Non-Ceruloplasmin Copper Identifies a Subtype of Alzheimer's Disease (CuAD): Characterization of the Cognitive Profile and Case of a CuAD Patient Carrying an RGS7 Stop-Loss Variant.

Alzheimer's disease (AD) is a type of dementia whose cause is incompletely defined. Copper (Cu) involvement in AD etiology was confirmed by a meta-analysis on about 6000 participants, showing that Cu levels were decreased in AD brain specimens, while Cu and non-bound ceruloplasmin Cu (non-Cp Cu) levels were increased in serum/plasma samples. Non-Cp Cu was advocated as a stratification add-on biomarker of a Cu subtype of AD (CuAD subtype). To further circumstantiate this concept, we evaluated non-Cp Cu reliability in classifying subtypes of AD based on the characterization of the cognitive profile. The stratification of the AD patients into normal AD (non-Cp Cu ≤ 1.6 µmol/L) and CuAD (non-Cp Cu > 1.6 µmol/L) showed a significant difference in executive function outcomes, even though patients did not differ in disease duration and severity. Among the Cu-AD patients, a 76-year-old woman showed significantly abnormal levels in the Cu panel and underwent whole exome sequencing. The CuAD patient was detected with possessing the homozygous (c.1486T > C; p.(Ter496Argext*19) stop-loss variant in the RGS7 gene (MIM*602517), which encodes for Regulator of G Protein Signaling 7. Non-Cp Cu as an add-on test in the AD diagnostic pathway can provide relevant information about the underlying pathological processes in subtypes of AD and suggest specific therapeutic options.

Non-Ceruloplasmin Copper Distincts Subtypes in Alzheimer's Disease: a Genetic Study of ATP7B Frequency.

Meta-analyses show that serum copper non-bound-to-ceruloplasmin (non-Cp-Cu) is higher in patients with Alzheimer's disease (AD). ATP7B gene variants associate with AD, modulating the size of non-Cp-Cu pool. However, a dedicated genetic study comparing AD patients after stratification for a copper biomarker to demonstrate the existence of a copper subtype of AD has not yet been carried out. An independent patient sample of 287 AD patients was assessed for non-Cp-Cu serum concentrations, rs1801243, rs1061472, and rs732774 ATP7B genetic variants and the APOE4 genotype. Patients were stratified into two groups based on a non-Cp-Cu cutoff (1.9 µM). Single-locus and haplotype-group analyses were performed to define their frequencies in dependence of the non-Cp-Cu group. The two AD subgroups did not differ regarding age, sex, MMSE score, or APOE4 frequency allele, while they did differ regarding non-Cp-Cu concentrations in serum, allele, genotype, and haplotype frequencies of rs1061472 A > G and rs732774 C > T after multiple testing corrections. AD patients with a GG genotype had a 1.76-fold higher risk of having a non-Cp-Cu higher than 1.9 µmol/L (p = 0.029), and those with a TT genotype for rs732774 C > T of 1.8-fold (p = 0.018). After 100,000 permutations for multiple testing corrections, the haplotype containing the AC alleles appeared more frequently in AD patients with normal non-Cp-Cu [43 vs. 33 %; Pm = 0.03], while the haplotype containing the GT risk alleles appeared more frequently in the higher non-Cp-Cu AD (66 vs. 55 %; Pm = 0.01). Genetic heterogeneity sustains a copper AD metabolic subtype; non-Cp-Cu is a marker of this copper AD.

Association Between Serum Ceruloplasmin Specific Activity and Risk of Alzheimer's Disease.

