Jessica Lowe

Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay

BACKGROUND Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disorder originating from exposure to bovine-spongiform-encephalopathy-like prions. Prion infections are associated with long and clinically silent incubations. The number of asymptomatic individuals with vCJD prion infection is unknown, posing risk to others via blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. We aimed to establish the sensitivity and specificity of a blood-based assay for detection of vCJD prion infection. METHODS We developed a solid-state binding matrix to capture and concentrate disease-associated prion proteins and coupled this method to direct immunodetection of surface-bound material. Quantitative assay sensitivity was assessed with a serial dilution series of 10⁻⁷ to 10⁻¹⁰ of vCJD prion-infected brain homogenate into whole human blood, with a baseline control of normal human brain homogenate in whole blood (10⁻⁶). To establish the sensitivity and specificity of the assay for detection of endogenous vCJD, we analysed a masked panel of 190 whole blood samples from 21 patients with vCJD, 27 with sporadic CJD, 42 with other neurological diseases, and 100 normal controls. Samples were masked and numbered by individuals independent of the assay and analysis. Each sample was tested twice in independent assay runs; only samples that were reactive in both runs were scored as positive overall. FINDINGS We were able to distinguish a 10⁻¹⁰ dilution of exogenous vCJD prion-infected brain from a 10⁻⁶ dilution of normal brain (mean chemiluminescent signal, 1·3×10⁵ [SD 1·1×10⁴] for vCJD vs 9·9×10⁴ [4·5×10³] for normal brain; p<0·0001)—an assay sensitivity that was orders of magnitude higher than any previously reported. 15 samples in the masked panel were scored as positive. All 15 samples were from patients with vCJD, showing an assay sensitivity for vCJD of 71·4% (95% CI 47·8–88·7) and a specificity of 100% (95% CIs between 97·8% and 100%). INTERPRETATION These initial studies provide a prototype blood test for diagnosis of vCJD in symptomatic individuals, which could allow development of large-scale screening tests for asymptomatic vCJD prion infection. FUNDING UK Medical Research Council.

Duplication of amyloid precursor protein (APP), but not prion protein (PRNP) gene is a significant cause of early onset dementia in a large UK series.

Amyloid precursor protein gene (APP) duplications have been identified in screens of selected probands with early onset familial Alzheimers disease (FAD). A causal role for copy number variation (CNV) in the prion protein gene (PRNP) in prion dementias is not known. We aimed to determine the prevalence of copy number variation in APP and PRNP in a large referral series, test a screening method for detection of the same, and expand knowledge of clinical phenotype. We used a 3-tiered screening assay for APP and PRNP duplication (exonic real-time quantitative polymerase chain reaction [exon-qPCR], fluorescent microsatellite quantitative PCR [fm-q-PCR], and Illumina array [Illumina Inc., San Diego, CA, USA]) for analysis of a heterogeneous referral series comprising 1531 probands. Five of 1531 probands screened showed APP duplication, a similar prevalence to APP missense mutation. Real-time quantitative PCR and fluorescent microsatellite quantitative PCR were similar individually but are theoretically complementary; we used Illumina arrays as our reference assay. Two of 5 probands were from an autosomal dominant early onset Alzheimers disease (familial Alzheimers disease) pedigree. One extensive, noncontiguous duplication on chromosome 21 was consistent with an unbalanced translocation not including the Downs syndrome critical region. Seizures were prominent in the other typical APP duplications. A range of imaging, neuropsychological, cerebrospinal fluid, and pathological findings are reported that extend the known phenotype. APP but not PRNP duplication is a significant cause of early onset dementia in the UK. The recognized phenotype may be expanded to include the possibility of early seizures and apparently sporadic disease which, in part, may be due to different mutational mechanisms. The pros and cons of our screening method are discussed.

Genome-wide association study in multiple human prion diseases suggests genetic risk factors additional to PRNP

