Large C9orf72 hexanucleotide repeat expansions are seen in multiple neurodegenerative syndromes and are more frequent than expected in the UK population.

Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800-4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.

Variants of PLCXD3 are not associated with variant or sporadic Creutzfeldt-Jakob disease in a large international study.

BACKGROUND: Human prion diseases are relentlessly progressive neurodegenerative disorders which include sporadic Creutzfeldt-Jakob disease (sCJD) and variant CJD (vCJD). Aside from variants of the prion protein gene (PRNP) replicated association at genome-wide levels of significance has proven elusive. A recent association study identified variants in or near to the PLCXD3 gene locus as strong disease risk factors in multiple human prion diseases. This study claimed the first non-PRNP locus to be highly significantly associated with prion disease in genomic studies. METHODS: A sub-study of a genome-wide association study with imputation aiming to replicate the finding at PLCXD3 including 129 vCJD and 2500 sCJD samples. Whole exome sequencing to identify rare coding variants of PLCXD3. RESULTS: Imputation of relevant polymorphisms was accurate based on wet genotyping of a sample. We found no supportive evidence that PLCXD3 variants are associated with disease. CONCLUSION: The marked discordance in vCJD genotype frequencies between studies, despite extensive overlap in vCJD cases, and the finding of Hardy-Weinberg disequilibrium in the original study, suggests possible reasons for the discrepancies between studies.

Genetic risk factors for the posterior cortical atrophy variant of Alzheimer's disease.

INTRODUCTION: The genetics underlying posterior cortical atrophy (PCA), typically a rare variant of Alzheimer's disease (AD), remain uncertain. METHODS: We genotyped 302 PCA patients from 11 centers, calculated risk at 24 loci for AD/DLB and performed an exploratory genome-wide association study. RESULTS: We confirm that variation in/near APOE/TOMM40 (P = 6 × 10(-14)) alters PCA risk, but with smaller effect than for typical AD (PCA: odds ratio [OR] = 2.03, typical AD: OR = 2.83, P = .0007). We found evidence for risk in/near CR1 (P = 7 × 10(-4)), ABCA7 (P = .02) and BIN1 (P = .04). ORs at variants near INPP5D and NME8 did not overlap between PCA and typical AD. Exploratory genome-wide association studies confirmed APOE and identified three novel loci: rs76854344 near CNTNAP5 (P = 8 × 10(-10) OR = 1.9 [1.5-2.3]); rs72907046 near FAM46A (P = 1 × 10(-9) OR = 3.2 [2.1-4.9]); and rs2525776 near SEMA3C (P = 1 × 10(-8), OR = 3.3 [2.1-5.1]). DISCUSSION: We provide evidence for genetic risk factors specifically related to PCA. We identify three candidate loci that, if replicated, may provide insights into selective vulnerability and phenotypic diversity in AD.

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.

BACKGROUND: Rare TREM2 variants are significant risk factors for Alzheimer's disease (AD). METHODS: We used next generation sequencing of the whole gene (n = 700), exon 2 Sanger sequencing (n = 2634), p.R47H genotyping (n = 3518), and genome wide association study imputation (n = 13,048) to determine whether TREM2 variants are risk factors or phenotypic modifiers in patients with AD (n = 1002), frontotemporal dementia (n = 358), sporadic (n = 2500), and variant (n = 115) Creutzfeldt-Jakob disease (CJD). RESULTS: We confirm only p.R47H as a risk factor for AD (odds ratio or OR = 2.19; 95% confidence interval or CI = 1.04-4.51; P = .03). p.R47H does not significantly alter risk for frontotemporal dementia (OR = 0.81), variant or sporadic CJD (OR = 1.06 95%CI = 0.66-1.69) in our cohorts. Individuals with p.R47H associated AD (n = 12) had significantly earlier symptom onset than individuals with no TREM2 variants (n = 551) (55.2 years vs. 61.7 years, P = .02). We note that heterozygous p.R47H AD is memory led and otherwise indistinguishable from "typical" sporadic AD. CONCLUSION: We find p.R47H is a risk factor for AD, but not frontotemporal dementia or prion disease.

Identifying the best PCR enzyme for library amplification in NGS.

