Coagulation Pathways in Neurological Diseases: Multiple Sclerosis

Significant progress has been made in understanding the complex interactions between the coagulation system and inflammation and autoimmunity. Increased blood-brain-barrier (BBB) permeability, a key event in the pathophysiology of multiple sclerosis (MS), leads to the irruption into the central nervous system of blood components that include virtually all coagulation/hemostasis factors. Besides their cytotoxic deposition and role as a possible trigger of the coagulation cascade, hemostasis components cause inflammatory response and immune activation, sustaining neurodegenerative events in MS. Early studies showing the contribution of altered hemostasis in the complex pathophysiology of MS have been strengthened by recent studies using methodologies that permitted deeper investigation. Fibrin(ogen), an abundant protein in plasma, has been identified as a key contributor to neuroinflammation. Perturbed fibrinolysis was found to be a hallmark of progressive MS with abundant cortical fibrin(ogen) deposition. The immune-modulatory function of the intrinsic coagulation pathway still remains to be elucidated in MS. New molecular details in key hemostasis components participating in MS pathophysiology, and particularly involved in inflammatory and immune responses, could favor the development of novel therapeutic targets to ameliorate the evolution of MS. This review article introduces essential information on coagulation factors, inhibitors, and the fibrinolytic pathway, and highlights key aspects of their involvement in the immune system and inflammatory response. It discusses how hemostasis components are (dys)regulated in MS, and summarizes histopathological post-mortem human brain evidence, as well as cerebrospinal fluid, plasma, and serum studies of hemostasis and fibrinolytic pathways in MS. Studies of disease-modifying treatments as potential modifiers of coagulation factor levels, and case reports of autoimmunity affecting hemostasis in MS are also discussed

C6orf10 low-frequency and rare variants in italian multiple sclerosis patients

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value <= 5 x 10(-6)). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) <= 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs 16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 x 10(-7) and p < 1 x 10(-20)). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10−6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10−7 and p < 1 × 10−20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS

Crosstalk between hemostasis inhibitors and cholesterol biomarkers in multiple sclerosis

The individual roles of cholesterol pathway biomarkers (CPB) and hemostasis inhibitors with neuroimaging outcomes were previously investigated in multiple sclerosis (MS). The purpose of this extension study was to investigate potential crosstalk between plasma CPB [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and apolipoproteins (Apo) ApoA-I, ApoAII, ApoB, ApoC-II and ApoE] and hemostasis inhibitors [heparin cofactor-II (HCII), protein C (PC), protein S (PS), thrombomodulin, ADAMTS13 and PAI-1] in a cohort of 127 MS patients, and 40 healthy individuals (HI). The associations were assessed with regressions. In MS patients, HCII was positively associated with TC, LDL-C, HDL-C and ApoA-I (p=0.028, 0.027, 0.002 and 0.027, respectively) but negatively associated with ApoCII (p=0.018). PC was positively associated with ApoC-II (p=0.001) and ApoB (p=0.016) whereas PS was associated with TC (p=0.024) and ApoE (p=0.003) in MS. The ApoC-II associations were not observed in HI. The negative association between ApoC-II and HCll was an exception amongst other positive associations between CPB and hemostasis inhibitors in MS. CPB do not modulate the PC associations with neurodegeneration in MS

Crosstalk between hemostasis inhibitors and cholesterol biomarkers in multiple sclerosis

The individual roles of cholesterol pathway biomarkers (CPB) and hemostasis inhibitors with neuroimaging outcomes were previously investigated in multiple sclerosis (MS). The purpose of this extension study was to investigate potential crosstalk between plasma CPB [total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and apolipoproteins (Apo) ApoA-I, ApoAII, ApoB, ApoC-II and ApoE] and hemostasis inhibitors [heparin cofactor-II (HCII), protein C (PC), protein S (PS), thrombomodulin, ADAMTS13 and PAI-1] in a cohort of 127 MS patients, and 40 healthy individuals (HI). The associations were assessed with regressions. In MS patients, HCII was positively associated with TC, LDL-C, HDL-C and ApoA-I (p=0.028, 0.027, 0.002 and 0.027, respectively) but negatively associated with ApoCII (p=0.018). PC was positively associated with ApoC-II (p=0.001) and ApoB (p=0.016) whereas PS was associated with TC (p=0.024) and ApoE (p=0.003) in MS. The ApoC-II associations were not observed in HI. The negative association between ApoC-II and HCll was an exception amongst other positive associations between CPB and hemostasis inhibitors in MS. CPB do not modulate the PC associations with neurodegeneration in MS

Protein S on the surface of plasma lipoproteins: a potential mechanism for protein S delivery to the atherosclerotic plaques?

