Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.     

Comparative performance of three TSE rapid tests for surveillance in healthy sheep affected by scrapie

Rapid tests specific for sheep and goats became part of European Union-wide active scrapie surveillance in 2006. Performance of three approved TSE rapid tests for the detection of sheep infected with scrapie in field cases in the pre-clinical stage of the disease was compared.The medulla oblongata of 969 asymptomatic sheep of various genotype and breed aged over 18 months from 23 Italian flocks affected with scrapie, were tested by the Bio-Rad TeSeE Sheep/Goat (A), the IDEXX HerdChek BSE-Scrapie Antigen Test Kit, EIA (B) and the Prionics^®-Check Western Small Ruminant (C) rapid tests.Of 136 positive samples of classical scrapie, as confirmed by Western blot assay, 132 were positive with test A (Se 97.06%, CI 95% 92.64-99.19); 135 with test B (Se 99.26%, 95% CI 95.97-99.98) and 128 with test C (Se 94.12%, 95% CI 88.74-97.43).Tests A and B showed the best performance on analytical sensitivity. All three systems demonstrated good reproducibility: being the intrarater and interrater kappa coefficients always over 0.83.The one available atypical scrapie sample was positive with tests A and B, negative with test C.Considering the discrepant results in the detection of low PrP^sc concentrations and of the atypical case, differences can be expected in the efficacy of an active surveillance system, depending on the test adopted.     

PrP gene polymorphism in sheep breeds in Slovakia and susceptibility to scrapie

We analyzed the prion protein (PrP) genotype based on the codons 136, 154 and 171 and assigned to five risk groups (R1-R5) in healthy and scrapie-affected sheep in Slovakia. In healthy (asymptomatic) population, 119 Merino, 106 Improved Valachian, 117 Tsigai, and 48 Suffolk breeds were tested. Among the asymptomatic sheep, the low-risk genotypes R1 and R2 were most abundant in Suffolk (94%) and Merino (84%) breeds, followed by Tsigai (58%) and Improved Valachian (40%) breeds. The medium-risk group R3 was most frequent in Improved Valachian (31%) breed, followed by Tsigai (21%), Merino (10%), and Suffolk (6%) breeds. The occurrence of high-risk groups R4 and R5 was none in Suffolk breed, followed by Merino (6%), Tsigai (21%), and Improved Valachian (30%) breeds. Since 2003, altogether 48 cases of scrapie have been confirmed in Tsigai (38), Merino (4), Improved Valachian (2), Improved Valachian x Tsigai (3), and Suffolk (1) breeds. Among sheep with scrapie, Merino breed belonged to the medium-risk group R3. The majority of scrapie-affected Tsigai sheep were classified into high-risk R5 (50%) and medium-risk R3 (42%) groups. We showed an association of scrapie with medium- and high-risk groups of PrP genotype in Slovakia. In particular, the glutamine at position 171 appears to be of major importance for the susceptibility to scrapie.     

Prion protein gene polymorphism and genetic risk evaluation for scrapie in all Turkish native sheep breeds

The aim of this study was to identify the prion protein (PrP) gene polymorphism in a total of 1,110 healthy sheep from 18 Turkish native sheep breeds. There were nine alleles and 22 genotypes observed based on codons 136, 154, and 171 of the PrP gene. The ARQ allele was predominant for all breeds. The most resistant allele to scrapie, ARR, was present in all breeds. The VRQ allele, associated with the highest susceptibility to scrapie, was detected at low frequencies in ?vesi (0.06), K?v?rc?k (0.021), Sak?z (0.010), Karayaka (0.011), Çine Çapar? (0.012), and Güneykaraman (0.017). In general, the ARQ/ARQ genotype was predominant in all breeds. The most resistant genotype to scrapie, ARR/ARR, was found with the frequency lower than 0.180. The most susceptible genotype, VRQ/VRQ, was found in only K?v?rc?k. The TRR and TRH alleles and the genotypes of ARR/TRR, ARR/ARK, and ARH/TRH have been found for the first time in Turkish native sheep breeds. According to these results, all breeds belong to risk group R3 followed by R2. It is propounded that the susceptibility to scrapie increased from eastern to western part of Turkey. Our findings of Turkish native sheep breeds with PrP gene polymorphisms will assist the sheep breeding program for selection of scrapie resistance genotypes to reduce the risk of scrapie.     

