Splice site SNPs of phospholipase PLCXD3 are significantly associated with variant and sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Variant Creutzfeldt-Jakob disease is an infectious, neurodegenerative, protein-misfolding disease, of the prion disease family, originally acquired through ingestion of meat products contaminated with bovine spongiform encephalopathy (BSE). Public health concern was increased by the discovery of human-to-human transmission via blood transfusion. This study has verified a novel genetic marker linked to disease risk. METHODS: SNP imputation and association testing indicated those genes that had significant linkage to disease risk and one gene was investigated further with Sanger resequencing. Results from variant Creutzfeldt-Jakob disease were compared with those from sporadic (idiopathic) Creutzfeldt-Jakob disease and published controls. RESULTS: The most significant disease risk, in addition to the prion protein gene, was for the phosphatidylinositol-specific phospholipase C, X domain containing 3 (PLCXD3) gene. Sanger resequencing of CJD patients across a region of PLCXD3 with known variants confirmed three SNPs associated with variant and sporadic CJD. CONCLUSIONS: These data provide the first highly significant confirmation of SNP allele frequencies for a novel CJD candidate gene providing new avenues for investigating these neurodegenerative prion diseases. The phospholipase PLCXD3 is primarily expressed in the brain and is associated with lipid catabolism and signal transduction.

Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt-Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking-induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD. METHODS: An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31-84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43-84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39-82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36-87 years; mean age, 63.5 ± 11.6 years). RESULTS: The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively. INTERPRETATION: This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests.

Inherited prion disease with 4-octapeptide repeat insertion: disease requires the interaction of multiple genetic risk factors.

Genetic factors are implicated in the aetiology of sporadic late-onset neurodegenerative diseases. Whether these genetic variants are predominantly common or rare, and how multiple genetic factors interact with each other to cause disease is poorly understood. Inherited prion diseases are highly heterogeneous and may be clinically mistaken for sporadic Creutzfeldt-Jakob disease because of a negative family history. Here we report our investigation of patients from the UK with four extra octapeptide repeats, which suggest that the risk of clinical disease is increased by a combination of the mutation and a susceptibility haplotype on the wild-type chromosome. The predominant clinical syndrome is a progressive cortical dementia with pyramidal signs, myoclonus and cerebellar abnormalities that closely resemble sporadic Creutzfeldt-Jakob disease. Autopsy shows perpendicular deposits of prion protein in the molecular layer of the cerebellum. Identity testing, PRNP microsatellite haplotyping and genealogical work confirm no cryptic close family relationships and suggests multiple progenitor disease haplotypes. All patients were homozygous for methionine at polymorphic codon 129. In addition, at a single nucleotide polymorphism upstream of PRNP thought to confer susceptibility to sporadic Creutzfeldt-Jakob disease (rs1029273), all patients were homozygous for the risk allele (combined P=5.9×10(-5)). The haplotype identified may also be a risk factor in other partially penetrant inherited prion diseases although it does not modify age of onset. Blood expression of PRNP in healthy individuals was modestly higher in carriers of the risk haplotype. These findings may provide a precedent for understanding apparently sporadic neurodegenerative diseases caused by rare high-risk mutations.

Validation of diagnostic criteria for variant Creutzfeldt-Jakob disease.