Meta-analyses demonstrate copper involvement in Alzheimer's disease (AD), and the systemic ceruloplasmin status in relation to copper is an emerging issue. To deepen this matter, we evaluated levels of ceruloplasmin concentration, ceruloplasmin activity, ceruloplasmin specific activity (eCp/iCp), copper, non-ceruloplasmin copper iron, transferrin, the ceruloplasmin/transferrin ratio, and the APOE genotype in a sample of 84 AD patients and 58 healthy volunteers. From the univariate logistic analyses we found that ceruloplasmin concentration, eCp/iCp, copper, transferrin, the ceruloplasmin/transferrin ratio, and the APOE genotype were significantly associated with the probability of AD. In the multivariable logistic regression analysis, we selected the best subset of biological predictors by the forward stepwise procedure. The analysis showed a decrease of the risk of having AD for eCp/iCp (p = 0.001) and an increase of this risk for non-ceruloplasmin copper (p = 0.008), age (p = 0.001), and APOE-ɛ4 allele (p <  0.001). The estimated model showed a good power in discriminating AD patients from healthy controls (area under curve: 88% ; sensitivity: 66% ; specificity 93%). These data strength the breakdown of copper homeostasis and propose eCp/iCp as a reliable marker of ceruloplasmin status.

Explorative genetic association study of GSTT2B copy number variant in complex disease risks.

BACKGROUND: Glutathione S-transferases (GSTs) are the main phase II enzymes involved in cellular detoxification. Through phase I and phase II detoxification reactions, the cell is able to detoxify endogenous and exogenous toxic compounds. AIMS: This study focused attention on the GSTT2B copy number variant (CNV) in order to explore its involvement in the genetic pre-disposition to asthma, Alzheimer's disease (AD), allergic rhinitis (AR), essential hypertension (EH), hypothyroidism and recurrent miscarriage (RM). METHODS: The study population consists of 1225 individuals divided into six case-control groups. The genotyping of the GSTT2B CNV was performed by using a duplex-PCR. Odds Ratios (ORs) were calculated, adjusting for the confounding variables, to estimate the association between GSTT2B CNV and the disease status. RESULTS: The χ(2)-test and ORs did not show any association between this genetic marker and pathological phenotypes. CONCLUSION: The data highlights that GSTT2B CNV is not associated with the investigated complex diseases in Italian patients. However, further investigations are necessary to replicate these findings in larger sample sizes and to explore other health-related phenotypes.

Altered metal metabolism in patients with HCV-related cirrhosis and hepatic encephalopathy.

Dysfunctional metal homeostasis contributes to oxidative stress and neuronal damage. These have been implicated in hepatic encephalopathy pathogenesis. To investigate whether altered metal metabolism is associated with hepatic encephalopathy. Twenty-one controls and 34 HCV-cirrhotic patients (ENC/NEC patients according to presence/absence of previous overt episodes of hepatic encephalopathy) and a control group were studied. Serum iron, copper, ceruloplasmin, ceruloplasmin activity, transferrin, and ceruloplasmin/transferrin ratio were determined. Neuropsychological tests were performed by the repeatable battery of neuropsychological status. Magnetic resonance assessed basal ganglia volumes and metal deposition (pallidal index and T2*). Cirrhotic patients performed worse than controls at cognitive tests, especially ENC patients,. At biochemical analysis copper concentrations, ceruloplasmin activity and transferrin levels were lower in ENC than in NEC patients and controls (p < 0.05 and p < 0.01, respectively). Ceruloplasmin/transferrin ratio was higher in ENC compared to NEC patients (p < 0.05), and controls (p < 0.01). By brain magnetic resonance, ENC patients showed reduced caudate and globus pallidus volumes compared to controls (p < 0.05), and ENC and NEC patients an increased pallidal index compared to controls (p < 0.01). In ENC patients, ceruloplasmin activity correlated with caudate volume and pallidal index (ρ = 0.773 and ρ = -0.683, p < 0.05). Altered metal metabolism likely contributes to cirrhotic hepatic encephalopathy.

Zinc in Alzheimer's Disease: A Meta-Analysis of Serum, Plasma, and Cerebrospinal Fluid Studies.