The anticoagulant protein S (PS) binds phospholipids with very high affinity, but PS interaction with lipoproteins and lipidrich atherosclerotic plaques remains still poorly defined. We investigated PS in plasma lipoproteins and in atherosclerotic plaques from ten patients undergoing endarterectomy. PS was detected by Western blotting after exposure of the necrotic core to liposomes and was found to maintain its ability to bind phosphatidylserine micelles. The amounts of PS bound to low/very low-density lipoproteins in patient' plasmas were higher and more variable than those detected in healthy subjects. A direct correlation between bound PS and low-density lipoproteins (LDL), plasma levels was found only in patients (r=0.921, p<0.001), thereby leading to hypothesize that the PS-phospholipids binding may increase by oxidative processes of LDL in atherosclerotic patients. The presence of the PS into the necrotic core of atherosclerotic plaques and on the surface of lipoproteins, particularly the atherogenic LDL, suggests a LDL-based delivery of PS to the atherosclerotic plaques and emphasizes the deep link between plasma lipids and coagulation in cardiovascular diseases

Genomic, vessel wall transcriptomic, and plasma proteomic approaches to investigate multiple sclerosis

This study was designed to investigate, by several experimental approaches, genes, and proteins associated with multiple sclerosis (MS), an inflammatory and demyelinating disease of the central nervous system (CNS). The study design was aimed to prioritize, by the investigation in patients, potential targets and biomarkers for future mechanistic studies. Through the genomic approach(chpt.10), selected families were investigated by WES for candidate genes from GWAS. The identified low-frequency variants were further investigated in unrelated MS patients. A number of rare and novel mutations were detected, and particularly null variants in the C6orf10 3’ region, in combination with both intra and extra locus low-frequency SNPs. These findings provide the bases for expression studies. The transcriptomic approach (chpt.6) was focused on the internal jugular vein wall, supported by the interaction between vascular and neurodegenerative mechanisms in MS. This original investigation produced a wealth of information on several biological pathways and permitted the combined transcriptome-protein analysis, which provided intriguing biological and clinical hints. Analysis at protein level was conducted in plasma by multiplex assays in relation to clinical MS phenotypes and brain MRI measures, as quantitative and “intermediate” phenotypes evaluating disease progression. Higher CCL18 plasma levels were associated with more severe neurodegenerative features, a noticeable finding(chpt.7). The contribution of adhesion molecules, suggested by the transcriptomic analysis, was similarly explored(chpt.8 and 9). Correlation between plasma levels of specific adhesion molecules in MS patients highlights the leukocyte adhesion process in disease. Increased blood-brain-barrier permeability, a key event in the MS pathophysiology, leads to the irruption of coagulation and hemostasis factors into the CNS, potentially causing an inflammatory response and immune activation. We investigated hemostasis components with main open questions in relation to MS. FXII, the key protease of the coagulation contact activation found deposited in patients’ brain, might participate in adaptive immunity during neuroinflammation. In plasma of MS patients(chpt.4), FXII protein levels were higher than activity, causing a decreased activity/antigen ratio. These findings, corroborated by specifically designed intrinsic thrombin generation assays, might support that FXII contribution in MS is not directly correlated with its “intrinsic” pro-coagulant activity. Negative regulators of hemostasis (TFPI, ADAMTS13, HCII, TM) with anti-inflammatory properties were also studied, which detected specific patterns of correlations (chpt.5 and 11). Positive association of TFPI with TM, observed in MS patients and not in healthy subjects, would imply that endothelium perturbation acts on multiple release mechanisms. In patients, PAI-1, the key fibrinolysis inhibitor, was positively associated with FXII, and negatively associated with HCII, which suggest disease mechanisms influencing their expression in different tissues with implications in fibrin generation/impaired fibrinolysis, important contributors to neuro-inflammation/degeneration. Correlations observed between hemostasis components plasma levels and MRI measures, of interest for brain disease mechanisms, did not overcome correction for multiple