Prion diseases are fatal neurodegenerative diseases of humans and animals caused by the misfolding and aggregation of prion protein (PrP). Mammalian prion diseases are under strong genetic control but few risk factors are known aside from the PrP gene locus (PRNP). No genome-wide association study (GWAS) has been done aside from a small sample of variant Creutzfeldt-Jakob disease (CJD). We conducted GWAS of sporadic CJD (sCJD), variant CJD (vCJD), iatrogenic CJD, inherited prion disease, kuru and resistance to kuru despite attendance at mortuary feasts. After quality control, we analysed 2000 samples and 6015 control individuals (provided by the Wellcome Trust Case Control Consortium and KORA-gen) for 491032-511862 SNPs in the European study. Association studies were done in each geographical and aetiological group followed by several combined analyses. The PRNP locus was highly associated with risk in all geographical and aetiological groups. This association was driven by the known coding variation at rs1799990 (PRNP codon 129). No non-PRNP loci achieved genome-wide significance in the meta-analysis of all human prion disease. SNPs at the ZBTB38-RASA2 locus were associated with CJD in the UK (rs295301, P = 3.13 × 10(-8); OR, 0.70) but these SNPs showed no replication evidence of association in German sCJD or in Papua New Guinea-based tests. A SNP in the CHN2 gene was associated with vCJD [P = 1.5 × 10(-7); odds ratio (OR), 2.36], but not in UK sCJD (P = 0.049; OR, 1.24), in German sCJD or in PNG groups. In the overall meta-analysis of CJD, 14 SNPs were associated (P < 10(-5); two at PRNP, three at ZBTB38-RASA2, nine at nine other independent non-PRNP loci), more than would be expected by chance. None of the loci recently identified as genome-wide significant in studies of other neurodegenerative diseases showed any clear evidence of association in prion diseases. Concerning common genetic variation, it is likely that the PRNP locus contains the only strong risk factors that act universally across human prion diseases. Our data are most consistent with several other risk loci of modest overall effects which will require further genetic association studies to provide definitive evidence.

R47H TREM2 variant increases risk of typical early-onset Alzheimer's disease but not of prion or frontotemporal dementia

Rare TREM2 variants are significant risk factors for Alzheimers disease (AD).

Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years.

Cases of iatrogenic CJD still occur in the UK 30 years after administration of human pituitary-derived growth hormone ceased. Rudge et al. report a change over time in genotype profile at polymorphic codon 129 of the human prion protein gene in UK patients, distinct from that seen in other countries.

The Medical Research Council Prion Disease Rating Scale: a new outcome measure for prion disease therapeutic trials developed and validated using systematic observational studies

Progress in therapeutics for rare disorders like prion disease is impeded by the lack of validated outcome measures and a paucity of natural history data derived from prospective observational studies. The first analysis of the U.K. National Prion Monitoring Cohort involved 1337 scheduled clinical assessments and 479 telephone assessments in 437 participants over 373 patient-years of follow-up. Scale development has included semi-quantitative and qualitative carer interviews, item response modelling (Rasch analysis), inter-rater reliability testing, construct analysis and correlation with several existing scales. The proposed 20-point Medical Research Council prion disease rating scale assesses domains of cognitive function, speech, mobility, personal care/feeding and continence, according to their relative importance documented by carer interviews. It is quick and simple to administer, and has been validated for use by doctors and nurses and for use over the telephone, allowing for frequent assessments that capture the rapid change typical of these diseases. The Medical Research Council Scale correlates highly with widely used cognitive and single item scales, but has substantial advantages over these including minimal floor effects. Three clear patterns of decline were observed using the scale: fast linear decline, slow linear decline (usually inherited prion disease) and in some patients, decline followed by a prolonged preterminal plateau at very low functional levels. Rates of decline and progress through milestones measured using the scale vary between sporadic, acquired and inherited prion diseases following clinical expectations. We have developed and validated a new functionally-oriented outcome measure and propose that future clinical trials in prion disease should collect data compatible with this scale, to allow for combined and comparative analyses. Such approaches may be advantageous in orphan conditions, where single studies of feasible duration will often struggle to achieve statistical power.

High levels of agreement between clinic-based ethyl glucuronide (EtG) immunoassays and laboratory-based mass spectrometry

Abstract Background: Immunoassay urine drug screening cups that detect use for two or more days are commonly used in addiction treatment settings. Until recently, there has been no comparable immunoassay test for alcohol use in these settings. Objectives: The aim of this study was to assess the agreement of a commercially available ethyl glucuronide immunoassay (EtG-I) test conducted at an outpatient addiction clinic and lab-based EtG mass spectrometry (EtG-MS) conducted at a drug testing laboratory at three cut-off levels. High agreement between these two measures would support the usefulness of EtG-I as a clinical tool for monitoring alcohol use. Methods: Forty adults with co-occurring alcohol dependence and serious mental illnesses submitted 1068 urine samples over a 16-week alcohol treatment study. All samples were tested using EtG-I on a benchtop analyzer and 149 were randomly selected for EtG-MS analysis at a local laboratory. Agreement was defined as the number of samples where EtG-I and EtG-MS were both above or below a specific cut-off level. Agreement was calculated at low cut-off levels (100 and 250 ng/ml), as well as at a higher cut-off level (500 ng/ml) recommended by most by commercial drug testing laboratories. Results: Agreement between EtG-I and EtG-MS was high across all cut-off levels (90.6% at 100 ng/ml, and 96.6% at 250 and 500 ng/ml). Conclusions: EtG immunoassays conducted at low cut-off levels in point-of-care testing settings have high agreement with lab-based EtG-MS. EtG-I can be considered a useful clinical monitoring tool for alcohol use in community-based addiction treatment settings.