To evaluate whether zinc levels in serum, plasma, and cerebrospinal fluid are altered in Alzheimer's disease (AD), we performed meta-analyses of 27 studies on the topic published from 1983 to 2014. The subjects' sample obtained by merging studies was a pooled total of 777 AD subjects and 1,728 controls for serum zinc studies, 287 AD subjects and 166 controls for plasma zinc, and of 292 AD subjects and 179 controls for CSF zinc. The main result of this meta-analysis is the very high heterogeneity among the studies either in demographic terms or in methodological approaches. Although we considered these effects in our analyses, the heterogeneity persisted and it has to be taken into account in the interpretation of the results. Our meta-analysis indicated that serum zinc appears significantly decreased in AD patients compared with healthy controls, and this result is confirmed when serum and plasma studies were analyzed together. If we considered the age-matched studies, the meta-analysis carried out on only six studies showed no significant difference in zinc levels between AD and healthy controls (SMD =-0.55, 95% CI (-1.18; 0.09); p = 0.094; I2 = 91%). In the light of these findings, we speculated about the possibility that the decreases observed could indicate a possible dietary zinc deficiency and we suggested that the possible involvement of zinc alterations in AD may have an interplay with copper metabolism.

Meta-analysis study on the role of bone-derived neurotrophic factor Val66Met polymorphism in Parkinson's disease.

To evaluate a possible involvement of the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism in susceptibility to Parkinson's disease (PD), we performed a meta-analysis of all studies on the topic published from 2002 to 2014. This article reviews and compares the data from two previous meta-analyses, including two studies not previously considered. We selected studies referring to a genetic comparison between PD patients and healthy controls, so 15 studies involving 3754 cases and 4026 controls were included in our meta-analysis. We found no association between the Val66Met polymorphism and the risk of developing PD in our overall analysis. The ethnicity-specific meta-analysis produced no significant association either. Our data do not support a major role for the BDNF Val66Met polymorphism in the pathogenesis of PD.

Val66Met BDNF gene polymorphism influences human motor cortex plasticity in acute stroke.

BACKGROUND: BDNF gene polymorphism impacts human motor cortex function and plasticity. OBJECTIVE/HYPOTHESIS: Using transcranial magnetic stimulation (TMS), we investigated whether BDNF polymorphism influences cortical plastic changes in acute stroke. METHODS: Twenty patients were recruited within 10 days of their first-ever ischemic stroke and genotyped for BDNF polymorphism. Blinded to the latter, we evaluated the excitability of the affected and unaffected hemisphere by measuring resting and active motor threshold and motor-evoked potential amplitude under baseline conditions and after intermittent theta burst stimulation, a protocol of repetitive TMS inducing LTP-like activity. We also computed laterality indexes to assess inter-hemispheric excitability imbalance. RESULTS: Demographics, threshold and amplitude of motor-evoked potentials did not differ between those with (8 patients) and without polymorphism. Excitability of the unaffected hemisphere was significantly higher than the excitability of the affected hemisphere as probed by each measure. This imbalance was exaggerated in those without polymorphism; laterality indexes of rest motor thresholds were 0.016 ± 0.050 and 0.139 ± 0.028 for patients with and without polymorphism [t = 2.270, P = 0.036]. Exaggerated hemispheric imbalance also persisted after intermittent theta burst stimulation, which failed to induce any difference between groups. CONCLUSIONS: Our results suggest that inter-hemispheric imbalance with greater excitability over unaffected hemisphere, is several times stronger in stroke patients without, as opposed to with, polymorphism.

In silico investigation of the ATP7B gene: insights from functional prediction of non-synonymous substitution to protein structure.

ATP7B is a copper-transporting ATPase that plays a key role in the regulation of copper homeostasis. Mutations in the ATP7B gene are causative for Wilson's disease, and recent reports have suggested that genetic variants are associated with susceptibility to Alzheimer's disease. Unfortunately, it is difficult to profile experimentally novel genetic variants in the ATP7B gene, because the human protein X-ray structure is not yet entirely understood. In order to investigate ATP7B non-synonymous substitutions, we used an in silico amino acid sequence-based approach. Specifically, we analyzed 337 ATP7B non-synonymous substitutions, which included Wilson's disease-causing mutations (DVs) and non Wilson's disease-causing variants (NDVs), with an algorithm that estimated a combined probability (cPdel) of an amino acidic change to be deleterious for the protein function. This approach appeared to reliably indentify the probability of DVs and NDVs to be deleterious and to profile still unknown gene variants. Specifically, after analyzing ATP7B protein domains with the cPdel method, we found results in line with the predicted-modeled domains and some new suggestions. In conclusion, a functional survey of amino acid changes in the ATP7B protein is provided herein, and we suggest that this bioinformatic method can furnish information about novel ATP7B mutations. Furthermore, the same approach can be applied to other uncharacterized proteins.