OBJECTIVE: Variant Creutzfeldt-Jakob disease (vCJD), a novel form of human prion disease, was recognized in 1996. The disease affected a younger cohort than sporadic CJD, and the early clinical course was dominated by psychiatric and sensory symptoms. In an attempt to aid diagnosis and establish standardization between surveillance networks, diagnostic criteria were established. These were devised from the features of a small number of cases and modified in 2000 as the clinical phenotype was established. Since then, only minor changes have been introduced; revalidation of the criteria in the current format is overdue. METHODS: Included in this study are autopsy/cerebral biopsy-proven cases of vCJD referred to the National CJD Surveillance Unit (NCJDSU) between 1995 and 2004 and suspect cases in which an alternative diagnosis was identified following autopsy/cerebral biopsy. RESULTS: Over the 10-year period, 106 definite cases of vCJD and 45 pathologically confirmed "noncases" were identified from the archives of the NCJDSU. The median age at onset of the cases was significantly younger than that of the noncases (27 years [range, 12-74 years] vs 43 years [range, 10-64 years]), and the median duration of illness was significantly shorter (14 months [range, 6-39 months] vs 22 months [range, 2-139 months]). The most commonly identified core clinical feature in cases was dementia; persistent painful sensory symptoms were the least frequent. Eighty-eight of 106 (83%) vCJD cases were retrospectively classified as probable in life, 6 cases were classified as possible. Most cases were classified as probable on the basis of core clinical features and brain magnetic resonance imaging. To date, the diagnostic criteria remain 100% specific, with no autopsy/cerebral biopsy-proven noncases classified as probable in life. INTERPRETATION: This study confirms that the diagnostic criteria for vCJD are sensitive and specific and provide a useful standard framework for case classification in a surveillance setting.

Cathepsin D (C224T) polymorphism in sporadic and genetic Creutzfeldt-Jakob disease.

Accumulation of cathepsin D immunoreactive lysosomes correlates with tissue pathology in sporadic Creutzfeldt-Jakob disease (CJD) brains. The C-to-T transition within exon 2 of the cathepsin D (CTSD) gene is associated with altered enzymatic activity. Possession of the TT genotype is a risk factor for variant CJD. To verify the association between the CTSD position 224T allele and the risk for and survival in sporadic and genetic CJD, we genotyped 540 sporadic, 101 genetic CJD, and 723 control individuals. Genotype data and duration of illness were compared using multiple logistic regression and Kruskal-Wallis test. Multivariate survival analysis was performed using Cox's regression model. The distribution of CTSD position 224 alleles was approximately the same in all groups. We observed a trend for shorter survival in sporadic CJD patients harboring the T allele at position 224 of the CTSD gene in particular in sporadic CJD patients with the prion protein gene position 129 MM genotype. We conclude that the CTSD position 224 polymorphism alone is not a significant risk or disease-modifying factor in sporadic or genetic CJD.

PRNP variation in UK sporadic and variant Creutzfeldt Jakob disease highlights genetic risk factors and a novel non-synonymous polymorphism.

BACKGROUND: Genetic analysis of the human prion protein gene (PRNP) in suspect cases of Creutzfeldt-Jakob disease (CJD) is necessary for accurate diagnosis and case classification. Previous publications on the genetic variation at the PRNP locus have highlighted the presence of numerous polymorphisms, in addition to the well recognised one at codon 129, with significant variability between geographically distinct populations. It is therefore of interest to consider their influence on susceptibility or the clinico-pathological disease phenotype. This study aimed to characterise the frequency and effect of PRNP open reading frame polymorphisms other than codon 129 in both disease and control samples sourced from the United Kingdom population. METHODS: DNA was extracted from blood samples and genetic data obtained by full sequence analysis of the prion protein gene or by restriction fragment length polymorphism analysis using restriction enzymes specific to the gene polymorphism under investigation. RESULTS: 147 of 166 confirmed cases of variant CJD (vCJD) in the UK have had PRNP codon 129 genotyping and all are methionine homozygous at codon 129; 118 have had full PRNP gene sequencing. Of the latter, 5 cases have shown other polymorphic loci: at codon 219 (2, 1.69%), at codon 202 (2, 1.69%), and a 24 bp deletion in the octapeptide repeat region (1, 0.85%). E219K and D202D were not found in sporadic CJD (sCJD) cases and therefore may represent genetic risk factors for vCJD.Genetic analysis of 309 confirmed UK sCJD patients showed codon 129 genotype frequencies of MM: 59.5% (n = 184), MV: 21.4% (n = 66), and VV: 19.1% (n = 59). Thirteen (4.2%) had the A117A polymorphism, one of which also had the P68P polymorphism, four (1.3%) had a 24 bp deletion, and a single patient had a novel missense variation at codon 167. As the phenotype of this latter case is similar to sCJD and in the absence of a family history of CJD, it is unknown whether this is a form of genetic CJD, or simply a neutral polymorphism. CONCLUSIONS: This analysis of PRNP genetic variation in UK CJD patients is the first to show a comprehensive comparison with healthy individuals (n = 970) from the same population, who were genotyped for the three most common variations (codon 129, codon 117, and 24 bp deletion). These latter two genetic variations were equally frequent in UK sCJD or vCJD cases and a normal (healthy blood donor) UK population.

