Diagnostic accuracy of cerebrospinal fluid biomarkers in genetic prion diseases.

Genetic prion diseases are a rare and diverse group of fatal neurodegenerative disorders caused by pathogenic sequence variations in the prion protein gene, PRNP. Data on CSF biomarkers in patients with genetic prion diseases are limited and conflicting results have been reported for unclear reasons. Here, we aimed to analyse the diagnostic accuracy of CSF biomarkers currently used in prion clinical diagnosis in 302 symptomatic genetic prion disease cases from 11 prion diagnostic centres, encompassing a total of 36 different pathogenic sequence variations within the open reading frame of PRNP. CSF samples were assessed for the surrogate markers of neurodegeneration, 14-3-3 protein (14-3-3), total-tau protein (t-tau) and α-synuclein and for prion seeding activity through the real-time quaking-induced conversion assay. Biomarker results were compared with those obtained in healthy and neurological controls. For the most prevalent PRNP pathogenic sequence variations, biomarker accuracy and associations between biomarkers, demographic and genetic determinants were assessed. Additionally, the prognostic value of biomarkers for predicting total disease duration from symptom onset to death was investigated. High sensitivity of the four biomarkers was detected for genetic Creutzfeldt-Jakob disease associated with the E200K and V210I mutations, but low sensitivity was observed for mutations associated with Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. All biomarkers showed good to excellent specificity using the standard cut-offs often used for sporadic Creutzfeldt-Jakob disease. In genetic prion diseases related to octapeptide repeat insertions, the biomarker sensitivity correlated with the number of repeats. New genetic prion disease-specific cut-offs for 14-3-3, t-tau and α-synuclein were calculated. Disease duration in genetic Creutzfeldt-Jakob disease-E200K, Gerstmann-Sträussler-Scheinker-P102L and fatal familial insomnia was highly dependent on PRNP codon 129 MV polymorphism and was significantly associated with biomarker levels. In a large cohort of genetic prion diseases, the simultaneous analysis of CSF prion disease biomarkers allowed the determination of new mutation-specific cut-offs improving the discrimination of genetic prion disease cases and unveiled genetic prion disease-specific associations with disease duration.

Cerebrospinal fluid neurofilament light levels in neurodegenerative dementia: Evaluation of diagnostic accuracy in the differential diagnosis of prion diseases.

INTRODUCTION: Neurofilament light (NFL) levels in the cerebrospinal fluid are increased in several neurodegenerative dementias. However, their diagnostic accuracy in the differential diagnostic context is unknown. METHODS: Cerebrospinal fluid NFL levels were quantified in nonprimarily neurodegenerative neurological and psychiatric diseases (n = 122), mild cognitive impairment (n = 48), Alzheimer's disease (n = 108), dementia with Lewy bodies/Parkinson's disease dementia (n = 53), vascular dementia (n = 46), frontotemporal dementia (n = 41), sporadic Creutzfeldt-Jakob disease (sCJD, n = 132), and genetic prion diseases (n = 182). RESULTS: The highest NFL levels were detected in sCJD, followed by vascular dementia, frontotemporal dementia, dementia with Lewy bodies/Parkinson's disease dementia, Alzheimer's disease, and mild cognitive impairment. In sCJD, NFL levels correlated with cerebrospinal fluid tau and disease duration. NFL levels were able to differentiate sCJD from nonprimarily neurodegenerative neurological and psychiatric diseases (area under the curve = 0.99, 95% confidence interval: 0.99-1) and from the other diagnostic groups showing cognitive impairment/dementia of a non-CJD etiology (area under the curve = 0.90, 95% confidence interval: 0.87-0.92). Compared to nonprimarily neurodegenerative neurological and psychiatric diseases, NFL was also elevated in genetic prion diseases associated with the E200K, V210I, P102L, and D178N prion protein gene mutations. DISCUSSION: Increased NFL levels are a common feature in neurodegenerative dementias.

Cerebrospinal Fluid Biomarker-Based Diagnosis of Sporadic Creutzfeldt-Jakob Disease: A Validation Study for Previously Established Cutoffs.