Influence of the prion protein gene,Prnp, on scrapie susceptibility in sheep

Natural scrapie in sheep occurs through a complex interplay between host genetic elements and various strains of the infectious scrapie agent. Scrapie-related polymorphisms in the coding region of the prion protein (PrP) gene, Prnp, have been studied in a number of breeds. The disease-promoting V136 allele, and the susceptibility-reducing R171 allele, have proved to be most important. However, variation in the coding region of Prnp cannot alone explain the diverse patterns of scrapie susceptibility in various breeds. For instance, in many breeds plagued with scrapie, the V136 allele appears to be a rarity. The R171 allele greatly reduces scrapie susceptibility This lays the molecular foundation for marker-assisted breeding for reduced scrapie susceptibility now underway in many countries. Although potentially important, and still under investigation, variable expression level and pattern of the ovine Prnp appears to be of little importance for the occurrence of natural scrapie. Studies of scrapie in mice also indicate that genetic elements other than Prnp may have a strong influence on scrapie incubation time, and hence susceptibility. Narrowing down the search to focus on these elements and identification of candidate genes are important tasks for future research in sheep scrapie.     

Distinction of scrapie phenotypes in sheep by lesion profiling

Major determinants of the pathological phenotype of natural scrapie are considered to be the agent strain and host prion protein (PrP) genotype, but the relationship between these is far from clear. Little is known about the strains that produce natural scrapie. A method of brain vacuolation profiling was developed which enables this aspect of disease phenotype to be characterized in detail. This method distinguished at least two distinct pathological phenotypes in sheep of a single genotype (ARQ/ARQ) from different flocks in the UK. Great similarity was also demonstrated between one of these phenotypes and the phenotype of sheep from a flock in Sardinia. The profile of four sheep of the same ARQ/ARQ genotype experimentally infected with bovine spongiform encephalopathy (BSE) was determined for comparison. It would appear from these preliminary observations that the application of lesion profiling techniques to ovine transmissible spongiform encephalopathy (TSE) may contribute to the definition of a particular scrapie phenotype within a flock. It may, therefore, have potential for improving our understanding of current TSE phenotypes in sheep, with regard to the possibility of identifying those of bovine origin.     

Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Population-based genetic testing of asymptomatic women for breast and ovarian cancer susceptibility

PurposeThe identification of carriers of hereditary breast and ovarian cancer (HBOC) gene variants through family cancer history alone is suboptimal, and most population-based genetic testing studies have been limited to founder mutations in high-risk populations. Here, we determine the clinical utility of identifying actionable variants in a healthy cohort of women.MethodsGermline DNA from a subset of healthy Australian women participating in the lifepool project was screened using an 11-gene custom sequencing panel. Women with clinically actionable results were invited to attend a familial cancer clinic (FCC) for post-test genetic counseling and confirmatory testing. Outcomes measured included the prevalence of pathogenic variants, and the uptake rate of genetic counseling, risk reduction surgery, and cascade testing.ResultsThirty-eight of 5908 women (0.64%) carried a clinically actionable pathogenic variant. Forty-two percent of pathogenic variant carriers did not have a first-degree relative with breast or ovarian cancer and 89% pursued referral to an FCC. Forty-six percent (6/13) of eligible women pursued risk reduction surgery, and the uptake rate of cascade testing averaged 3.3 family members per index case.ConclusionWithin our cohort, HBOC genetic testing was well accepted, and the majority of high-risk gene carriers identified would not meet eligibility criteria for genetic testing based on their existing family history.     

Biological and biochemical characterization of sheep scrapie in Japan

Due to the apparent absence of an agent-specific nucleic acid genome, scrapie strains cannot be classified by genome characterization, which is commonly used for the classification of many viruses. However, scrapie strains can be distinguished to some extent by biological properties such as transmissibility to experimental animals and distribution of neuropathological lesions and by biochemical properties such as the molecular mass and relative protease-resistance of the disease-specific isoform of prion protein (PrP(Sc)). In order to preliminarily characterize the scrapie strains that are prevalent in Japan, we analyzed the transmissibility of sheep scrapie isolates to mice and the relative proteinase K (PK) resistance of the corresponding PrP(Sc). The results indicate that Japanese scrapie strains can be divided into at least three groups based on biological and biochemical properties. The first group includes isolates which cause disease in mice with an incubation period of approximately 400 days and possess PrP(Sc) with relatively high PK resistance. Isolates of the second group contain PrP(Sc) that is highly resistant to PK digestion but transmit poorly to mice. The final group consists of isolates that cause disease in mice with an incubation period of less than 300 days and are associated with PrP(Sc) with reduced PK resistance. Sheep scrapie has occurred sporadically in Japan since1982, with only approximately 60 officially reported cases so far. However, the diversity of scrapie strains in the field suggested by our data raises the concern that a scrapie strain similar to the parental agent of bovine spongiform encephalopathy could exist or emerge in Japan. Thus, continuous surveillance for scrapie will be required to prevent the further spread of scrapie, not only among the sheep population but also to other species, and to eliminate any potential risk of sheep scrapie to public health.     

