enrolment in March 2010. Participants were prospectively recruited from three collaborating Dutch Alzheimer Centers, with three annual follow-up assessments after baseline. Extensive neuropsychological assessments, and detailed profiling of comorbidities, health and frailty at each follow up were the key features of the 4C study. As such, the 4C study was designed to study if and how patients' comorbidities and frailty are associated with the course of MCI and dementia measured with a comprehensive and multidimensional set of outcomes including cognition, daily functioning, quality of life, behavioral disturbances, caregiver burden, institutionalization and death and whether the effects of medical health and frailty differ between MCI and dementia stages of cognitive disorders., Conclusion: Sampled in a clinical setting, the 4C study complements population-based studies on neurodegenerative disorders in terms of the type of assessment (e.g. comorbidity, frailty, and functional status were repeatedly assessed). The 4C study complements available clinical cohorts of MCI and dementia patients, because the exclusion criteria were kept to a minimum, to obtain a sample that is representative for the average patient visiting a memory clinic.

Objective: Early differentiation between psychiatric disorders and behavioral variant frontotemporal dementia (bvFTD) is of paramount importance in patients with the late-onset frontal lobe syndrome. As bvFTD in patients will deteriorate, psychiatric disorders are treatable. To date, misdiagnosis often occurs due to an overlap of symptoms and lack of specific biomarkers. The aim of our study was to investigate whether specific symptoms could separate bvFTD from psychiatric disorders., Methods: In a naturalistic, prospective, multicenter study, 137 patients (aged 45-75 years, 72% male) with a late-onset frontal lobe syndrome were included based on their scores on the Frontal Behavioral Inventory (FBI) and the Stereotypy Rating Inventory (SRI) from April 2011 to June 2013. In a multidisciplinary consensus meeting, diagnoses were established based on elaborate neuropsychological testing, magnetic resonance imaging, fludeoxyglucose F 18 positron emission tomography, cerebrospinal fluid biomarkers, and clinical examination by a neurologist and a psychiatrist based on the International bvFTD Criteria Consortium for bvFTD and DSM-IV-TR criteria for psychiatric disorders., Results: Forty-four subjects (32.8%) were diagnosed with a psychiatric disorder, 10 (7.3%) with possible bvFTD, and 45 (32.8%) with probable bvFTD. A logistic regression analysis was performed with "psychiatry or bvFTD" as dependent variable and clinical variables (Montgomery-Asberg Depression Rating Scale [MADRS], SRI, FBI) and demographics as independent variables. A positive history of psychiatric illness, male gender, lower SRI scores and higher MADRS scores were predictive of psychiatric disorders, explaining 65.2% of the variance in diagnosis of psychiatry versus bvFTD (χ²₅ = 60.04, P < .001). On the FBI, symptom level verbal apraxia/aphasia and impulsivity were predictive of bvFTD, whereas irritability was predictive of psychiatric disorders., Conclusions: In daily clinical practice, specific subtyping of clinical symptoms in patients with late-onset frontal lobe syndrome may aid in differentiating bvFTD patients from psychiatric patients and may provide guidance in patient management., (© Copyright 2016 Physicians Postgraduate Press, Inc.)

Objectives: Although bipolar disorder has been understood classically as a cyclic disease with full recovery between mood episodes, in the last decade, evidence has accumulated supporting progressive features. The clinical picture of advanced or end-stage bipolar disorder is heterogeneous with possible deficits in cognition and behavior, as illustrated by our case series., Cases: From our neuropsychiatric outpatient clinic, we describe four cases with bipolar disorder gradually developing a clinical syndrome, including apathy, disinhibition, loss of empathy, stereotypical behavior, and compulsiveness, fulfilling the criteria for possible behavioral variant frontotemporal dementia. All cases were diagnosed with bipolar 1 disorder at least 10 years before the onset of the current symptoms, which were not due to recent mood episodes or switches of medication. In all cases, 3-7 years of follow-up yielded no progression. Repeated neuroimaging was within normal limits. Cerebrospinal fluid biomarker studies were not supportive of underlying neurodegenerative pathology. C9orf72 mutation status was negative in all cases., Conclusion: Symptoms fitting the criteria for possible behavioral variant frontotemporal dementia may be present in end-stage of bipolar disorder. An alternative neurodegenerative nature seems unlikely based on repeated normal neuroimaging and the absence of clinical progression. Functional involvement of the frontal-subcortical networks might play a role.