The cognitive profile of prion disease: a prospective clinical and imaging study

Prion diseases are dementing illnesses with poorly defined neuropsychological features. This is probably because the most common form, sporadic Creutzfeldt‐Jakob disease, is often rapidly progressive with pervasive cognitive decline making detailed neuropsychological investigation difficult. This study, which includes patients with inherited, acquired (iatrogenic and variant) and sporadic forms of the disease, is the only large‐scale neuropsychological investigation of this patient group ever undertaken and aimed to define a neuropsychological profile of human prion diseases.

Predictors of stimulant abuse treatment outcomes in severely mentally ill outpatients.

BACKGROUND Severe mental illness is often exclusionary criteria for studies examining factors that influence addiction treatment outcome. Therefore, little is known about predictors of treatment response of individuals receiving psychosocial treatments for addictions who suffer from co-occurring severe mental illness. METHODS The impact of demographic, substance abuse severity, psychiatric severity, and service utilization variables on in-treatment performance (i.e., longest duration of abstinence) in a 12-week contingency management (CM) intervention for stimulant abuse in 96 severely mentally ill adults was investigated. A 4-step linear regression was used to identify independent predictors of in-treatment abstinence. RESULTS This model accounted for 37.4% of variance in the longest duration of abstinence outcome. Lower levels of stimulant use (i.e., stimulant-negative urine test) and psychiatric severity (i.e., lower levels of psychiatric distress), as well as higher rates of outpatient treatment utilization at study entry were independently associated with longer duration of drug abstinence. CONCLUSION These data suggest that individuals with low levels of stimulant use and psychiatric severity, as well as those actively engaged in services are most likely to succeed in a typical CM intervention. For others, modifications to CM interventions, such as increasing the value of reinforcement or adding CM to evidence based psychiatric interventions may improve treatment outcomes.

Clinical Trial Simulations Based on Genetic Stratification and the Natural History of a Functional Outcome Measure in Creutzfeldt-Jakob Disease.

IMPORTANCE A major challenge for drug development in neurodegenerative diseases is that adequately powered efficacy studies with meaningful end points typically require several hundred participants and long durations. Prion diseases represent the archetype of brain diseases caused by protein misfolding, the most common subtype being sporadic Creutzfeldt-Jakob disease (sCJD), a rapidly progressive dementia. There is no well-established trial method in prion disease. OBJECTIVE To establish a more powerful and meaningful clinical trial method in sCJD. DESIGN, SETTING, AND PARTICIPANTS A stratified medicine and simulation approach based on a prospective interval-cohort study conducted from October 2008 to June 2014. This study involved 598 participants with probable or definite sCJD followed up over 470 patient-years at a specialist national referral service in the United Kingdom with domiciliary, care home, and hospital patient visits. We fitted linear mixed models to the outcome measurements, and simulated clinical trials involving 10 to 120 patients (no dropouts) with early to moderately advanced prion disease using model parameters to compare the power of various designs. MAIN OUTCOMES AND MEASURES A total of 2681 assessments were done using a functionally orientated composite end point (Medical Research Council Scale) and associated with clinical investigations (brain magnetic resonance imaging, electroencephalography, and cerebrospinal fluid analysis) and molecular data (prion protein [PrP] gene sequencing, PrPSc type). RESULTS Of the 598 participants, 273 were men. The PrP gene sequence was significantly associated with decline relative to any other demographic or investigation factors. Patients with sCJD and polymorphic codon 129 genotypes MM, VV, and MV lost 10% of their function in 5.3 (95% CI, 4.2-6.9), 13.2 (95% CI, 10.9-16.6), and 27.8 (95% CI, 21.9-37.8) days, respectively (P < .001). Simulations indicate that an adequately powered (80%; 2-sided α = .05) open-label randomized trial using 50% reduction in Medical Research Council Scale decline as the primary outcome could be conducted with only 120 participants assessed every 10 days and only 90 participants assessed daily, providing considerably more power than using survival as the primary outcome. Restricting to VV or MV codon 129 genotypes increased power even further. Alternatively, single-arm intervention studies (half the total sample size) could provide similar power in comparison to the natural history cohort. CONCLUSIONS AND RELEVANCE Functional end points in neurodegeneration need not require long and very large clinical studies to be adequately powered for efficacy. Patients with sCJD may be an efficient and cost-effective group for testing disease-modifying therapeutics. Stratified medicine and natural history cohort approaches may transform the feasibility of clinical trials in orphan diseases.