Population structure and migration in the Eastern Highlands of Papua New Guinea, a region impacted by the kuru epidemic.

Populations of the Eastern Highlands of Papua New Guinea (EHPNG, area 11,157 km2) lived in relative isolation from the rest of the world until the mid-20th century, and the region contains a wealth of linguistic and cultural diversity. Notably, several populations of EHPNG were devastated by an epidemic prion disease, kuru, which at its peak in the mid-twentieth century led to some villages being almost depleted of adult women. Until now, population genetic analyses to learn about genetic diversity, migration, admixture, and the impact of the kuru epidemic have been restricted to a small number of variants or samples. Here, we present a population genetic analysis of the region based on genome-wide genotype data of 943 individuals from 21 linguistic groups and 68 villages in EHPNG, including 34 villages in the South Fore linguistic group, the group most affected by kuru. We find a striking degree of genetic population structure in the relatively small region (average FST between linguistic groups 0.024). The genetic population structure correlates well with linguistic grouping, with some noticeable exceptions that reflect the clan system of community organization that has historically existed in EHPNG. We also detect the presence of migrant individuals within the EHPNG region and observe a significant excess of females among migrants compared to among non-migrants in areas of high kuru exposure (p = 0.0145, chi-squared test). This likely reflects the continued practice of patrilocality despite documented fears and strains placed on communities as a result of kuru and its associated skew in female incidence.

From Kuru to Alzheimer: A personal outlook.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.

A naturally occurring variant of the human prion protein completely prevents prion disease.

Mammalian prions, transmissible agents causing lethal neurodegenerative diseases, are composed of assemblies of misfolded cellular prion protein (PrP). A novel PrP variant, G127V, was under positive evolutionary selection during the epidemic of kuru--an acquired prion disease epidemic of the Fore population in Papua New Guinea--and appeared to provide strong protection against disease in the heterozygous state. Here we have investigated the protective role of this variant and its interaction with the common, worldwide M129V PrP polymorphism. V127 was seen exclusively on a M129 PRNP allele. We demonstrate that transgenic mice expressing both variant and wild-type human PrP are completely resistant to both kuru and classical Creutzfeldt-Jakob disease (CJD) prions (which are closely similar) but can be infected with variant CJD prions, a human prion strain resulting from exposure to bovine spongiform encephalopathy prions to which the Fore were not exposed. Notably, mice expressing only PrP V127 were completely resistant to all prion strains, demonstrating a different molecular mechanism to M129V, which provides its relative protection against classical CJD and kuru in the heterozygous state. Indeed, this single amino acid substitution (G→V) at a residue invariant in vertebrate evolution is as protective as deletion of the protein. Further study in transgenic mice expressing different ratios of variant and wild-type PrP indicates that not only is PrP V127 completely refractory to prion conversion but acts as a potent dose-dependent inhibitor of wild-type prion propagation.

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.

Epidemiological mechanisms of genetic resistance to kuru.

Transmissible spongiform encephalopathies (TSEs), such as kuru, are invariably fatal neurodegenerative conditions caused by a malformation of the prion protein. Heterozygosity of codon 129 of the prion protein gene has been associated with increased host resistance to TSEs, although the mechanism by which this resistance is achieved has not been determined. To evaluate the epidemiological mechanism of human resistance to kuru, we developed a model that combines the dynamics of kuru transmission and the population genetics of human resistance. We fitted our model to kuru data from the epidemic that occurred in Papua New Guinea over the last hundred years. To elucidate the epidemiological mechanism of human resistance, we estimated the incubation period and transmission rate of kuru for codon 129 heterozygotes and homozygotes using kuru incidence data and human genotype frequency data from 1957 to 2004. Our results indicate that human resistance arises from a combination of both a longer incubation period and reduced susceptibility to infection. This work provides evidence for balancing selection acting on a human population and the mechanistic basis for the heterozygote resistance to kuru.

Prion disease: a tale of folds and strains.