Intronic rs2147363 variant in ATP7B transcription factor-binding site associated with Alzheimer's disease.

Copper homeostasis abnormalities have been shown to be associated with Alzheimer's disease (AD), possibly by accelerating amyloid-ß toxicity and plaque formation. The ATP7B gene plays a key role in controlling body copper balance. Our previous studies showed an association between ATP7B variants and AD risk. Among these variants, an intronic single nucleotide polymorphism, rs2147363, was associated with AD risk. In order to understand this intronic association, we screened a population of 286 AD patients and 283 healthy controls, and verified the presence of other functional coding variants in linkage disequilibrium (LD). Then we searched for a regulatory function region close to rs2147363. An LD analysis revealed the presence of an LD between rs2147363 and a Wilson's disease-causing variant, rs7334118. However, this mutation did not explain the observed genetic association. Conversely, in silico analyses of rs2147363 functionality highlighted that this variant is located in a binding site of a transcription factor, and is, consequently, associated with regulatory function. These data suggest that the genetic variation in cis-regulatory elements located in non-coding regions can have a role in determining ATP7B functionality and account for some of the AD missing hereditability.

ATP7B variants as modulators of copper dyshomeostasis in Alzheimer's disease.

To understand the role of the key copper-regulating gene, ATP7B, in copper dyshomeostasis associated with Alzheimer's disease (AD), we analyzed the serum levels of copper, ceruloplasmin and 'free' (i.e., non-ceruloplasmin bound) copper in 399 patients with AD and 303 elderly healthy controls. We also performed analyses of informative variants of ATP7B. AD patients had higher levels of copper and free copper than controls. Individuals with free copper levels higher than 1.6 µmol/L (the upper value of the normal reference range) were more frequent among cases (p < 0.001). Among these individuals, those who were carriers of the ATP7B variants accounted for a large proportion of the free copper levels, specifically in the AD group (p < 0.01). Our results suggest the existence of a 'copper dysfunction' phenotype of sporadic AD which has a genetic basis. They also suggest that free copper is a risk factor for this disorder, modulating additional pathways leading to the disease cascade.

Linkage disequilibrium and haplotype analysis of the ATP7B gene in Alzheimer's disease.

Copper dyshomeostasis leading to a labile Cu(2+) not bound to ceruloplasmin ("free" copper) may influence Alzheimer's disease (AD) onset or progression. To investigate this hypothesis, we investigated ATP7B, the gene that controls copper excretion through the bile and concentrations of free copper in systemic circulation. Our study analyzed informative ATP7B single-nucleotide polymorphisms (SNPs) in a case-control population (n=515). In particular, we evaluated the genetic structure of the ATP7B gene using the HapMap database and carried out a genetic association investigation. Linkage disequilibrium (LD) analysis highlighted that our informative SNPs and their LD SNPs covered 96% of the ATP7B gene sequence, distinguishing two "strong LD" blocks. The first LD block contains the gene region encoding for transmembrane and copper-binding, whereas the second LD block encodes for copper-binding domains. The genetic association analysis showed significant results after multiple testing correction for all investigated variants (rs1801243, odds ratio [OR]=1.52, 95% confidence interval [CI]=1.10-2.09, p=0.010; rs2147363, OR=1.58, 95% CI=1.11-2.25, p=0.010; rs1061472, OR=1.73, 95% CI=1.23-2.43, p=0.002; rs732774, OR=2.31, 95% CI=1.41-3.77, p<0.001), indicating that SNPs in transmembrane domains may have a stronger association with AD risk than variants in copper-binding domains. Our study provides novel insights that confirm the role of ATP7B as a potential genetic risk factor for AD. The analysis of ATP7B informative SNPs confirms our previous hypothesis about the absence of ATP7B in the significant loci of genome-wide association studies of AD and the genetic association study suggests that transmembrane and adenosine triphosphate (ATP) domains in the ATP7B gene may harbor variants/haplotypes associated with AD risk.