Introduction: The genetics underlying posterior cortical atrophy (PCA), typically a rare variant of Alzheimer's disease (AD), remain uncertain., Methods: We genotyped 302 PCA patients from 11 centers, calculated risk at 24 loci for AD/DLB and performed an exploratory genome-wide association study., Results: We confirm that variation in/near APOE/TOMM40 (P = 6 × 10(-14)) alters PCA risk, but with smaller effect than for typical AD (PCA: odds ratio [OR] = 2.03, typical AD: OR = 2.83, P = .0007). We found evidence for risk in/near CR1 (P = 7 × 10(-4)), ABCA7 (P = .02) and BIN1 (P = .04). ORs at variants near INPP5D and NME8 did not overlap between PCA and typical AD. Exploratory genome-wide association studies confirmed APOE and identified three novel loci: rs76854344 near CNTNAP5 (P = 8 × 10(-10) OR = 1.9 [1.5-2.3]); rs72907046 near FAM46A (P = 1 × 10(-9) OR = 3.2 [2.1-4.9]); and rs2525776 near SEMA3C (P = 1 × 10(-8), OR = 3.3 [2.1-5.1])., Discussion: We provide evidence for genetic risk factors specifically related to PCA. We identify three candidate loci that, if replicated, may provide insights into selective vulnerability and phenotypic diversity in AD., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Pathogenic mutations in the granulin gene (GRN) are causative in 5-10% of patients with frontotemporal dementia (FTD), mostly leading to reduced progranulin protein (PGRN) levels. Upcoming therapeutic trials focus on enhancing PGRN levels., Methods: Fluctuations in plasma PGRN (n = 41) and its relationship with cerebrospinal fluid (CSF, n = 32) and specific single nucleotide polymorphisms were investigated in pre- and symptomatic GRN mutation carriers and controls., Results: Plasma PGRN levels were lower in carriers than in controls and showed a mean coefficient of variation of 5.3% in carriers over 1 week. Although plasma PGRN correlated with CSF PGRN in carriers (r = 0.54, p = 0.02), plasma only explained 29% of the variability in CSF PGRN. rs5848, rs646776 and rs1990622 genotypes only partly explained the variability of PGRN levels between subjects., Conclusions: Plasma PGRN is relatively stable over 1 week and therefore seems suitable for treatment monitoring of PGRN-enhancing agents. Since plasma PGRN only moderately correlated with CSF PGRN, CSF sampling will additionally be needed in therapeutic trials.

Objective: To evaluate cerebrospinal fluid (CSF) and serum neurofilament light chain (NfL) levels in genetic frontotemporal dementia (FTD) as a potential biomarker in the presymptomatic stage and during the conversion into the symptomatic stage. Additionally, to correlate NfL levels to clinical and neuroimaging parameters., Methods: In this multicenter case-control study, we investigated CSF NfL in 174 subjects (48 controls, 40 presymptomatic carriers and 86 patients with microtubule-associated protein tau (MAPT), progranulin (GRN), and chromosome 9 open reading frame 72 (C9orf72) mutations), and serum NfL in 118 subjects (39 controls, 44 presymptomatic carriers, 35 patients). In 55 subjects both CSF and serum was determined. In two subjects CSF was available before and after symptom onset (converters). Additionally, NfL levels were correlated with clinical parameters, survival, and regional brain atrophy., Results: CSF NfL levels in patients (median 6762 pg/mL, interquartile range 3186-9309 pg/mL) were strongly elevated compared with presymptomatic carriers (804 pg/mL, 627-1173 pg/mL, P < 0.001), resulting in a good diagnostic performance to discriminate both groups. Serum NfL correlated highly with CSF NfL (r s = 0.87, P < 0.001) and was similarly elevated in patients. Longitudinal samples in the converters showed a three- to fourfold increase in CSF NfL after disease onset. Additionally, NfL levels in patients correlated with disease severity, brain atrophy, annualized brain atrophy rate and survival., Interpretation: NfL in both serum and CSF has the potential to serve as a biomarker for clinical disease onset and has a prognostic value in genetic FTD.