BACKGROUND: Several biomarkers have been proposed to discriminate sporadic Creutzfeldt-Jakob disease (sCJD) from other dementias and control cases. However, their clinical accuracy depends on the PRNP codon 129 genotype, leaving it unclear how well established markers behave in untested conditions. METHODS: We analyzed 14-3-3, tau, p-tau levels, and the p-tau/tau ratio in a population sample collected from Polish hospitals including nondementia, dementia, and definite sCJD cases and validated their parameters according to previously established cutoffs. Additionally, the correlation between biomarkers and disease duration as well as the influence of the PRNP129 polymorphism are reported. RESULTS: The tau levels and p-tau/tau ratios differed considerably between sCJD and clinically characterized non-CJD cases (p < 0.001). p-tau was only elevated in sCJD when compared to cases without dementia (p < 0.05). Tau and the p-tau/tau ratio showed a sensitivity of 95 and 100%, respectively, in detecting sCJD cases. A negative correlation between tau levels and disease duration, but not the timing of lumbar puncture was observed. CONCLUSION: The present findings confirmed the value of the p-tau/tau ratio as a robust sCJD biomarker and suggest a role for tau as prognostic marker.

Evaluation of α-synuclein as a novel cerebrospinal fluid biomarker in different forms of prion diseases.

INTRODUCTION: Accurate diagnosis of prion diseases and discrimination from alternative dementias gain importance in the clinical routine, but partial overlap in cerebrospinal fluid (CSF) biomarkers impedes absolute discrimination in the differential diagnostic context. METHODS: We established the clinical parameters for prion disease diagnosis for the quantification of CSF α-synuclein in patients with sporadic (n = 234) and genetic (n = 56) prion diseases, in cases with cognitive impairment/dementia or neurodegenerative disease (n = 278), and in the neurologic control group (n = 111). RESULTS: An optimal cutoff value of 680 pg/mL α-synuclein results in 94% sensitivity and 96% specificity when diagnosing sporadic Creutzfeldt-Jakob disease (CJD). Genetic CJD cases showed increased CSF α-synuclein values. No increased α-synuclein levels were detected in non-CJD cases with rapid progression course. DISCUSSION: Detection of α-synuclein in the CSF of patients with suspected CJD is a valuable diagnostic test reaching almost full discrimination from non-prion disease cases. These data highlight the utility of CSF α-synuclein quantification in front of classical CSF biomarkers in clinical routine.

Mitochondrial DNA differentiates Alzheimer's disease from Creutzfeldt-Jakob disease.

INTRODUCTION: Low content of cell-free mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) is a biomarker of early stage Alzheimer's disease (AD), but whether mtDNA is altered in a rapid neurodegenerative dementia such as Creutzfeldt-Jakob disease is unknown. METHODS: CSF mtDNA was measured using digital polymerase chain reaction (dPCR) in two independent cohorts comprising a total of 112 patients diagnosed with sporadic Creutzfeldt-Jakob disease (sCJD), probable AD, or non-Alzheimer's type dementia. RESULTS: Patients with AD exhibit low mtDNA content in CSF compared with patients diagnosed with sCJD or with non-Alzheimer's type dementias. The CSF concentration of mtDNA does not correlate with Aß, t-tau, p-tau, and 14-3-3 protein levels in CSF. DISCUSSION: Low-CSF mtDNA is not a consequence of brain damage and allows the differential diagnosis of AD from sCJD and other dementias. These results support the hypothesis that mtDNA in CSF is a pathophysiological biomarker of AD.

High variability of clinical symptoms in a Polish family with a novel THAP1 mutation.

BACKGROUND: Mutations in the THAP1 gene are associated with a broad spectrum of dystonia including focal and generalized forms. Missense, nonsense and frameshift mutations, including small insertions/deletions within the THAP1 gene, have been reported and majority of them cause autosomal dominant disease with limited penetrance of approximately 60%. Here, we describe a novel THAP1 mutation. MATERIALS AND METHODS: Blood samples were collected from consenting family members for extraction of genomic DNA. As controls, we analyzed 150 individuals without neurological disorders. THAP1 coding sequences were amplified with PCR and sequenced. RESULTS: We describe a Polish family with a novel heterozygous substitution: c.167A>G (p.Glu56Gly) in THAP1 exon 2. This is the largest reported family with the mutation in THAP1 exon 2. The mutation was found in four of five genetically studied family members, including two clinically affected male individuals and two asymptomatic carriers (male and female). Data on one deceased male symptomatic subject were available and two assumed carriers were identified. The substitution was not present in any of the analyzed healthy controls. The high variability of phenotype included age of onset, localization of the initial symptom as well as the rate and degree of generalization. CONCLUSIONS: Our findings strongly suggest the role of other genetic factors or environmental triggers in the pathogenesis of dystonia related to mutations in THAP1 gene.