Fe and Cu do not differ in Parkinson's disease: a replication study plus meta-analysis.

To evaluate whether iron and copper levels in serum, plasma, and cerebrospinal fluid are disarranged in Parkinson's disease (PD), we performed meta-analyses of 33 studies on the topic published from 1987 to 2011 and contextually carried out a replication study in serum by ourselves as well. We found no variation in metals between PD patients and healthy controls, according to our replication study. The metaregression for sex revealed that serum copper differences found in some studies could be referred to the different percentage of women in the PD sample. Transferrin and transferrin saturation levels found increased in PD subjects underline the concept to extend the iron study in PD to iron master proteins.

Copper in Alzheimer's disease: a meta-analysis of serum, plasma, and cerebrospinal fluid studies.

This contribution reviews and corrects data from our previous meta-analysis, which appeared in the Journal of Alzheimer's Disease in 2011 concerning the role of copper in Alzheimer's disease. We repeated the meta-analysis after excluding four of the five studies from our laboratory to avoid possible bias in the result. In addition, we included two studies on serum copper levels in Alzheimer's disease not previously considered. The results indicate higher levels of copper in Alzheimer's disease patients than in controls, confirming our previous conclusion.

Association of K832R and R952K SNPs of Wilson's disease gene with Alzheimer's disease.

Copper homeostasis appears abnormal in Alzheimer's disease (AD) patients. The aim of this study was to assess whether loci of susceptibility for AD lie in the Wilson's disease (WD) ATP7B gene. We studied single nucleotide polymorphisms (SNPs) K832R (c.2495 A>G, rs1061472) and R952K (c. 2855 G>A, rs732774) of the WD gene in 251 AD patients and 201 healthy controls. We also evaluated their relation with apolipoprotein E (ApoE) ε4 allele frequency. R allele in K832R [adjusted Odds Ratio (OR) = 1.71 (1.12-2.60); p = 0.012] and the K allele in R952K [adjusted OR = 1.82 (1.19-2.80); p = 0.006] ATP7B SNPs were associated with an increased risk of developing AD, as well as the haplotype R832/K952, containing the 2 risk alleles (X2 = 4.85; p = 0.028). Conversely, the K832/R952 haplotype appeared to confer protection against the disease (X2 = 7.21; p = 0.007). No difference in the frequency of the ATP7B alleles between carriers and non-carriers of the ApoE ε4 variant was revealed. The linkage disequilibrium (LD) analysis revealed an association between K832R and R952K substitutions in both AD patients (D' = 0.79) and controls (D' = 0.81). A high LD between K832R and R952K was also confirmed in all HapMap populations. Our investigation demonstrated the presence of loci of susceptibility for AD in the WD ATP7B gene, supporting a role of copper dysfunction in contributing or accelerating neurodegenerative processes leading to AD.

Effects of hemochromatosis and transferrin gene mutations on iron dyshomeostasis, liver dysfunction and on the risk of Alzheimer's disease.

It is now accepted that transition metals, such as iron and copper, are involved in the pathogenesis of the Alzheimer's disease (AD) through their participation in toxic oxidative phenomena. In this context, hemochromatosis (Hfe) and transferrin (Tf) genes are of particular importance, since they play a key role in iron homeostasis. Also, signs of liver distress which accompany metal dysmetabolisms have been shown to be linked to AD. In order to investigate whether and how all these factors are interconnected, in this study we have explored the relationship of the gene variants of Hfe H63D and C282Y and of Tf C2 with serum markers of iron status (iron, ferritin, TF, TF-saturation, ceruloplasmin -CP-, CP and TF serum concentrations (CP/TF) ratio), and of liver function (albumin, transaminases, prothrombin time-prothrombin time (PT)) in a sample of 160 AD patients and 79 healthy elderly controls. Albumin resulted in lower, PT longer and AST/ALT higher ratios in AD patients than in controls, indicating a distress of the liver. Also TF was lower and ferritin higher in AD. Multiple logistic regression backward analyses, performed to evaluate the effects of our biochemical variables upon the probability of developing AD, revealed that a one-unit TF serum-decrease increases the probability of AD by 80%, a one-unit albumin serum-decrease reduces this probability by 20%, and a one-unit increase of AST/ALT ratio generates a 4-fold probability increase. Patients who were carriers of the H63D mutation showed higher levels of iron, lower levels of TF and CP and higher CP/TF ratios, a panel resembling hemochromatosis. This picture was found neither in H63D non-carrier patients, nor in healthy controls. Our results suggest the existence of a link between Hfe mutations and iron abnormalities that increases the probability of developing AD when accompanied by a distress of the liver.