Research on prions, the infectious agents of devastating neurological diseases in humans and animals, has been in the forefront of developing the concept of protein aggregation diseases. Prion diseases are distinguished from other neurodegenerative diseases by three peculiarities. First, prion diseases, in addition to being sporadic or genetic like all other neurodegenerative diseases, are infectious diseases. Animal models were developed early on (a long time before the advent of transgenic technology), and this has made possible the discovery of the prion protein as the infectious agent. Second, human prion diseases have true equivalents in animals, such as scrapie, which has been the subject of experimental research for many years. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonosis caused by bovine spongiform encephalopathy (BSE) prions. Third, they show a wide variety of phenotypes in humans and animals, much wider than the variants of any other sporadic or genetic neurodegenerative disease. It has now become firmly established that particular PrP(Sc) isoforms are closely related to specific human prion strains. The variety of human prion diseases, still an enigma in its own right, is a focus of this article. Recently, a series of experiments has shown that the concept of aberrant protein folding and templating, first developed for prions, may apply to a variety of neurodegenerative diseases. In the wake of these discoveries, the term prion has come to be used for Aß, α-synuclein, tau and possibly others. The self-propagation of alternative conformations seems to be the common denominator of these "prions," which in future, in order to avoid confusion, may have to be specified either as "neurodegenerative prions" or "infectious prions."

Human prion diseases: from Kuru to variant Creutzfeldt-Jakob disease.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are the names given to the group of fatal neurodegenerative disorders that includes kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal and sporadic familial insomnia and the novel prion disease variable protease-sensitive prionopathy (PSPr) in humans. Kuru was restricted to natives of the Foré linguistic group in Papua New Guinea and spread by ritualistic endocannibalism. CJD appears as sporadic, familial (genetic or hereditary) and infectious (iatrogenic) forms. Variant CJD is a zoonotic CJD type and of major public health importance, which resulted from transmission from bovine spongiform encephalopathy (BSE) through ingestion of contaminated meat products. GSS is a slowly progressive hereditary autosomal dominant disease and the first human TSE in which a mutation in a gene encoding for prion protein (PrP) was discovered. The rarest human prion disease is fatal insomnia, which may occur, in genetic and sporadic form. More recently a novel prion disease variable protease-sensitive prionopathy (PSPr) was described in humans.TSEs are caused by a still incompletely defined infectious agent known as a "prion" which is widely regarded to be an aggregate of a misfolded isoform (PrP(Sc)) of a normal cellular glycoprotein (PrP(c)). The conversion mechanism of PrP(c) into PrP(Sc) is still not certain.

Kuru: the first prion disease.

Kuru disease is linked with the name of D. Carleton Gajdusek and he was the first to show that this human neurodegenerative disease can be transmitted to chimpanzees and subsequently classified as a transmissible spongiform encephalopathy (TSE), or slow unconventional virus disease. It was first reported to Western world in 1957 by Gajdusek and Vincent Zigas,(1,2) and in 1975 a complete bibliography of kuru was published by Alpers et al.(3) "Kuru" in the Fore language in Papua New Guinea means to shiver from fever and cold. The disease has been found to spread through ritualistic cannibalism and is an invariably fatal cerebellar ataxia accompanied by tremor, choreiform and athetoid movements. Neuropathologically, kuru is characterized by the presence of amyloid "kuru" plaques.

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.

Kuru: genes, cannibals and neuropathology.

Kuru was the first human transmissible spongiform encephalopathy (TSE) or prion disease identified, occurring in the Fore linguistic group of Papua New Guinea. Kuru was a uniformly fatal cerebellar ataxic syndrome, usually followed by choreiform and athetoid movements. Kuru imposed a strong balancing selection on the Fore population, with individuals homozygous for the 129 Met allele of the gene (PRNP) encoding for prion protein (PrP) being the most susceptible. The decline in the incidence of kuru in the Fore has been attributed to the exhaustion of the susceptible genotype and ultimately by discontinuation of exposure via cannibalism. Neuropathologically, kuru-affected brains were characterized by widespread degeneration of neurons, astroglial and microglial proliferation, and the presence of amyloid plaques. These early findings have been confirmed and extended by recent immunohistochemical studies for the detection of the TSE-specific PrP (PrP). Confocal laser microscopy also showed the concentration of glial fibrillary acidic protein-positive astrocytic processes at the plaque periphery. The fine structure of plaques corresponds to that described earlier by light microscopy. The successful experimental transmission of kuru led to the awareness of its similarity to Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease and formed a background against which the recent epidemics of iatrogenic and variant Creutzfeldt-Jakob disease could be studied.

An overview of human prion diseases.