Selection of reference genes for gene expression studies in astrocytomas.

This study was aimed to test a panel of six housekeeping genes (GAPDH, HPRT1, POLR2A, RPLP0, ACTB, and H3F) so as to identify and validate the most suitable reference genes for expression studies in astrocytomas. GAPDH was the most stable and HPRT1 was the least stable reference gene. The effect of reference gene selection on quantitative real-time polymerase chain reaction data interpretation was demonstrated, normalizing the expression data of a selected gene of interest. Thus, GAPDH may be recommended for data normalization in gene expression studies in astrocytomas. Nevertheless, a preliminary validation of reference gene stability is required prior to every study.

Identification of novel risk loci and causal insights for sporadic Creutzfeldt-Jakob disease: a genome-wide association study.

BACKGROUND: Human prion diseases are rare and usually rapidly fatal neurodegenerative disorders, the most common being sporadic Creutzfeldt-Jakob disease (sCJD). Variants in the PRNP gene that encodes prion protein are strong risk factors for sCJD but, although the condition has similar heritability to other neurodegenerative disorders, no other genetic risk loci have been confirmed. We aimed to discover new genetic risk factors for sCJD, and their causal mechanisms. METHODS: We did a genome-wide association study of sCJD in European ancestry populations (patients diagnosed with probable or definite sCJD identified at national CJD referral centres) with a two-stage study design using genotyping arrays and exome sequencing. Conditional, transcriptional, and histological analyses of implicated genes and proteins in brain tissues, and tests of the effects of risk variants on clinical phenotypes, were done using deep longitudinal clinical cohort data. Control data from healthy individuals were obtained from publicly available datasets matched for country. FINDINGS: Samples from 5208 cases were obtained between 1990 and 2014. We found 41 genome-wide significant single nucleotide polymorphisms (SNPs) and independently replicated findings at three loci associated with sCJD risk; within PRNP (rs1799990; additive model odds ratio [OR] 1·23 [95% CI 1·17-1·30], p=2·68 × 10-15; heterozygous model p=1·01 × 10-135), STX6 (rs3747957; OR 1·16 [1·10-1·22], p=9·74 × 10-9), and GAL3ST1 (rs2267161; OR 1·18 [1·12-1·25], p=8·60 × 10-10). Follow-up analyses showed that associations at PRNP and GAL3ST1 are likely to be caused by common variants that alter the protein sequence, whereas risk variants in STX6 are associated with increased expression of the major transcripts in disease-relevant brain regions. INTERPRETATION: We present, to our knowledge, the first evidence of statistically robust genetic associations in sporadic human prion disease that implicate intracellular trafficking and sphingolipid metabolism as molecular causal mechanisms. Risk SNPs in STX6 are shared with progressive supranuclear palsy, a neurodegenerative disease associated with misfolding of protein tau, indicating that sCJD might share the same causal mechanisms as prion-like disorders. FUNDING: Medical Research Council and the UK National Institute of Health Research in part through the Biomedical Research Centre at University College London Hospitals National Health Service Foundation Trust.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors.

BACKGROUND: Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. METHODS: Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. RESULTS: In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. CONCLUSION: Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP.