Background: Neuroimaging has a reasonable accuracy to differentiate behavioral variant frontotemporal dementia (bvFTD) from other neurodegenerative disorders, its value for the differentiation of bvFTD among subjects with acquired behavioral disturbances is unknown., Objective: To determine the diagnostic accuracy of MRI, additional [18F]FDG-PET, and their combination for bvFTD among subjects with late onset behavioral changes., Methods: Patients with late onset behavioral changes referred to a memory clinic or psychiatric services were included. At baseline, 111 patients had a brain MRI scan and 74 patients received an additional [18F]FDG-PET when the MRI was inconclusive. The consensus diagnosis after two-year-follow-up was used as the gold standard to calculate sensitivity and specificity for baseline neuroimaging., Results: 27 patients had probable/definite bvFTD and 84 patients had a non-bvFTD diagnosis (primary psychiatric diagnosis or other neurological disorders). MRI had a sensitivity of 70% (95% CI 52-85%) with a specificity of 93% (95% CI 86-97%). Additional [18F]FDG-PET had a sensitivity of 90% (95% CI 66-100%) with a specificity of 68% (95% CI 56-79%). The sensitivity of combined neuroimaging was 96% (95% CI 85-100%) with a specificity of 73% (95% CI 63-81%). In 66% of the genetic FTD cases, MRI lacked typical frontotemporal atrophy. 40% of cases with a false positive [18F]FDG-PET scan had a primary psychiatric diagnosis., Conclusion: A good diagnostic accuracy was found for MRI and additional [18F]FDG-PET for bvFTD in patients with late onset behavioral changes. Caution with the interpretation of neuroimaging results should especially be taken in cases with a genetic background and in cases with a primary psychiatric differential diagnosis where [18F]FDG-PET is the only abnormal investigation.

Purpose To investigate the diagnostic accuracy of an image-based classifier to distinguish between Alzheimer disease (AD) and behavioral variant frontotemporal dementia (bvFTD) in individual patients by using gray matter (GM) density maps computed from standard T1-weighted structural images obtained with multiple imagers and with independent training and prediction data. Materials and Methods The local institutional review board approved the study. Eighty-four patients with AD, 51 patients with bvFTD, and 94 control subjects were divided into independent training (n = 115) and prediction (n = 114) sets with identical diagnosis and imager type distributions. Training of a support vector machine (SVM) classifier used diagnostic status and GM density maps and produced voxelwise discrimination maps. Discriminant function analysis was used to estimate suitability of the extracted weights for single-subject classification in the prediction set. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were calculated for image-based classifiers and neuropsychological z scores. Results Training accuracy of the SVM was 85% for patients with AD versus control subjects, 72% for patients with bvFTD versus control subjects, and 79% for patients with AD versus patients with bvFTD (P ≤ .029). Single-subject diagnosis in the prediction set when using the discrimination maps yielded accuracies of 88% for patients with AD versus control subjects, 85% for patients with bvFTD versus control subjects, and 82% for patients with AD versus patients with bvFTD, with a good to excellent AUC (range, 0.81-0.95; P ≤ .001). Machine learning-based categorization of AD versus bvFTD based on GM density maps outperforms classification based on neuropsychological test results. Conclusion The SVM can be used in single-subject discrimination and can help the clinician arrive at a diagnosis. The SVM can be used to distinguish disease-specific GM patterns in patients with AD and those with bvFTD as compared with normal aging by using common T1-weighted structural MR imaging. (©) RSNA, 2015.

Objectives: The impact of the dementia might be more severe for caregivers of people with young onset dementia (YOD) compared with those who care for someone with late onset dementia (LOD), as a young age among caregivers has been identified as a predictor of increased burden. The present study compares well-being between LOD and YOD caregivers longitudinally because this knowledge is essential in order to develop adequate support programs., Design, Setting, and Participants: 220 YOD and 108 LOD patient-caregiver dyads were included from two prospective cohorts with a 2-year follow up. To assess well-being we used the Short Sense of Competence Questionnaire, the RAND-36, the Symptom Checklist 90, and the Montgomery Asberg Depression Rating Scale. The severity and the course of the different measures used to describe caregiver burden were analyzed with linear mixed models., Results: Caregivers in both groups experienced high levels of physical and psychological complaints, mild depressive symptoms, lower health-related quality of life (HRQoL), and decreased feelings of competence. The severity and the course of most measures were similar in both groups, although HRQoL on both the physical and the mental domain was lower for the YOD caregivers., Conclusions: The number of actual psychological and physical complaints does not differ between YOD and LOD caregivers. YOD caregivers have greater perceived difficulties in daily life because of these complaints, however., (Copyright © 2015 American Association for Geriatric Psychiatry. Published by Elsevier Inc. All rights reserved.)

