Silvia Bagnoli

A Pan-European Study of the C9orf72 Repeat Associated with FTLD: Geographic Prevalence, Genomic Instability, and Intermediate Repeats

We assessed the geographical distribution of C9orf72 G4C2 expansions in a pan‐European frontotemporal lobar degeneration (FTLD) cohort (n = 1,205), ascertained by the European Early‐Onset Dementia (EOD) consortium. Next, we performed a meta‐analysis of our data and that of other European studies, together 2,668 patients from 15 Western European countries. The frequency of the C9orf72 expansions in Western Europe was 9.98% in overall FTLD, with 18.52% in familial, and 6.26% in sporadic FTLD patients. Outliers were Finland and Sweden with overall frequencies of respectively 29.33% and 20.73%, but also Spain with 25.49%. In contrast, prevalence in Germany was limited to 4.82%. In addition, we studied the role of intermediate repeats (7–24 repeat units), which are strongly correlated with the risk haplotype, on disease and C9orf72 expression. In vitro reporter gene expression studies demonstrated significantly decreased transcriptional activity of C9orf72 with increasing number of normal repeat units, indicating that intermediate repeats might act as predisposing alleles and in favor of the loss‐of‐function disease mechanism. Further, we observed a significantly increased frequency of short indels in the GC‐rich low complexity sequence adjacent to the G4C2 repeat in C9orf72 expansion carriers (P < 0.001) with the most common indel creating one long contiguous imperfect G4C2 repeat, which is likely more prone to replication slippage and pathological expansion.

Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients

We have measured the levels of typical end products of the processes of lipid peroxidation, protein oxidation, and total antioxidant capacity (TAC) in skin fibroblasts and lymphoblasts taken from patients with familial Alzheimers disease (FAD), sporadic Alzheimers disease (AD), and age-matched healthy controls. Compared to controls, the fibroblasts and lymphoblasts carrying amyloid precursor protein (APP) and presenilin-1 (PS-1) gene mutations showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In contrast, the antioxidant defenses of cells from FAD patients were lower than those from normal subjects. Lipoperoxidation and antioxidant capacity in lymphoblasts from patients affected by sporadic AD were virtually indistinguishable from the basal values of normal controls. An oxidative attack on protein gave rise to greater protein carbonyl content in FAD patients than in age-matched controls. Furthermore, ADP ribosylation levels of poly(ADP-ribose) polymerase (PARP) nuclear substrates were significantly raised, whereas the PARP content did not differ significantly between fibroblasts carrying gene mutations and control cells. These results indicate that peripheral cells carrying APP and PS-1 gene mutations show altered levels of oxidative markers even though they are not directly involved in the neurodegenerative process of AD. These results support the hypothesis that oxidative damage to lipid, protein, and DNA is an important early event in the pathogenesis of AD.

Heterozygous TREM2 mutations in frontotemporal dementia

A causative association was recently demonstrated between homozygous TREM2 mutations and frontotemporal dementia (FTD)-like syndrome and between heterozygous TREM2 exon2 genetic variations and late-onset Alzheimers disease (AD). The objective of this study was to evaluate whether heterozygous TREM2 genetic variations might be associated to the risk of FTD. TREM2 exon 2 was sequenced in a group of 1030 subjects-namely, 352 patients fulfilling clinical criteria for FTD, 484 healthy control subjects (HCs), and 194 patients with AD. The mutation frequency and the associated clinical characteristics were analyzed. We identified 8 missense and nonsense mutations in TREM2 exon 2 in 24 subjects. These mutations were more frequent in patients with FTD than in HCs (4.0% vs. 1.0%, p = 0.005). In particular, TREM2 Q33X, R47H, T66M, and S116C mutations were found in FTD and were absent in HCs. These mutations were associated with either the semantic variant of primary progressive aphasia or the behavioral variant FTD phenotypes. The FTD and AD groups were not significantly different with regard to TREM2 genetic variation frequency (AD: 2.6%, p = 0.39). Heterozygous TREM2 mutations modulate the risk of FTD in addition to increasing susceptibility to AD. Additional studies are warranted to investigate the possible role of these mutations in the pathogenesis of neurodegenerative disorders.