comparisons. Extravascular leakage of blood components in MS patients, measured as cerebral microbleeds (CMBs) by MRI, was investigated in relation to plasma levels of hemostasis components. Interestingly, lower ADAMTS13 levels were detected in the MS cohort, in particular in patients with CMBs (chpt.5), who also showed higher VAP-1 levels(chpt.9). These novel findings support the investigation of the plasma protease ADAMTS13, and the amino oxidase/adhesion protein VAP-1, in relation to CMBs. This study provides novel MS disease biomarkers as well as potential drug targetsQuesto studio è stato progettato per indagare attraverso diversi approcci sperimentali i geni e le proteine associate alla sclerosi multipla (SM), una malattia infiammatoria e demielinizzante del sistema nervoso centrale (SNC). L’obiettivo era individuare mediante indagini su pazienti, potenziali bersagli e biomarcatori per futuri studi meccanicistici. Mediante l'approccio genomico(cap.10), le famiglie selezionate sono state studiate attraverso WES per geni candidati da GWAS. Gli SNPs identificati a bassa frequenza sono stati ulteriormente studiati in pazienti indipendenti con SM. L’indagine ha rilevato varianti rare e nuove, tra cui le nulle della regione 3' di C6orf10 in combinazione con SNPs a bassa frequenza a livello intra ed extra locus, fornendo le basi per studi di espressione. L'approccio trascrittomico(cap.6) focalizzato sulla parete interna della vena giugulare, era supportato dall'interazione tra i meccanismi vascolari e quelli neurodegenerativi nella SM. Questa indagine ha prodotto una grande quantità di informazioni su diversi percorsi biologici e ha permesso l'analisi combinata trascrittoma-proteine. L'analisi a livello proteico è stata condotta nel plasma mediante saggi multiplex in relazione ai fenotipi clinici di SM e alle misure cerebrali MRI considerate come fenotipi quantitativi e "intermedi" della progressione della malattia. I livelli plasmatici più alti di CCL18 erano associati a caratteristiche neurodegenerative più gravi(cap.7). Il contributo delle molecole di adesione, suggerito dall'analisi trascrittomica, è stato esplorato in modo analogo(cap.8 e 9). La correlazione tra i livelli plasmatici di specifiche molecole di adesione nei pazienti ha evidenziato il processo di adesione dei leucociti nella malattia. L'aumento della permeabilità della barriera emato-encefalica, evento chiave nella fisiopatologia della SM, porta all’irruzione di fattori emostatici nel SNC, causando una risposta infiammatoria e l’attivazione immunitaria. I componenti dell'emostasi con le principali domande aperte in relazione alla SM sono stati investigati. Il FXII, la proteasi attivatrice della coagulazione da contatto trovata depositata nel cervello dei pazienti, potrebbe partecipare all'immunità adattativa durante la neuroinfiammazione. Nel plasma di pazienti(cap.4) i livelli di proteina del FXII erano superiori all'attività, causando un ridotto rapporto attività/antigene. I risultati corroborati dai saggi di generazione intrinseca di trombina, supporterebbero il contributo del FXII nella SM non attraverso la sua attività pro-coagulante. Lo studio di alcuni inibitori dell'emostasi (TFPI, ADAMTS13, HCII, TM) con proprietà antinfiammatorie, ha rivelato specifici schemi di correlazione(cap.5 e 11). L'associazione positiva di TFPI con TM, osservata nei pazienti e non in soggetti sani, implicherebbe che la perturbazione dell'endotelio agisca su più meccanismi di rilascio. Nei pazienti il PAI-1, l'inibitore chiave della fibrinolisi, era associato positivamente al FXII e negativamente all'HCII, suggerendo meccanismi patologici che influenzano la loro espressione in diversi tessuti con implicazioni nella generazione di fibrina e nella compromissione della fibrinolisi. Le correlazioni osservate tra i livelli plasmatici dei componenti dell'emostasi con le misure di MRI, non hanno superato la correzione per confronti multipli. La perdita extravascolare di sangue misurata come micro sanguinamenti cerebrali (MSC) attraverso MRI è stata studiata nei pazienti in relazione ai livelli plasmatici di componenti dell'emostasi. Livelli più bassi di ADAMTS13 sono stati rilevati nella coorte di SM ed in particolare nei pazienti con MSC(cap.5) che mostravano anche livelli più alti di VAP-1(cap.9). Queste nuove scoperte supportano l'analisi della proteasi ADAMTS13 e l’aminossidasi/proteina di adesione VAP-1 in relazione ai MSC. Questo studio fornisce nuovi biomarcatori della SM e potenziali bersagli farmacologic

Novel gnomAD missense mutations in the Ceruloplasmin gene result in ferroxidase impairment and are consistent with significant underdiagnosis of Aceruloplasminemia