Background. PCR amplification is a necessary step in many next-generation sequencing (NGS) library preparation methods [1, 2]. Whilst many PCR enzymes are developed to amplify single targets efficiently, accurately and with specificity, few are developed to meet the challenges imposed by NGS PCR, namely unbiased amplification of a wide range of different sizes and GC content. As a result PCR amplification during NGS library prep often results in bias toward GC neutral and smaller fragments. As NGS has matured, optimized NGS library prep kits and polymerase formulations have emerged and in this study we have tested a wide selection of available enzymes for both short-read Illumina library preparation and long fragment amplification ahead of long-read sequencing.We tested over 20 different hi-fidelity PCR enzymes/NGS amplification mixes on a range of Illumina library templates of varying GC content and composition, and find that both yield and genome coverage uniformity characteristics of the commercially available enzymes varied dramatically. Three enzymes Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) 'Equinox' and Takara Ex Premier were found to give a consistent performance, over all genomes, that mirrored closely that observed for PCR-free datasets. We also test a range of enzymes for long-read sequencing by amplifying size fractionated S. cerevisiae DNA of average size 21.6 and 13.4 kb, respectively.The enzymes of choice for short-read (Illumina) library fragment amplification are Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) 'Equinox' and Takara Ex Premier, with RepliQa also being the best performing enzyme from the enzymes tested for long fragment amplification prior to long-read sequencing.

A novel protective prion protein variant that colocalizes with kuru exposure.

BACKGROUND: Kuru is a devastating epidemic prion disease that affected a highly restricted geographic area of the Papua New Guinea highlands; at its peak, it predominantly affected adult women and children of both sexes. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism. METHODS: We performed genetic and selected clinical and genealogic assessments of more than 3000 persons from Eastern Highland populations, including 709 who participated in cannibalistic mortuary feasts, 152 of whom subsequently died of kuru. RESULTS: Persons who were exposed to kuru and survived the epidemic in Papua New Guinea are predominantly heterozygotes at the known resistance factor at codon 129 of the prion protein gene (PRNP). We now report a novel PRNP variant--G127V--that was found exclusively in people who lived in the region in which kuru was prevalent and that was present in half of the otherwise susceptible women from the region of highest exposure who were homozygous for methionine at PRNP codon 129. Although this allele is common in the area with the highest incidence of kuru, it is not found in patients with kuru and in unexposed population groups worldwide. Genealogic analysis reveals a significantly lower incidence of kuru in pedigrees that harbor the protective allele than in geographically matched control families. CONCLUSIONS: The 127V polymorphism is an acquired prion disease resistance factor selected during the kuru epidemic, rather than a pathogenic mutation that could have triggered the kuru epidemic. Variants at codons 127 and 129 of PRNP demonstrate the population genetic response to an epidemic of prion disease and represent a powerful episode of recent selection in humans.

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.

HECTD2 is associated with susceptibility to mouse and human prion disease.

Prion diseases are fatal transmissible neurodegenerative disorders, which include Scrapie, Bovine Spongiform Encephalopathy (BSE), Creutzfeldt-Jakob Disease (CJD), and kuru. They are characterised by a prolonged clinically silent incubation period, variation in which is determined by many factors, including genetic background. We have used a heterogeneous stock of mice to identify Hectd2, an E3 ubiquitin ligase, as a quantitative trait gene for prion disease incubation time in mice. Further, we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases, vCJD and kuru. We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease. Although the substrate of HECTD2 is unknown, these data highlight the importance of proteosome-directed protein degradation in neurodegeneration. This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases. Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods.

PRNP allelic series from 19 years of prion protein gene sequencing at the MRC Prion Unit.

Mutation of the human prion protein gene (PRNP) open reading frame (ORF) accounts for almost all reported familial concurrence of prion disease. The more common mutations globally: octapeptide repeat insertions, P102L, D178N, E200K, and V210I have occurred in large multigenerational pedigrees and display autosomal dominant inheritance, however, many rare genetic changes have been reported that are of uncertain pathogenicity. Based on 19 years of PRNP sequencing at the MRC Prion Unit, London, and analysis of 3664 samples from patients referred with suspected prion disease and healthy populations, we present novel allele combinations, healthy control population data, results of screening the PRNP ORF in DNA from the entire referral series and the CEPH human genome diversity cell line panel. Of the 10 alleles detected in patients for which detailed cases histories are presented, 4 are unreported (G54S, D167N, V209M, Q212PP), two changes are thought to be pathogenic but have not been described in our regions (P105L from the UK, G114V from India and Turkey), and the remainder reported in healthy control populations or in trans to known pathogenic mutations suggesting non- or low pathogenicity (G54S, 1-OPRI, G142S, N171S, V209M, E219K). New genotype-phenotype correlations and population frequencies presented will help the diagnosis and genetic counselling of those with suspected inherited prion disease.

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.

Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study.