Risk factors for sporadic Creutzfeldt-Jakob disease.

OBJECTIVE: Although surgical transmission of Creutzfeldt-Jakob disease (CJD) has been demonstrated, these iatrogenic cases account for only a small proportion of all CJD cases. The majority are sporadic CJD (sCJD) cases of unknown cause. This study investigated whether some cases classified as sCJD might have an unrecognized iatrogenic basis through surgical or other medical procedures METHODS: This study compared medical risk factors from 431 sCJD cases referred 1998 to 2006 with 454 population control subjects. Possible geographic and temporal links between neurological and gynecological operations in 857 sCJD cases referred from 1990 to 2006 were investigated RESULTS: A reported history of ever having undergone surgery was associated with increased risk for sCJD (odds ratio, 2.0; 95% confidence interval, 1.3-2.1; p = 0.003). Increased risk was not associated with surgical categories chosen a priori but was confined to the residual category "other surgery," in which the increase in risk appeared most marked for three subcategories: skin stitches, nose/throat operations, and removal of growths/cysts/moles. No convincing evidence was found of links (same hospital, within 2 years) between cases undergoing neurosurgery or gynecological surgery INTERPRETATION: It is unlikely that a high proportion of UK sCJD cases are the result of transmission during surgery, but we cannot exclude the possibility that such transmission occurs occasionally. A study based on accurate surgical histories obtained from medical records is required to determine whether the increased risk associated with reported surgical history reflects a causal association or recall bias.

Cathepsin D SNP associated with increased risk of variant Creutzfeldt-Jakob disease.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) originally resulted from the consumption of foodstuffs contaminated by bovine spongiform encephalopathy (BSE) material, with 163 confirmed cases in the UK to date. Many thousands are likely to have been exposed to dietary infection and so it is important (for surveillance, epidemic modelling, public health and understanding pathogenesis) to identify genetic factors that may affect individual susceptibility to infection. This study looked at a polymorphism in the cathepsin D gene (refSNP ID: rs17571) previously examined in Alzheimer's disease (AD). METHODS: Blood samples taken from 110 vCJD patients were tested for the C-T base change, and genotype data were compared with published frequencies for a control population using multiple logistic regression. RESULTS: There was a significant excess of the cathepsin D polymorphism TT genotype in the vCJD cohort compared to controls. The TT genotype was found to have a 9.75 fold increase in risk of vCJD compared to the CT genotype and a 10.92 fold increase compared to the CC genotype. CONCLUSION: This mutation event has been observed to alter the protease activity of the cathepsin D protein and has been linked to an increase in amyloid beta plaque formation in AD. vCJD neuropathology is characterised by the presence of amyloid plaques, formed from the prion protein, and therefore alterations in the amyloid processing activity of cathepsin D may affect the neuropathogenesis of this disease.

No evidence for association between tau gene haplotypic variants and susceptibility to Creutzfeldt-Jakob disease.

