Genome Wide Association Study and Next Generation Sequencing: A Glimmer of Light Toward New Possible Horizons in Frontotemporal Dementia Research

Frontotemporal Dementia (FTD) is a focal neurodegenerative disease, with a strong genetic background, that causes early onset dementia. The present knowledge about the risk loci and causative mutations of FTD mainly derives from genetic linkage analysis, studies of candidate genes, Genome-Wide Association Studies (GWAS) and Next-Generation Sequencing (NGS) applications. In this review, we report recent insights into the genetics of FTD, and, specifically, the results achieved thanks to GWAS and NGS approaches. Linkage studies of large FTD pedigrees have prompted the identification of causal mutations in different genes: mutations in C9orf72, MAPT, and GRN genes explain the large majority of cases with a high family history of the disease. In cases with a less clear inheritance, GWAS and NGS have contributed to further understand the genetic picture of FTD. GWAS identified several common genetic variants with a modest risk effect. Of interest, many of these variants are in genes belonging to the endo-lysosomal pathway, the immune response and neuronal survival. On the opposite, the NGS approach allowed the identification of rare variants with a strong risk effect. These variants were identified in known FTD-associated genes and again in genes involved in the endo-lysosomal pathway and in the immune response. Interestingly, both approaches demonstrated that several genes are associated to multiple neurodegenerative disorders including FTD. Thanks to these complementary approaches, the genetic picture of FTD is becoming more clear and novel key molecular processes are emerging. This will foster opportunities to move toward prevention and therapy for this incurable disease

molecular mechanisms in cognitive frailty potential therapeutic targets for oxygen ozone treatment

Abstract In the last decade, cognitive frailty has gained great attention from the scientific community. It is characterized by high inflammation and oxidant state, endocrine and metabolic alterations, mitochondria dysfunctions and slowdown in regenerative processes and immune system, with a complex and multifactorial aetiology. Although several treatments are available, challenges regarding the efficacy and the costs persist. Here, we proposed an alternative non-pharmacological, non-side-effect, low cost therapy based on anti-inflammation, antioxidant, regenerative and anti-pathogens properties of ozone (O3), through the activation of several molecular mechanisms (Nrf2-ARE, NF-κB, NFAT, AP-1, HIFα). We highlighted how these specific processes could be implicated in cognitive frailty to identify putative therapeutic targets for its treatment. The O2-O3 therapy has never been tested for cognitive frailty. This work provides thus wide scientific background to build a consistent rationale for testing for the first time this therapy, that could modulate the immune, inflammatory, oxidant, metabolic, endocrine, microbiota and regenerative processes impaired in cognitive frailty. Although insights are needed, the O2-O3 therapy could represent a faster, easier, inexpensive monodomain intervention, working in absence of side effects, for cognitive frailty

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

The Missing Heritability of Sporadic Frontotemporal Dementia: New Insights from Rare Variants in Neurodegenerative Candidate Genes

Frontotemporal dementia (FTD) is a common form of dementia among early-onset cases. Several genetic factors for FTD have been revealed, but a large proportion of FTD cases still have an unidentified genetic origin. Recent studies highlighted common pathobiological mechanisms among neurodegenerative diseases. In the present study, we investigated a panel of candidate genes, previously described to be associated with FTD and/or other neurodegenerative diseases by targeted next generation sequencing (NGS). We focused our study on sporadic FTD (sFTD), devoid of disease-causing mutations in GRN, MAPT and C9orf72. Since genetic factors have a substantially higher pathogenetic contribution in early onset patients than in late onset dementia, we selected patients with early onset (<65 years). Our study revealed that, in 50% of patients, rare missense potentially pathogenetic variants in genes previously associated with Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis and Lewy body dementia (GBA, ABCA7, PARK7, FUS, SORL1, LRRK2, ALS2), confirming genetic pleiotropy in neurodegeneration. In parallel, a synergic genetic effect on FTD is suggested by the presence of variants in five different genes in one single patient. Further studies employing genome-wide approaches might highlight pathogenic variants in novel genes that explain the still missing heritability of FTD

Can APOE and MTHFR polymorphisms have an influence on the severity of cardiovascular manifestations in Italian Pseudoxanthoma elasticum affected patients?