Autosomal Dominant Frontotemporal Lobar Degeneration Due to the C9ORF72 Hexanucleotide Repeat Expansion: Late-Onset Psychotic Clinical Presentation

BACKGROUND A hexanucleotide repeat expansion in the first intron of C9ORF72 has been shown to be responsible for a high number of familial cases of amyotrophic lateral sclerosis or frontotemporal lobar degeneration (FTLD). Atypical presentations have been described, particularly psychosis. METHODS We determined the frequency of the hexanucleotide repeat expansions in a population of 651 FTLD patients and compared the clinical characteristics of carriers and noncarriers. In addition, we genotyped 21 patients with corticobasal syndrome, 31 patients with progressive supranuclear palsy, and 222 control subjects. RESULTS The pathogenic repeat expansion was detected in 39 (6%) patients with FTLD (17 male and 22 female subjects); however, it was not detected in any corticobasal syndrome and progressive supranuclear palsy patients or controls. Twenty-four of 39 carriers had positive family history for dementia and/or amyotrophic lateral sclerosis (61.5%), whereas only 145 of 612 noncarriers had positive family history (23.7%; p<.000001). Clinical phenotypes of carriers included 29 patients with the behavioral variant frontotemporal dementia (bvFTD; 5.2% of all bvFTD cases), 8 with bvFTD/motor neuron disease (32% bvFTD/motor neuron disease cases), 2 with semantic dementia (5.9% of patients with semantic dementia), and none with progressive nonfluent aphasia. The presentation with late-onset psychosis (median age = 63 years) was more frequent in carriers than noncarriers (10/33 vs. 3/37, p = .029), as well as the presence of cognitive impairment at onset (15/33 vs. 5/37; p = .0039). CONCLUSIONS The repeat expansion in C9ORF72 is a common cause of FTLD and often presents with late-onset psychosis or memory impairment.

Cholesteryl ester transfer protein (CETP) I405V polymorphism and longevity in Italian centenarians.

A common polymorphism (I405V) in exon 14 of the cholesteryl ester transfer protein (CETP) gene has been recently associated to healthy aging in Ashkenazi Jewish. In order to study this genetic effect in long-lived individuals with a different ethnicity, we analyzed the allele and genotype distributions of the CETP polymorphism a sample of Italian centenarians. Our result does not confirm the association between the I405V CETP variation and the healthy aging phenotype described in the Ashkenazi Jewish population and suggests that other gene environment interactions contribute to longevity.

Brain-derived neurotrophic factor, apolipoprotein E genetic variants and cognitive performance in Alzheimer's disease.

Since greater attention has been paid to the direct link of genetic variation to cognition and memory performance, apolipoprotein E (ApoE) and brain-derived neurotrophic factor (BDNF) have been the two most frequently studied genes. To investigate the effect of BDNF and ApoE polymorphisms on the cognitive profile of mild-moderate Alzheimers disease (AD) cases, AD patients, genotyped for ApoE and BDNF polymorphisms, underwent extensive neuropsychological investigation. The effect of either ApoE epsilon4 allele and BDNF genetic variant on the neuropsychological pattern of mental impairment was examined both in terms of group differences in performance on the neuropsychological tests between carriers and non-carriers of each variant and by selecting the best predictor of cognitive performance among demographic and genetic factors by means of a multiple regression analysis. Our data confirm a specific effect caused by the presence and amount of ApoE epsilon4 allele, while they suggest that BDNF genetic variants are not a susceptibility factor to AD.

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts.

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimers disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Abeta 1-40 and Abeta 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

The C677T methylenetetrahydrofolate reductase mutation is not associated with Alzheimer's disease.