BACKGROUND: A polymorphism at codon 129 of the prion protein gene (PRNP) is the only well-known genetic risk factor for Creutzfeldt-Jakob disease (CJD). However, there is increasing evidence that other loci outside the PRNP open reading frame might play a role in CJD aetiology as well. METHODS: We studied tau protein gene (MAPT) haplotypic variations in a population of sporadic and variant CJD patients. We tested 6 MAPT haplotype tagging SNPs (htSNPs) in a Dutch population-based sample of sporadic CJD (sCJD) patients and a cognitively normal control group of similar age distribution. We genotyped the same polymorphisms in two other sample groups of sCJD cases from Italy and the UK. In addition, we compared MAPT haplotypes between sCJD and variant CJD (vCJD) patients. RESULTS: Single locus and haplotype analyses did not detect any significant difference between sCJD cases and controls. When we compared MAPT haplotypes between sCJD and variant CJD (vCJD) patients, we found that two of them were represented differently (H1f: 8% in sCJD versus 2% in vCJD; H1j:1% in sCJD versus 7% in vCJD). However, these two haplotypes were rare in both groups of patients, and taking the small sample sizes into account, we cannot exclude that the differences are due to chance. None of the p-values remained statistically significant after applying a multiple testing correction. CONCLUSION: Our study shows no evidence for an association between MAPT gene variations and sCJD, and some weak evidence for an association to vCJD.

Mutations of the prion protein gene phenotypic spectrum.

Prion diseases are inherited in 5-15 % of cases. They are classified according to changes in the prion protein gene ( PRNP) or conventionally according to phenotype as Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), or familial Creutzfeldt-Jakob disease (fCJD). Point mutations and insertions within PRNP form the genetic background. We report the results of a systematic analysis of over 500 case reports of patients with PRNP abnormalities. We compare clinical, neuropathological and molecular data in five groups, namely GSS, FFI, fCJD, base pair insertion (BPI), and all cases collectively. Clinical presentation overlaps between mutations, but some have characteristic features (e. g. P105L, D178N-129M, T183A). Some mutations, especially in the lack of sufficient family history, in earlier phases tend to resemble other neurodegenerative disorders like multiple system atrophy, corticobasal degeneration or familial diseases such as late-onset spinocerebellar ataxia, spastic paraparesis, frontotemporal dementia, or Alzheimer's disease. The codon 129 polymorphism has a phenotypic influence in inherited prion diseases, as in non-genetic forms, but additional factors might be considered as background for phenotypic variability.

14-3-3 in the cerebrospinal fluid of patients with variant and sporadic Creutzfeldt-Jakob disease measured using capture assay able to detect low levels of 14-3-3 protein.

A protein capture assay was used to measure 14-3-3 (-isoform) in the cerebrospinal fluid (CSF) of patients with either variant or sporadic Creutzfeldt-Jakob disease (CJD). The results were compared with those obtained using Western blotting. Elevated levels of 14-3-3 were found in 58% of variant CJD (vCJD) patients and 82% of sporadic CJD (spCJD) patients using the protein capture assay. Using a Western blotting technique, the presence of CSF 14-3-3 was detected in 58% of vCJD patients and in 89% of spCJD patients. When the results from the protein capture assay and the Western blot were combined, 14-3-3 was detected in 77% of vCJD patients and in 91% of spCJD patients. These results suggest that although analysis of CSF 14-3-3 is not as useful in vCJD as it is in the sporadic form of the disease, a combination of these two techniques results in increased sensitivity of 14-3-3 for the diagnosis of vCJD.

A genome wide association study links glutamate receptor pathway to sporadic Creutzfeldt-Jakob disease risk.