Cerebrospinal fluid neurofilament light in suspected sporadic Creutzfeldt-Jakob disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease. It is invariably fatal and displays a short clinical disease stage. The key event in sCJD is the propagation of a beta-sheet rich conformer of the physiological PrPC protein, known as PrPSc. Neuropathological disease characteristics include gliosis, neuronal loss and spongiform degeneration; disease clinical manifestations refer to mental and visual disabilities, cognitive impairment, gait or limb ataxia, myoclonus and mutism. Definite sCJD diagnosis requires post-mortem brain material histopathological examination. However, highly certain pre-mortem differential diagnosis is desired to exclude other treatable disorders and to reduce disease transmission risks. Detection and/or quantification of cerebrospinal fluid (CSF) biomarkers reflecting neuronal damage and PrPC misfolding in the diseased brain significantly enhance pre-mortem diagnosis. Previously established and newly identified biomarkers are used towards this direction. Increased CSF Neurofilament light chain (NFL) concentrations have been reported in several neurological disorders, including prion diseases. In the present study, we analyzed CSF NFL levels in two independent patient cohorts, consisting of highly suspected sCJD cases that were further classified as sCJD or non-CJD according to established diagnostic criteria. CSF NFL concentrations were increased in sCJD compared to non-CJD cases in both cohorts (area under the curve (with 95% confidence interval) equal to 0.89 (0.82 to 0.97) and 0.86 (0.77 to 0.96), respectively. CSF NFL was associated neither to age nor to sex but correlated with total-tau concentrations in both cohorts. Overall, our data provide independent validation of CSF NFL utility in sCJD differential diagnosis.

Analysis of APBB2 gene polymorphisms in sporadic Alzheimer's disease.

The accumulation of beta-amyloid (Abeta) in the brain plays a central role in the pathogenesis of Alzheimer's disease (AD). The processing of Abeta precursor protein to Abeta is modulated by binding proteins including APBB2 [amyloid beta precursor protein-binding family B member 2, FE65-like, FE65L1]. We investigated two intronic SNPs within the APBB2 gene: rs13133980 and hCV1558625 (rs17443013), among Polish AD patients and healthy controls (n=213, 171). The frequencies of rs13133980 alleles and genotypes did not differ between cases and controls, irrespective of age of onset or APOE epsilon4 carrier status. The hCV1558625 G allele was over-represented in patients with onset under age 70 compared to controls in the same age range (57% vs. 43%, p=0.03). The association between the hCV1558625 G allele and susceptibility for AD at relatively young ages needs to be confirmed in other samples.

[Cerebrospinal fluid markers of prion diseases]. / Markery chorób wywolanych przez priony w plynie mózgowo-rdzeniowym.

There are no non-invasive tests allowing for definite premortem diagnosis of Creutzfeldt-Jakob disease (CJD). The paper presents the current knowledge about laboratory tests supporting CJD diagnosis. 14-3-3 protein in the cerebrospinal fluid is the only biochemical marker included in the diagnostic criteria for CJD approved by WHO. However, the test for 14-3-3 protein is useful only when considered in an appropriate clinical context, together with other diagnostic criteria. In the paper we also present current research in the use of other proteins detected in the cerebrospinal fluid (tau, S100b, neuron-specific enolase, transthyretin, and beta-amyloid1-42 as potential diagnostic markers. We discuss advantages and drawbacks of these markers.

Validation of α-Synuclein as a CSF Biomarker for Sporadic Creutzfeldt-Jakob Disease.

The analysis of cerebrospinal fluid (CSF) biomarkers gains importance in the differential diagnosis of prion diseases. However, no single diagnostic tool or combination of them can unequivocally confirm prion disease diagnosis. Electrochemiluminescence (ECL)-based immunoassays have demonstrated to achieve high diagnostic accuracy in a variety of sample types due to their high sensitivity and dynamic range. Quantification of CSF α-synuclein (a-syn) by an in-house ECL-based ELISA assay has been recently reported as an excellent approach for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), the most prevalent form of human prion disease. In the present study, we validated a commercially available ECL-based a-syn ELISA platform as a diagnostic test for correct classification of sCJD cases. CSF a-syn was analysed in 203 sCJD cases with definite diagnosis and in 445 non-CJD cases. We investigated reproducibility and stability of CSF a-syn and made recommendations for its analysis in the sCJD diagnostic workup. A sensitivity of 98% and a specificity of 97% were achieved when using an optimal cut-off of 820 pg/mL a-syn. Moreover, we were able to show a negative correlation between a-syn levels and disease duration suggesting that CSF a-syn may be a good prognostic marker for sCJD patients. The present study validates the use of a-syn as a CSF biomarker of sCJD and establishes the clinical and pre-analytical parameters for its use in differential diagnosis in clinical routine. Additionally, the current test presents some advantages compared to other diagnostic approaches: it is fast, economic, requires minimal amount of CSF and a-syn levels are stable along disease progression.