Ethical issues in susceptibility genetic testing for late‐onset neurodegenerative diseases

Genome‐wide association studies have revolutionized our understanding of the genetic architecture of complex traits and diseases over the last decade. This knowledge is enabling clinicians, researchers, and direct‐to‐consumer genetics companies to conduct disease susceptibility testing based on powerful methods such as polygenic risk scoring. However, these technologies raise a set of complex ethical, legal, social, and policy considerations. Here we review and discuss a series of ethical dilemmas associated with susceptibility genetic testing for the two most common late‐onset neurodegenerative diseases, Alzheimer's and Parkinson's disease, including testing in asymptomatic individuals. Among others, these include informed consent, disclosure of results and unexpected findings, mandatory screening, privacy and confidentiality, and stigma and genetic discrimination. Importantly, appropriate counseling is a deciding factor for the ethical soundness of genetic testing, which poses a challenge for the regulation of these tests and the training of healthcare professionals. As genetic knowledge about these diseases continues growing and genetic testing becomes more widespread, it is increasingly important to raise awareness among researchers, medical practitioners, genetic counselors, and decision makers about the ethical, legal, and social issues associated with genetic testing for polygenic diseases.     

Early and late pathogenesis of natural scrapie infection in sheep

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.     

Active Surveillance for Scrapie by Third Eyelid Biopsy and Genetic Susceptibility Testing of Flocks of Sheep in Wyoming

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP^Sc, the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP^Sc assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP^Sc-positive sheep were identified through the passive surveillance program, and an additional three PrP^Sc-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP^Sc-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.     

Public interest in predictive genetic testing, including direct-to-consumer testing, for susceptibility to major depression: preliminary findings

The past decade has seen rapid advances in the identification of associations between candidate genes and a range of common multifactorial disorders. This paper evaluates public attitudes towards the complexity of genetic risk prediction in psychiatry involving susceptibility genes, uncertain penetrance and gene–environment interactions on which successful molecular-based mental health interventions will depend. A qualitative approach was taken to enable the exploration of the views of the public. Four structured focus groups were conducted with a total of 36 participants. The majority of participants indicated interest in having a genetic test for susceptibility to major depression, if it was available. Having a family history of mental illness was cited as a major reason. After discussion of perceived positive and negative implications of predictive genetic testing, nine of 24 participants initially interested in having such a test changed their mind. Fear of genetic discrimination and privacy issues predominantly influenced change of attitude. All participants still interested in having a predictive genetic test for risk for depression reported they would only do so through trusted medical professionals. Participants were unanimously against direct-to-consumer genetic testing marketed through the Internet, although some would consider it if there was suitable protection against discrimination. The study highlights the importance of general practitioner and public education about psychiatric genetics, and the availability of appropriate treatment and support services prior to implementation of future predictive genetic testing services.     

The impact of familial environment on depression scores after genetic testing for cancer susceptibility

The associations between characteristics of family relationships and family trends in cancer worry and the psychological adjustment of recipients of genetic testing for cancer susceptibility were investigated. Data provided by 178 individuals from 24 families with Lynch syndrome who participated in a cohort study investigating psychological and behavioral outcomes of genetic testing were used. Responses from multiple family members were aggregated to construct family trends representing norms and departure from norms in cancer worry. Lower perceived family cohesion at baseline and decrease in this variable at 6 months after receipt of test results were associated with higher depression scores at 12 months. More variability in cancer worry among family members at baseline was also associated with higher depression scores at 12 months. Increase in family conflict was associated with decrease in depression scores among individuals from families with higher levels of cancer worry on average and less variability among the members. Family relationships and family trends in levels of cancer worry may play important roles in the psychological adjustment of genetic test recipients. The findings highlight the complexity of familial environment surrounding individuals that undergo genetic testing and suggest the benefits of considering these factors when providing genetic services.     