Background: Aceruloplasminemia (ACP) is a ultra-rare autosomal recessive disease caused by mutations on the Ceruloplasmin (CP) gene, encoding the key plasma ferroxidase protein in mammals. The disease is characterized by the accumulation of iron in various organs including the brain, resulting in a clinical landscape characterized by anemia with hyperferritinemia, diabetes, neurological impairment and neurodegeneration. Importantly, although the reported prevalence is 0.5x106 (based on a single report on a very small Japanese population from 1999) the availability of recent population genomic datasets allows for a real-world assessment of pathogenic and potentially pathogenic Cp variants. Aims: To perform a real-world assessment of pathogenic and potentially pathogenic missense CP variants using gnomAD, identifying them and functionally characterizing their effect on CP structure and function, thus improving prevalence estimates for ACP. Methods: literature data was reviewed to identify CP residues crucial for CP function as well as ACP missense mutations. CP missense variants present in the latest gnomAD release (4.0) were identified and compared with literature-derived CP residues of interest. Candidate new pathogenic missense variants were identified and prioritized for further investigation based on allele frequency reported on the gnomAD database. Expression plasmids containing wild-type (WT) recombinant (r) human CP cDNA or rCP mutants encoding the selected variants (and known pathogenic variants as controls) were transiently transfected into HEK293T cell-line, followed by biochemical and functional characterization. Results: A number of novel gnomAD-derived CP missense variants displayed robust impairment of CP ferroxidase activity and protein expression/secretion. The prevalence (actual or predicted) of these variants in the homozygous state in the human population is significantly higher than current ACP estimates, with compound heterozygotes potentially further contributing to increase ACP prevalence. Summary/Conclusion: Within the human population, we identified new heterozygous and homozygous missense alleles in the CP gene which strongly impact CP ferroxidase activity and its biology, thus representing novel pathogenic variants. The actual or predicted prevalence of these variants in the homozygous condition is considerably higher than previous prevalence estimates for ACP, suggesting that a large number of individuals at risk of developing ACP remains undiagnosed

NEXT-GENERATION SEQUENCING GENE DISCOVERY STUDIES IN EARLY-ONSET DEMENTIA

Background: Major breakthroughs in dementia research were realized in studying large families with an early disease onset. Although early-onset dementia (EOD) is relatively rare, these findings are at the basis of our current understanding of the disease biology of neurodegenerative dementias and at the foundation of current drug development strategies. Methods: To further elucidate the missing heritability in dementia we apply whole exome sequencing (WES) on selected EOD patients with high genetic load. This project builds on an impressive collection of EOD patients (> 5000)/families ascertained within the framework of the European Early-Onset Dementia consortium. Patients of all dementia phenotypes are selected based on onset age < 65 years. We prioritize the WES studies on families with DNA for 2-5 affected relatives and isolated patients with onset age < 55 years and, particularly for Alzheimer patients, APOE E4 minus carriers. Selected patients are profiled for mutations in known genes using a NGS gene panel of 30 genes associated with neurodegenerative dementia and related diseases. Results: At present we selected 11 families with DNA of 2-3 patients. Except for one recessive pedigree all families were consistent with dominant inheritance. Further, we included 272 familial or sporadic isolated patients with disease onset below 55 years. Phenotypes include AD, FTLD, FTLD-ALS, ALS, CBD and ANCL. In one family WES we already identified a genetic variant that most probably explains disease. The pedigree includes a sibship of 5 of whom 4 patients and 2 unaffected parents, consistent with recessive disease. The patients suffer from a mixed phenotype of cognitive deterioration, speech problems, extrapyramidal symptoms and epilepsy presenting at age 50-60 years. The candidate variant is homozygous in the 3 tested patients and heterozygous in the unaffected mother. The unaffected sib is homozygous for the wild-type allele. Conclusions: A better understanding of the genetics and biology of dementia will improve classification of patients based on their molecular profile rather than on clinico-pathological symptomatology, which is expected to drastically improve development of effective diagnostic tools, biomarkers and targeted therapies, both for early- and the more common late-onset forms of dementia

Hemostasis gene expression of the internal jugular and saphenous veins

No abstract availabl

Soluble neural cell adhesion molecule and behavioural recovery in minimally conscious patients undergoing transcranial direct current stimulation

Transcranial direct current stimulation (tDCS) is used for therapeutic purpose in severely brain-injured patients. The relationship between the recovery after tDCS and potential biomarkers in plasma has been limitedly investigated in patients with minimal conscious state (MCS)