Diverse molecular pattern in a bihemispheric glioblastoma (butterfly glioma) in a 16-year-old boy.

Glioblastoma multiforme (GBM), the most common malignant brain tumor of adults, is relatively rare in children. In a GBM affecting a 16-year-old boy, the tumor spread across the corpus callosum (butterfly glioma). This type of bilateral hemispheric growth has previously been thought to result from spread along the white matter tracts. Two samples obtained from opposite sides of the same tumor were analyzed comprehensively for loss of heterozygosity (LOH) and microsatellite instability (MSI). Amplification of EGFR and MDM2 was studied by means of multiplex polymerase chain reaction. Exons 5, 6, 7, and 8 of TP53 were screened for mutations by sequencing. In neither specimen were molecular alterations found in the EGFR, MDM2, or TP53 genes. The specimen obtained from the right hemisphere exhibited a high level of MSI and LOH in chromosome arms 5q, 9p, and 13q. The specimen from the left hemisphere exhibited LOH in chromosome arms 3p, 5q, 9p, 9q, 10p, 10q, and 13q. Here we propose four plausible hypothetical scenarios underlying the tumorigenesis of this GBM.

Regulatory sequences of the PRNP gene influence susceptibility to sporadic Creutzfeldt-Jakob disease.

The prion diseases are fatal neurodegenerative disorders that afflict both humans and animals. They comprise kuru, Creutzfeldt-Jakob disease (CJD), Gerstmman-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). Both GSS, FFI and approximately 10% of CJD cases are genetically linked disorders, whereas 90% of CJD cases are not associated with mutations in the PRNP coding region, therefore other factors must be involved in pathogenesis of these forms of CJD. There is strong evidence that in transgenic mice the level of PrP gene expression influences the initiation and progression of the prion diseases. Moreover, in in vitro experiments demonstrated that mutations in the regulatory region of PRNP gene altered gene expression, therefore it may be expected that PrP expression level influences the susceptibility to CJD. In order to investigate whether single nucleotide polymorphisms within regulatory region of PRNP may modulate genetic susceptibility to sporadic CJD we examined an association of the C/G polymorphism at position -101 with the sCJD. In our study -101G polymorphism is over-represented among sCJD PRNP codon 129M/V cases compared with the control group. Our data suggest that polymorphism at position -101 in the regulatory region of PRNP may be a risk factor for sCJD among codon 129 heterozygotes.

KCTD11 expression in medulloblastoma is lower than in adult cerebellum and higher than in neural stem cells.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood, and the most frequent associated genetic alteration is loss of heterozygosity on chromosome region 7p13. Two genes mapping to this region, KCTD11 (alias REN) and HIC1, have been proposed as involved in MB pathogenesis. We used real-time polymerase chain reaction in 20 tissue samples of primary MB to examine the transcriptional level of the two genes, with reference to two types of controls: adult cerebellum and fetal neural stem cells. A significant reduction of KCTD11 expression relative to adult normal cerebellum was detected in 14 of 20 (70%) of MB samples. Neural stem cells had even lower levels of KCTD11 expression than did MB. HIC1 gene expression was low ( approximately 100 times lower than KCTD11 expression) in MB, and low also in both adult cerebellum and neural stem cells. Hypermethylation of the 5'UTR or the central region of HIC1 (or both) was detected in a significant number of MB samples, as well as in cerebellum and neural stem cells. Our data suggest that KCTD11 may play an important role in MB tumorigenesis, but do not support the role of HIC1 in this tumor development. We argue that recognition of the gene or genes important in MB tumorigenesis depends in part on defining an appropriate control.

Regulation of PrPC expression: nerve growth factor (NGF) activates the prion gene promoter through the MEK1 pathway in PC12 cells.