We investigated whether the functional connectivity and network topology in 69 Alzheimer's disease (AD), 48 behavioral variant of frontotemporal dementia (bvFTD) patients, and 64 individuals with subjective cognitive decline are different using resting-state electroencephalography recordings. Functional connectivity between all pairs of electroencephalography channels was assessed using the phase lag index (PLI). We subsequently calculated PLI-weighted networks, from which minimum spanning trees (MSTs) were constructed. Finally, we investigated the hierarchical clustering organization of the MSTs. Functional connectivity analysis showed frequency-dependent results: in the delta band, bvFTD showed highest whole-brain PLI; in the theta band, the whole-brain PLI in AD was higher than that in bvFTD; in the alpha band, AD showed lower whole-brain PLI compared with bvFTD and subjective cognitive decline. The MST results indicate that frontal networks appear to be selectively involved in bvFTD against the background of preserved global efficiency, whereas parietal and occipital loss of network organization in AD is accompanied by global efficiency loss. Our findings suggest different pathophysiological mechanisms in these 2 separate neurodegenerative disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recently, the diagnostic criteria for the behavioral variant of frontotemporal dementia were revised. Although these criteria offer a relatively high sensitivity, their specificity is yet unknown. We describe a 54-year-old woman fulfilling criteria for both late-onset schizophrenia and probable behavioral variant frontotemporal dementia. Following an initial presentation with psychosis, she developed progressive apathy, compulsiveness, and executive dysfunction. Moreover, bilateral frontotemporal hypometabolism was seen on [(18)F]fludeoxyglucose-positron emission tomography. A post-mortem diagnosis of schizophrenia was established, given the clinical picture combined with the pathological exclusion of a neurodegenerative cause. Our case suggests that patients with other brain disorders may meet the current diagnostic criteria for probable frontotemporal dementia. Further clinicopathological validation of these criteria is needed to determine their exact specificity.

Progranulin (PGRN) levels in blood and cerebrospinal fluid (CSF) are increasingly studied as potential markers for neurodegenerative disorders. We aimed to 1) characterize two commercially available PGRN ELISAs on several assay validation parameters, 2) assess the stability of PGRN in serum and CSF under pre-analytical conditions, and 3) compare stability in the two assays. Intra- and inter-assay variation, inter-lot variation, linearity, lower limit of detection, and kit correlations were assessed for the Adipogen and R&D PGRN ELISA kits. Blood and serum samples were experimentally exposed to ≤9 freeze/thaw cycles, delayed processing for ≤24 h at room temperature and 4°C, and to temperature stability tests for ≤3 weeks at -20°C, 4°C, room temperature, and 37°C. Both commercial PGRN ELISA kits showed acceptable ranges for intra- and inter-assay variation, where the R&D kit performed more accurate than the Adipogen kit, especially for inter-assay variation (intra-assay serum: 6.7 and 8.3%, respectively; inter-assay serum: 9.2 and 21.0%; intra-assay CSF: 3.6 and 12.0%; inter-assay CSF: 16.0 and 44.5%). Absolute serum PGRN concentrations were 1.9-fold higher in Adipogen than R&D (p < 0.001) and strongly correlated between both kits (ρ= 0.86, p < 0.0001) and CSF PGRN levels were on the borderline of detection in both kits. PGRN was typically stable under all pre-analytical conditions addressed, although two weeks at 37°C resulted in decreased PGRN concentrations in CSF, only when using the Adipogen kit. These results support further examination of PGRN as a potential marker in neurodegenerative diseases, since PGRN is stable in serum and CSF and can be measured using ELISA kits from several providers.