BACKGROUND: Human and animal prion diseases are under genetic control, but apart from PRNP (the gene that encodes the prion protein), we understand little about human susceptibility to bovine spongiform encephalopathy (BSE) prions, the causal agent of variant Creutzfeldt-Jakob disease (vCJD). METHODS: We did a genome-wide association study of the risk of vCJD and tested for replication of our findings in samples from many categories of human prion disease (929 samples) and control samples from the UK and Papua New Guinea (4254 samples), including controls in the UK who were genotyped by the Wellcome Trust Case Control Consortium. We also did follow-up analyses of the genetic control of the clinical phenotype of prion disease and analysed candidate gene expression in a mouse cellular model of prion infection. FINDINGS: The PRNP locus was strongly associated with risk across several markers and all categories of prion disease (best single SNP [single nucleotide polymorphism] association in vCJD p=2.5 x 10(-17); best haplotypic association in vCJD p=1 x 10(-24)). Although the main contribution to disease risk was conferred by PRNP polymorphic codon 129, another nearby SNP conferred increased risk of vCJD. In addition to PRNP, one technically validated SNP association upstream of RARB (the gene that encodes retinoic acid receptor beta) had nominal genome-wide significance (p=1.9 x 10(-7)). A similar association was found in a small sample of patients with iatrogenic CJD (p=0.030) but not in patients with sporadic CJD (sCJD) or kuru. In cultured cells, retinoic acid regulates the expression of the prion protein. We found an association with acquired prion disease, including vCJD (p=5.6 x 10(-5)), kuru incubation time (p=0.017), and resistance to kuru (p=2.5 x 10(-4)), in a region upstream of STMN2 (the gene that encodes SCG10). The risk genotype was not associated with sCJD but conferred an earlier age of onset. Furthermore, expression of Stmn2 was reduced 30-fold post-infection in a mouse cellular model of prion disease. INTERPRETATION: The polymorphic codon 129 of PRNP was the main genetic risk factor for vCJD; however, additional candidate loci have been identified, which justifies functional analyses of these biological pathways in prion disease.

Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms.

Highly accurate and high-throughput SNP genotyping platforms are increasingly popular but the performance of suboptimal DNA samples remains unclear. The aim of our study was to determine the best platform, amplification technique, and loading concentration to maximize genotype accuracy and call rate using degraded samples. We amplified high-molecular weight genomic DNA samples recently extracted from whole blood and degraded DNA samples extracted from 50-year-old patient sera. Two whole-genome amplification (WGA) methodologies were used: an isothermal multiple displacement amplification method (MDA) and a fragmentation-PCR-based method (GenomePlex [GPLEX]; Sigma-Aldrich, St. Louis, MO). Duplicate runs were performed on genome-wide dense SNP arrays (Nsp-Mendel; Affymetrix) and custom SNP platforms based on molecular inversion probes (Targeted Genotyping [TG]; Affymetrix) and BeadArray technology (Golden Gate [GG]; Illumina). Miscalls and no-calls on Mendel arrays were correlated with each other, with confidence scores from the Bayesian calling algorithm, and with average probe intensity. Degraded DNA amplified with MDA gave low call rates and concordance across all platforms at standard loading concentrations. The call rate with MDA on GG was improved when a 5 x concentration of amplified DNA was used. The GPLEX amplification gave high call rate and concordance for degraded DNA at standard and higher loading concentrations on both TG and GG platforms. Based on these analyses, after standard filtering for SNP and sample performance, we were able to achieve a mean call rate of 99.7% and concordance 99.7% using degraded samples amplified by GPLEX on GG technology at 2 x loading concentration. These findings may be useful for investigators planning case-control association studies with patient samples of suboptimal quality.

Inherited mtDNA variations are not strong risk factors in human prion disease.

Aside from variation in the prion protein gene, genetic risk factors for sporadic Creutzfeldt-Jakob disease remain elusive. Given emerging evidence implicating mitochondrial dysfunction in the pathogenesis of the disorders, we studied the role of inherited mitochondrial DNA variation in a 2255 sporadic prion disease cases and 3768 controls. Our analysis indicates that inherited mitochondrial DNA variation does not have a major role in the risk of developing the disorder.

Rare structural genetic variation in human prion diseases.

Prion diseases are a diverse group of neurodegenerative conditions, caused by the templated misfolding of prion protein. Aside from the strong genetic risk conferred by multiple variants of the prion protein gene (PRNP), several other variants have been suggested to confer risk in the most common type, sporadic Creutzfeldt-Jakob disease (sCJD) or in the acquired prion diseases. Large and rare copy number variants (CNVs) are known to confer risk in several related disorders including Alzheimer's disease (at APP), schizophrenia, epilepsy, mental retardation, and autism. Here, we report the first genome-wide analysis for CNV-associated risk using data derived from a recent international collaborative association study in sCJD (n = 1147 after quality control) and publicly available controls (n = 5427). We also investigated UK patients with variant Creutzfeldt-Jakob disease (n = 114) and elderly women from the Eastern Highlands of Papua New Guinea who proved highly resistant to the epidemic prion disease kuru, who were compared with healthy young Fore population controls (n = 395). There were no statistically significant alterations in the burden of CNVs >100, >500, or >1000 kb, duplications, or deletions in any disease group or geographic region. After correction for multiple testing, no statistically significant associations were found. A UK blood service control sample showed a duplication CNV that overlapped PRNP, but these were not found in prion disease. Heterozygous deletions of a 3' region of the PARK2 gene were found in 3 sCJD patients and no controls (p = 0.001, uncorrected). A cell-based prion infection assay did not provide supportive evidence for a role for PARK2 in prion disease susceptibility. These data are consistent with a modest impact of CNVs on risk of late-onset neurologic conditions and suggest that, unlike APP, PRNP duplication is not a causal high-risk mutation.