The methylenetetrahydrofolate reductase (MTHFR) gene has been recently considered as a candidate gene for Alzheimers disease (AD). MTHFR is a key enzyme in the metabolism of homocysteine and elevated levels of that amino acid have been associated to Vascular Dementia and AD. A T-->C transition at codon 677 produces a thermolabile type of the enzyme. However, contrasting results on the distribution of the MTHFR C677T common polymorphism in AD have been published. We analyzed the distribution of the MTHFR and apolipoprotein E (APOE) polymorphisms in Italian patients with sporadic AD. The distribution of the C677T polymorphism did not differ in AD and controls. Our data suggest that the MTHFR polymorphism does not contribute to genetic susceptibility in Italian sporadic AD and does not mitigate the effect of ApoE epsilon4 allele on AD risk.

KIBRA gene variants are associated with episodic memory performance in subjective memory complaints.

The KIBRA gene encodes a cytoplasmatic protein, a member of the signal transduction protein family, expressed mainly in the brain. Recent studies have implicated the involvement of a genetic variation in the KIBRA gene (T allele) in human memory in normal subjects and in the risk of developing Alzheimers disease (AD). We report here the distribution of the KIBRA genetic variant and the Apolipoprotein E (ApoE) epsilon4 allele and their association with neuropsychological measures in older adults reporting problems with everyday memory (subjective memory complaints, SMC). We found that SMC subjects with the CT/TT genotype performed more poorly than those with the CC genotype on long-term memory tests. Thus, in our opinion, these data suggest that the KIBRA genotype could affect memory performance in a different way in those that complain of memory deficits compared to those that do not.

Brain-derived neurotrophic factor genetic variants are not susceptibility factors to Alzheimer's disease in Italy

Several lines of evidences have suggested an involvement of the brain-derived neurotrophic factor (BDNF, 11p13) in the pathogenesis of neurodegenerative disorders, in particular, in Alzheimer’s disease (AD). Recently, positive associations between two BDNF polymorphisms (the Val66Met[G196A] and 270C/T) and AD have suggested a possible effect of BDNF genetic variations on the risk of AD. In light of these findings, we analyzed the segregation of the Val66Met(G196A) and 270C/T BDNF and apolipoprotein E (ApoE) genes polymorphisms in Italian patients with AD. We analyzed a sample of 128 Italian patients with sporadic AD (45 men and 83 women; mean age, 71.1 8.5; age at onset, 65.7 8.6 mean SD, with age of onset defined as the onset of first cognitive changes) enrolled at the Department of Neurology, University of Florence and 97 healthy controls (36 men and 61 women; mean age, 72.9 24 years). All subjects were carefully assessed with a rigorous diagnostic evaluation to exclude diagnosis of any neurological disorder. Clinical assessments of patients were done according to published guidelines and the AD diagnosis fulfilled the National Institute of Neurological and Communicative Disorders and Stroke Alzheimer’s disease and related Disorders Association criteria for probable AD. The local ethical committee approved the protocol of the study and the written consent for genetic screening was obtained from all subjects or, where appropriate, their relative or legal representative. All the gene polymorphisms were analyzed by polymerase chain reaction followed by appropriate restriction enzyme digestion. Allele frequencies were estimated by gene counting. Comparisons of genotype and allele frequencies distribution between patients and controls were assessed by 2 test. Both polymorphisms followed a distribution in Hardy– Weinberg equilibrium and did not significantly differ from that of controls (p 0.1) (Table). In addition, no correlation was observed between age at onset, sex, or any BDNF genotype. Stratification of data irrespective of the ApoE status (data not shown) did not show an epistatic effect of the two genes. It could be speculated that, in previous studies, the analyzed populations were not matched for their ethnic background, a major confounding factor when studying polymorphisms. Several nonmutually exclusive factors can produce conflicting results in association studies of common complex diseases including the presence of genetic, clinical, and population heterogeneity. Our data suggest that BDNF genetic variants are not a susceptibility factor for AD, nor do they mitigate the effect of ApoE ε4 allele on AD risk.