Background: The clinical phenotype of Pseudoxanthoma elasticum (PXE) affected patients, although progressive with age, is very heterogeneous, even in the presence of identical ABCC6 mutations, thus suggesting the occurrence of modifier genes. Beside typical skin manifestations, the cardiovascular (CV) system, and especially the peripheral vasculature, is frequently and prematurely compromised. Methods and results: A cohort of 119 Italian PXE patients has been characterized for apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms by PCR. The severity of the clinical phenotype has been quantified according to the Phenodex PXE International score system. Statistical analysis (chi2 test, odd ratio, regression analysis, analysis of variance) were done by GraphPad. Data demonstrate that the frequency of APOE alleles is similar in PXE patients and in healthy subjects and that the allelic variant E2 confers a protection against the age-related increase of CV manifestations. By contrast, PXE patients are characterized by high frequency of the MTHFR-T677T polymorphism. With age, CV manifestations in T677T, but also in C677T, patients are more severe than those associated with the C677C genotype. Interestingly, compound heterozygosity for C677T and A1298C polymorphisms is present in 70% of PXE patients. Conclusions: PXE patients may be screened for these polymorphisms in order to support clinicians for a better management of disease-associated CV complications

The Heritability of Frontotemporal Lobar Degeneration: Validation of Pedigree Classification Criteria in a Northern Italy Cohort

A large portion of frontotemporal lobar degeneration (FTLD) patients has a family history of disease and the presence of a pathogenic mutation confirms the clinical diagnosis. Recently, standardized criteria to evaluate FTLD pedigree, based on first- and second-degree affected relatives, their age at onset, and clinical phenotype, were proposed and validated in an American cohort. Herein we applied these criteria to 402 Italian FTLD pedigrees and assessed mutation frequencies in GRN, C9orf72, and MAPT genes with the aim of validating these criteria. Moreover, we evaluated whether genetic counseling requests reflect the estimated family risk. 12.4% of pedigrees had high family history, 6.5% medium, 15.4% low; 39% were apparent sporadic cases and 26.6% had family history of unknown significance. Mutations frequencies were in line with the categorization proposed: the highest rate was found in the most at-risk families (74%) and decreased in other categories (medium: 15.4%; low: 9.7%; sporadic: 1.3%). Mutation carriers with unknown family history (5.6%) were mostly early-onset patients. Detected mutation frequency was comparable with the US-cohort (13.7%), but mutations distribution among genes was different, with higher frequency of GRN mutations (9.4%) in our cohort. An elevated proportion of FTLD patients belonging to "high risk" pedigrees asked for genetic counseling (42%); requests decreased according to the estimated family risk (medium: 26.9%; low: 17.7%; sporadic: 5.1%). In conclusion, the proposed pedigree classification criteria, herein further validated, should be incorporated in the FTLD diagnostic work-up. Moreover, our data suggest to extend genetic screening to early-onset patients with unknown family history

Next Generation Sequencing Analysis in Early Onset Dementia Patients.

BackgroundEarly onset dementias (EOD) are rare neurodegenerative dementias that present before 65 years. Genetic factors have a substantially higher pathogenetic contribution in EOD patients than in late onset dementia.ObjectiveTo identify known and/or novel rare variants in major candidate genes associated to EOD by high-throughput sequencing. Common-risk variants of apolipoprotein E (APOE) and prion protein (PRNP) genes were also assessed.MethodsWe studied 22 EOD patients recruited in Memory Clinics, in the context of studies investigating genetic forms of dementia. Two methodological approaches were applied for the target-Next Generation Sequencing (NGS) analysis of these patients. In addition, we performed progranulin plasma dosage, C9Orf72 hexanucleotide repeat expansion analysis, and APOE genotyping.ResultsWe detected three rare known pathogenic mutations in the GRN and PSEN2 genes and eleven unknown-impact mutations in the GRN, VCP, MAPT, FUS, TREM2, and NOTCH3 genes. Six patients were carriers of only common risk variants (APOE and PRNP), and one did not show any risk mutation/variant. Overall, 69% (n = 9) of our early onset Alzheimer's disease (EAOD) patients, compared with 34% (n = 13) of sporadic late onset Alzheimer's disease (LOAD) patients and 27% (n = 73) of non-affected controls (ADNI, whole genome data), were carriers of at least two rare/common risk variants in the analyzed candidate genes panel, excluding the full penetrant mutations.ConclusionThis study suggests that EOD patients without full penetrant mutations are characterized by higher probability to carry polygenic risk alleles that patients with LOAD forms. This finding is in line with recently reported evidence, thus suggesting that the genetic risk factors identified in LOAD might modulate the risk also in EOAD