Validation of next-generation sequencing technologies in genetic diagnosis of dementia.

Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. Here we assess the use of next generation sequencing (NGS) technologies as a quick, accurate and cost effective method to determine genetic diagnosis in EOD. We developed gene panel based technologies to assess 16 genes known to harbour mutations causal of dementia and combined these with PCR based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP. In a blinded study of 95 samples we show very high sensitivity and specificity are achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. The MRC Dementia Gene Panel and similar technologies are likely to be transformational in EOD diagnosis with a significant impact on the proportion of patients in whom a genetic cause is identified.

A novel exon 2 I27V VCP variant is associated with dissimilar clinical syndromes.

Mutations in valosin-containing protein (VCP) are associated with a syndromic constellation of inclusion body myositis, Paget's disease of bone and frontotemporal dementia. Here we describe the case reports of two patients with a novel variation (p.I27V) in the VCP gene that was not identified in a healthy control population. One patient presented with a frontotemporal dementia syndrome associated with raised serum alkaline phosphatase and a family history of progressive muscle disease and behavioural decline, while the second patient presented with isolated progressive dysarthria. Together these cases suggest a potential for the same VCP mutation to produce distinct patterns of brain damage, underlining the clinical heterogeneity of VCP-associated disease.

Genetic susceptibility, evolution and the kuru epidemic.

The acquired prion disease kuru was restricted to the Fore and neighbouring linguistic groups of the Papua New Guinea highlands and largely affected children and adult women. Oral history documents the onset of the epidemic in the early twentieth century, followed by a peak in the mid-twentieth century and subsequently a well-documented decline in frequency. In the context of these strong associations (gender, region and time), we have considered the genetic factors associated with susceptibility and resistance to kuru. Heterozygosity at codon 129 of the human prion protein gene (PRNP) is known to confer relative resistance to both sporadic and acquired prion diseases. In kuru, heterozygosity is associated with older patients and longer incubation times. Elderly survivors of the kuru epidemic, who had multiple exposures at mortuary feasts, are predominantly PRNP codon 129 heterozygotes and this group show marked Hardy-Weinberg disequilibrium. The deviation from Hardy-Weinberg equilibrium is most marked in elderly women, but is also significant in a slightly younger cohort of men, consistent with their exposure to kuru as boys. Young Fore and the elderly from populations with no history of kuru show Hardy-Weinberg equilibrium. An increasing cline in 129V allele frequency centres on the kuru region, consistent with the effect of selection in elevating the frequency of resistant genotypes in the exposed population. The genetic data are thus strikingly correlated with exposure. Considering the strong coding sequence conservation of primate prion protein genes, the number of global coding polymorphisms in man is surprising. By intronic resequencing in a European population, we have shown that haplotype diversity at PRNP comprises two major and divergent clades associated with 129M and 129V. Kuru may have imposed the strongest episode of recent human balancing selection, which may not have been an isolated episode in human history.

Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice.

Prion diseases are fatal neurodegenerative disorders of humans and animals, which include bovine spongiform encephalopathy (BSE) and its human form, variant Creutzfeldt-Jakob disease (vCJD). They are characterized by a prolonged incubation period, which is known to be influenced by polymorphisms in the prion protein gene. Previous studies of inbred mice have demonstrated that additional genetic loci also contribute to the observed variation in incubation period. However, a substantial transmission barrier between cow and mouse complicates studies using BSE. As a result, primary transmissions display large variations in incubation period and not all animals develop clinical signs of disease. To identify quantitative trait loci for BSE without the presence of a transmission barrier, we analysed 124 animals from an F2 intercross between CAST/Ei and NZW/OlaHsd mice and challenged them intracerebrally with a strain of BSE that was passaged twice through C57BL/6OlaHsd mice. Interval mapping identified two highly significant linked regions on chromosomes 2 and 11 with peak lod scores of 6.34 and 4.77, respectively. Composite interval mapping suggests that chromosome 2 includes three linked quantitative trait loci. Loci in the same position on chromosomes 2 and 11 were also identified in a previous study using the same mouse cross but infected with Chandler/RML scrapie prions. If these are the same loci, it suggests that these loci may be influencing incubation time independently of prion strain. This provides hope that it may be possible to identify human quantitative trait loci for prion incubation time using mouse models that may allow identification of at-risk individuals and the discovery of novel therapeutic targets.