Association between the c. 2495 A>G ATP7B Polymorphism and Sporadic Alzheimer's Disease.

Nonceruloplasmin-bound copper ("free") is reported to be elevated in Alzheimer's disease (AD). In Wilson's disease (WD) Cu-ATPase 7B protein tightly controls free copper body levels. To explore whether the ATP7B gene harbours susceptibility loci for AD, we screened 180 AD chromosomes for sequence changes in exons 2, 5, 8, 10, 14, and 16, where most of the Mediterranean WD-causing mutations lie. No WD mutation, but sequence changes corresponding to c.1216 T>G Single-Nucleotide Polymorphism (SNP) and c.2495 A>G SNP were found. Thereafter, we genotyped 190 AD patients and 164 controls for these SNPs frequencies estimation. Logistic regression analyses revealed either a trend for the c.1216 SNP (P = .074) or a higher frequency for c.2495 SNP of the GG genotype in patients, increasing the probability of AD by 74% (P = .028). Presence of the GG genotype in ATP7B c.2495 could account for copper dysfunction in AD which has been shown to raise the probability of the disease.

Copper in Alzheimer's disease: a meta-analysis of serum,plasma, and cerebrospinal fluid studies.

There is an ongoing debate on the involvement of systemic copper (Cu) dysfunctions in Alzheimer's disease (AD), and clinical studies comparing Cu levels in serum, plasma, and cerebrospinal fluid (CSF) of AD patients with those of healthy controls have delivered non-univocal and often conflicting results. In an attempt to evaluate whether Cu should be considered a potential marker of AD, we applied meta-analysis to a selection of 26 studies published in the literature. Meta-analysis is a quantitative method that combines the results of independent reports to distinguish between small effects and no effects, random variations, variations in sample used, or in different analytical approaches. The subjects' sample obtained by merging studies was a pooled total of 761 AD subjects and 664 controls for serum Cu studies, 205 AD subjects and 167 controls for plasma Cu, and of 116 AD subjects and 129 controls for CSF Cu. Our meta-analysis of serum data showed that AD patients have higher levels of serum Cu than healthy controls. Plasma data did not allow conclusions, due to their high heterogeneity, but the meta-analysis of the combined serum and plasma studies confirmed higher Cu levels in AD. The analysis of CSF data, instead, revealed no difference between AD patients and controls.

Copper status abnormalities and how to measure them in neurodegenerative disorders.

Copper is essential for life. It plays a pivotal role in the central nervous system, in which a low concentration of copper results in incomplete development, whereas an excess of copper is injurious. Redox reactions are at the basis of copper toxicity: in fact, it catalyses the production of reactive oxygen species in Fenton or Haber-Weiss reactions. Abnormalities of copper homeostasis in neurodegenerative disorders were discovered decades ago. The steady increase in reports from the literature demonstrating copper involvement in neurodegenerative disorders coincides with the improvement, reliability and low cost devices which measure copper markers in biological samples. These devices also demonstrate increasing relevance in diagnosis and in therapy monitoring as well. Methods and new perspectives for the analysis of copper markers status are discussed herein, weighing pros and cons of application to a specific neurological disorder. In particular, it have been introduced three patents regarding a new apparatus for measuring levels of metal in biological samples, employing a current measuring device. A mention of recent patents concerning new derivatives of curcumin has been done considering its metal chelating and multi-functional properties that make these compounds interesting candidates for treatment of some neurodegenerative disorders.