We performed a genome-wide association (GWA) study in 434 sporadic Creutzfeldt-Jakob disease (sCJD) patients and 1939 controls from the United Kingdom, Germany and The Netherlands. The findings were replicated in an independent sample of 1109 sCJD and 2264 controls provided by a multinational consortium. From the initial GWA analysis we selected 23 SNPs for further genotyping in 1109 sCJD cases from seven different countries. Five SNPs were significantly associated with sCJD after correction for multiple testing. Subsequently these five SNPs were genotyped in 2264 controls. The pooled analysis, including 1543 sCJD cases and 4203 controls, yielded two genome wide significant results: rs6107516 (p-value=7.62x10-9) a variant tagging the prion protein gene (PRNP); and rs6951643 (p-value=1.66x10-8) tagging the Glutamate Receptor Metabotropic 8 gene (GRM8). Next we analysed the data stratifying by country of origin combining samples from the pooled analysis with genotypes from the 1000 Genomes Project and imputed genotypes from the Rotterdam Study (Total n=12967). The meta-analysis of the results showed that rs6107516 (p-value=3.00x10-8) and rs6951643 (p-value=3.91x10-5) remained as the two most significantly associated SNPs. Rs6951643 is located in an intronic region of GRM8, a gene that was additionally tagged by a cluster of 12 SNPs within our top100 ranked results. GRM8 encodes for mGluR8, a protein which belongs to the metabotropic glutamate receptor family, recently shown to be involved in the transduction of cellular signals triggered by the prion protein. Pathway enrichment analyses performed with both Ingenuity Pathway Analysis and ALIGATOR postulates glutamate receptor signalling as one of the main pathways associated with sCJD. In summary, we have detected GRM8 as a novel, non-PRNP, genome-wide significant marker associated with heightened disease risk, providing additional evidence supporting a role of glutamate receptors in sCJD pathogenesis.

Genome-wide study links MTMR7 gene to variant Creutzfeldt-Jakob risk.

The aim of our study was to discover genomic variations related to variant Creutzfeldt-Jakob disease (vCJD) susceptibility. A genome-wide association analysis with most vCJD samples available in the world was performed. A series of 93 vCJD UK patients and 1504 UK controls were included in the discovery stage. Our best findings were replicated in an independent population of 22 UK and 20 French vCJD cases. Post hoc analysis to assess our main results included 5711 French controls, 445 Dutch controls, and 446 sporadic Creutzfeldt-Jakob disease (CJD) cases. We found 2 genome wide significant variants tagging PRNP: rs6107516 (p = 2.6 × 10(-18)) and rs2065706 (p = 8.8 × 10(-14)). Two other single nucleotide polymorphisms (SNPs) (rs4921542 and rs7565981) were successfully replicated in independent samples and reached genome-wide significance after pooling discovery and replication populations. Rs4921542 (p = 1.6 × 10(-8)) is an intronic variant in the myotubularin related protein 7 gene (MTMR7), which is specifically expressed in the central nervous system (CNS) and dephosphorylates phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate. Rs7565981 (p = 4.2 × 10(-8)) is in an intergenic region upstream of the neuronal PAS (per-ARNT-sim) domain-containing protein 2 gene (NPAS2), a regulatory gene belonging to a family of transcription factors that has been implicated in memory, seasonal affective disorder, and the molecular clock in the mammalian forebrain. A proxy of rs7565981 (rs17024792; r(2) = 1.0) has been found to regulate the phospholipase C-delta-3 gene (PLCD3) in trans. This enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Our study reveals 2 new genome-wide significant markers for vCJD outside PRNP and provides evidence supporting a role of the phosphatidylinositol pathway in vCJD susceptibility.

Systematic review of therapeutic interventions in human prion disease.

BACKGROUND: The potential threat of a large outbreak of variant Creutzfeldt-Jakob disease initiated a proliferation of research into the understanding and treatment of human prion disease. However, clinical research is at an early stage with a pressing need for objective evaluation of treatments to inform the design of future studies. METHODS: We aimed to summarize existing research on outcomes of patients with prion disease, considering any published clinical study and patient series with data on disease progression. Methods were prespecified in a protocol and studies were identified from systematic searches of multiple sources. RESULTS: One randomized trial was identified. Many studies were flawed or poorly reported, and therefore interpreted cautiously. One hundred forty published patient series revealed wide ranges in disease duration for each of the prion diseases. Thirty-three studies described the use of 14 drugs, 10 which were reported in single studies of three or fewer patients and one which was reported for two individual cases. Effects of four drugs were examined in more detail, with mixed results. The only current reliable evidence is from the single randomized trial suggesting that flupirtine may slow cognitive decline. Based on published information identified by this review, survival of most treated patients is within the ranges reported in the untreated patient series. CONCLUSIONS: Thirty years of clinical investigation of patients with prion disease has resulted in little progress in either defining or evaluating potential treatments. Disease course and treatment of all patients must be evaluated within a structured framework, preferably within randomized controlled trials.