A high expression of PrP(C) in cells is one factor that increases the risk of conversion to the misfolded, disease-associated form (PrP(Sc)) characteristic of transmissible spongiform encephalopathies. Thus, developing a method to control the level of PrP(C) expression in cells could be one way to delay or prevent the onset of clinical signs of these diseases. In this study the mechanisms controlling the expression of the Prnp gene in PC12 cells and in rat brain were examined. We observed a slight activation of a cloned fragment of the human PRNP gene promoter using the luciferase reporter system in PC12 cells stimulated with nerve growth factor (NGF). The activating effect of NGF was enhanced by mitogen-activated protein kinase (MEK1) and suppressed by myristylated serine/threonine kinase (myrAKT). These results suggest that MEK1 is a positive activator of the PRNP promoter that inhibits the AKT pathway. Independent experiments suggested that high expression of PrP(C) in the brain depends on the rate of translation and/or the efficiency of PrP(C) stabilization. We also investigated the epigenic status of the Prnp promoter. We observed no increase of PrP(C) or Prnp mRNA levels in PC12 cells after treatment with the DNA-demethylating agent. The Prnp promoter did not display methylation either in NGF-treated and untreated PC12 cells, or in the rat brain. These results improve the understanding of the regulation of the Prnp gene promoter, a DNA regulatory element controlling the expression of PrP(C), a protein involved in several neurological diseases.

Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/µL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.

CYP46: a risk factor for Alzheimer's disease or a coincidence?

Excess cholesterol is removed from the brain via hydroxylation mediated by cholesterol 24S-hydroxylase (CYP46). Although serum and cerebrospinal fluid (CSF) concentrations of 24S-hydroxycholesterol are altered during the progress of Alzheimer's disease, studies carried out to date in different populations on the association of CYP46 gene polymorphisms and risk of AD have been inconclusive. In this report, we analyzed CYP46 polymorphisms in 215 Polish AD cases and 173 healthy individuals. A fragment of CYP46 intron 2 was amplified by PCR reaction and sequenced. We discovered a new single nucleotide substitution in CYP46 intron 2, but found no difference in particular genotype or allele frequencies between AD patients and controls. However, the GG genotype of the known rs754203 polymorphic site might be a risk factor for AD, especially in APOE varepsilon4 carriers. Interestingly, in AD patients the rs754203 G allele was more frequent in males than in females. However, considering the extreme divergence of results obtained by different authors, a clear connection between the CYP46 gene and AD is questionable.

[Analyses of 14-3-3 protein in the cerebrospinal fluid in Creutzfeldt-Jakob disease. Preliminary report]. / Badania bialka 14-3-3 w plynie mózgowo-rdzeniowym w chorobie Creutzfeldta-Jakoba. Doniesienie wstepne.

BACKGROUND AND PURPOSE: Definitive diagnosis of Creutzfeldt-Jakob disease (CJD) is possible only after neuropathological confirmation; there are no non-invasive tests which allow to definitely diagnose CJD pre mortem. The only one biochemical marker included in the diagnostic criteria for CJD approved by WHO is an elevated level of 14-3-3 protein in the cerebrospinal fluid (CSF). The aim of the study was to introduce 14-3-3 analysis for a routine diagnostics of CJD in Poland and to optimise the Western blotting technique used in the Reference Centre of Prion Diseases in Lodz. MATERIAL AND METHODS: We analysed 33 samples of CSF: 17 from patients suspected of CJD (2 definitive, 2 probable, 4 possible, 11 others), and 16 controls (from patients with other neurological diseases). CSF samples were analysed using the Western blotting method and commercially available antibodies (Santa Cruz). We used 2 methods of CSF preparation: with and without lyophilization, and 2 different anti-14-3-3 antibodies: rabbit polyclonal and mouse monoclonal antibodies. RESULTS: We found 14-3-3 protein in both definitive sCJD cases and in one probable case. Among other samples, we obtained a positive result only in one patient with alcohol abuse history and in samples of CSF contaminated with erythrocytes. CONCLUSIONS: The Western blotting test for 14-3-3 protein in CSF is useful for diagnosis of CJD, but only when considered in an appropriate clinical context, together with other diagnostic criteria. Contamination of CSF with erythrocytes can cause false positive results. Analysis of a non-lyophilised CSF and detection of 14-3-3 protein using the mouse monoclonal antibody gave the most reliable results.