Women's interest in genetic testing for breast cancer susceptibility may be based on unrealistic expectations

We report on results of an interview study assessing women's attitudes toward and hypothetical interest in genetic susceptibility testing for breast cancer. Data are from 246 interviews with women of varying ethnicity (African American, European American, Native American, and Ashkenazi Jewish), family history of breast cancer (negative, positive, and borderline), and educational level. Semistructured interviews included questions on general health beliefs; attitudes, experiences, and concerns about breast cancer; and hypothetical interest in genetic testing. Influence of specific test characteristics was assessed with 14 Likert scales varying negative and positive predictive value, timing of disease, possible medical interventions following a positive result. Results reported include both statistical and qualitative analysis. We found that women had a high level of interest in testing which, in general, did not vary by ethnicity, level of education, or family history. Interest in testing appeared to be shaped by an exaggerated sense of vulnerability to breast cancer, limited knowledge about genetic susceptibility testing, and generally positive views about information provided through medical screening. However, study participants were most interested in a test that didn't exist (high positive predictive value followed by effective, noninvasive, preventive therapy) and least interested in the test that does exist (less than certain positive predictive value, low negative predictive value, and limited, invasive, and objectionable therapeutic options). Our data suggest that without a careful counseling process, women could easily be motivated toward interest in a test which will not lead to the disease prevention they are seeking.     

Search for healthy carriers of scrapie: an assessment of subclinical infection of sheep in an Icelandic scrapie flock by three diagnostic methods and correlation with PrP genotypes

Summary.  Subclinical infection in scrapie of sheep, characterized by a long incubation period, may be of importance for the spread of the disease. We screened brain samples from all 65 sheep in a scrapie-affected flock for subclinical infection and correlated with results of PrP genotyping, which is of relevance for the epidemiology and the question, whether by breeding for resistant genotypes one would be breeding for healthy carriers. The sensitivity of three methods was compared, i.e. histopathological examination for vacuoles (HP), immunohistochemical staining (IHC) and Western blotting (WB) for PrP^Sc. Five sheep showed definite clinical signs and histological scrapie lesions, and signs of infection were detected in 25 of 60 asymptomatic sheep, by HP and/or IHC and WB. The IHC was slightly more sensitive than HP and WB. Sheep with subclinical infection were, with one exception, either homo- or heterozygotes for 136-V, as were four of the five sheep with clinical scrapie. The incidence of the VRQ allelic variant in the flock was unusually high compared to the Icelandic sheep population probably contributing to the high prevalence of both clinical and subclinical infection in the flock. Neither sheep with definite scrapie nor detectable subclinical infection, were of the resistant AHQ genotype, indicating that Icelandic AHQ sheep are not healthy carriers of scrapie infection. Received September 7, 2001 Accepted December 13, 2001     

Consumers' use of web-based information and their decisions about multiplex genetic susceptibility testing

Background

Few data exist to inform concerns raised by online direct-to-consumer marketing of genetic susceptibility tests, such as those offered by commercial entities like 23andme, Navigenics, and DNA Direct. The Multiplex Initiative, a population-based study of healthy adults, provides the first opportunity to evaluate how use of a Web-based decision tool that conveyed information about a genetic susceptibility test influenced individuals’ test decisions.

Objective

To inform the ongoing debate over whether individuals offered genetic susceptibility testing without the involvement of a health care provider (eg, through direct-to-consumer testing) can make informed decisions about testing when guided by online decision aids.

Methods

Participants were 526 members of a large health maintenance organization aged 25 to 40 years old who visited a study website. Multivariate logistic regression models were tested to examine the association of website usage with downstream test decisions.

Results

Participants viewed an average of 2.9 of the 4 pages introducing the multiplex test, 2.2 of the 8 pages describing the health conditions, and 3.2 of the 15 pages describing the genes. For each page viewed, participants were more likely to describe their decision-making as easy (odds ratio [OR] 1.04, 95% confidence interval [CI] 1.01-1.07) and to decide to be tested (OR 1.08, 95% CI 1.05-1.11).

Conclusions

Healthy adults in this study perceived Web-based genomic information presented using evidence-based communications approaches to be helpful in supporting both decisions to test and not to test. Continued research is needed to ensure that these results generalize to target groups with lower literacy and less Internet savvy.