Risk factors for variant Creutzfeldt-Jakob disease: a case-control study.

OBJECTIVE: To investigate the potential risk factors for variant Creutzfeldt-Jakob disease (VCJD) in the United Kingdom. METHODS: Definite and probable vCJD cases (n = 136) were residing in Great Britain at disease onset, and were referred between May 1995 and November 2003. Control subjects (n = 922) were recruited between 2002 and 2003, from 100 randomly selected geographical clusters sampled to represent the geographical distribution of vCJD. RESULTS: Reported frequent consumption of beef and beef products thought likely to contain mechanically recovered or head meat, or both, including burgers and meat pies, was associated with increased risk for vCJD, as was reported frequent chicken consumption. Surgical operations were generally similarly reported for cases and control subjects, with the exception of a small group of minor operations, possibly attributable to underreporting in control subjects. Cases and control subjects had similar reported occupational histories and exposure to animals. INTERPRETATION: These findings are consistent with dietary exposure to contaminated beef products being the main route of infection of vCJD, but recall bias cannot be excluded. There was no convincing evidence of increased risk through medical, surgical, or occupational exposure or exposure to animals.

First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features.

OBJECTIVE: To describe the early psychiatric and neurological features of variant Creutzfeldt-Jakob disease. DESIGN: Cohort study. SETTING: National surveillance system for Creutzfeldt-Jakob disease in the United Kingdom. PARTICIPANTS: The first 100 cases of variant Creutzfeldt-Jakob disease identified in the United Kingdom. MAIN OUTCOME MEASURES: The timing and nature of early psychiatric and neurological symptoms in variant Creutzfeldt-Jakob disease. RESULTS: The early stages of variant Creutzfeldt-Jakob disease are dominated by psychiatric symptoms, but neurological symptoms precede psychiatric symptoms in 15% of cases and are present in combination with psychiatric symptoms in 22% of cases from the onset of disease. Common early psychiatric features include dysphoria, withdrawal, anxiety, insomnia, and loss of interest. No common early neurological features exist, but a significant proportion of patients do exhibit neurological symptoms within 4 months of clinical onset, including poor memory, pain, sensory symptoms, unsteadiness of gait, and dysarthria. CONCLUSIONS: Although the diagnosis of variant Creutzfeldt-Jakob disease may be impossible in the early stages of the illness, particular combinations of psychiatric and neurological features may allow early diagnosis in an appreciable proportion of patients.

Apolipoprotein E and other cerebrospinal fluid proteins differentiate ante mortem variant Creutzfeldt-Jakob disease from ante mortem sporadic Creutzfeldt-Jakob disease.

The ability to perform an ante mortem differential diagnosis of Creutzfeld-Jakob disease (CJD) is aided by several clinical and molecular tests. There is a need for molecular tests which can reliably distinguish ante mortem variant CJD (vCJD) from ante mortem sporadic CJD (spCJD). A proteomics approach employing two-dimensional protein electrophoresis is applied to the study of ante mortem CSF samples obtained in collaboration with the CJD Surveillance Unit and the National Hospital for Neurology and Neurosurgery. The sample set includes two cases of vCJD, three cases of spCJD and three neurologic controls. Preliminary data using a panel of seven molecular markers is able to distinguish vCJD from spCJD using a heuristic clustering algorithm. One of the molecular markers has been identified as apolipoprotein E which appears to be upregulated in the cerebrospiral fluid (CSF) of patients with vCJD as compared to spCJD. Analysis of ante mortem CSF may help to differentiate patients with vCJD from those patients with spCJD.