Prion diseases are transmissible, progressive and invariably fatal neurodegenerative conditions associated with misfolding and aggregation of a host-encoded cellular prion protein, PrP(C). They have occurred in a wide range of mammalian species including human. Human prion diseases can arise sporadically, be hereditary or be acquired. Sporadic human prion diseases include Cruetzfeldt-Jacob disease (CJD), fatal insomnia and variably protease-sensitive prionopathy. Genetic or familial prion diseases are caused by autosomal dominantly inherited mutations in the gene encoding for PrP(C) and include familial or genetic CJD, fatal familial insomnia and Gerstmann-Sträussler-Scheinker syndrome. Acquired human prion diseases account for only 5% of cases of human prion disease. They include kuru, iatrogenic CJD and a new variant form of CJD that was transmitted to humans from affected cattle via meat consumption especially brain. This review presents information on the epidemiology, etiology, clinical assessment, neuropathology and public health concerns of human prion diseases. The role of the PrP encoding gene (PRNP) in conferring susceptibility to human prion diseases is also discussed.

The prion diseases.

The prion diseases are a family of rare neurodegenerative disorders that result from the accumulation of a misfolded isoform of the prion protein (PrP), a normal constituent of the neuronal membrane. Five subtypes constitute the known human prion diseases; kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal insomnia (FI), and variant CJD (vCJD). These subtypes are distinguished, in part, by their clinical phenotype, but primarily by their associated brain histopathology. Evidence suggests these phenotypes are defined by differences in the pathogenic conformation of misfolded PrP. Although the vast majority of cases are sporadic, 10% to 15% result from an autosomal dominant mutation of the PrP gene (PRNP). General phenotype-genotype correlations can be made for the major subtypes of CJD, GSS, and FI. This paper will review some of the general background related to prion biology and detail the clinical and pathologic features of the major prion diseases, with a particular focus on the genetic aspects that result in prion disease or modification of its risk or phenotype.

Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease.

Six clinico-pathological phenotypes of sporadic Creutzfeldt-Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrP(TSE), present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrP(TSE) type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt-Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt-Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt-Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt-Jakob disease MM1 and genetic Creutzfeldt-Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt-Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt-Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt-Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt-Jakob disease of the VV2 or MV2 subtype.

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.

Review. Lessons of kuru research: background to recent studies with some personal reflections.

The widespread exposure of the UK population to bovine spongiform encephalopathy prions, and the potential consequences for public health, led to a renewed interest in kuru, the principal example of epidemic human prion disease. Kuru research in Papua New Guinea was expanded to study the range of incubation periods possible in human prion infection, to investigate maternal and other possible natural routes of transmission, to characterize genetic susceptibility and resistance factors and to gain insights into the peripheral pathogenesis of orally acquired prion disease in humans. Although now essentially over, the kuru epidemic continues to provide important lessons.

Review. The neuropathology of kuru and variant Creutzfeldt-Jakob disease.

A comparison of the pathological profiles of two spongiform encephalopathies with a similar presumptive route of infection was performed. Archival kuru and recent variant Creutzfeldt-Jakob disease (vCJD) cases reveal distinct lesional differences, particularly with respect to prion protein, suggesting that the strain of agent is important in determining the phenotype. Genotype analysis of the polymorphism on codon 129 reveals (in conjunction with updated information from more kuru cases) that all three genotypes (VV, MV and MM (where M is methionine and V is valine)) are detected in kuru with some preference for MM homozygosity. The presence of valine does not therefore appear to determine peripheral selection of PrPCJD. vCJD remains restricted to date to MM homozygosity on codon 129. It remains to be determined whether this genotype is dictating a shorter incubation period.

A clinical study of kuru patients with long incubation periods at the end of the epidemic in Papua New Guinea.

Kuru is so far the principal human epidemic prion disease. While its incidence has steadily declined since the cessation of its route of transmission, endocannibalism, in Papua New Guinea in the 1950s, the arrival of variant Creutzfeldt-Jakob disease (vCJD), also thought to be transmitted by dietary prion exposure, has given kuru a new global relevance. We investigated all suspected cases of kuru from July 1996 to June 2004 and identified 11 kuru patients. There were four females and seven males, with an age range of 46-63 years at the onset of disease, in marked contrast to the age and sex distribution when kuru was first investigated 50 years ago. We obtained detailed histories of residence and exposure to mortuary feasts and performed serial neurological examination and genetic studies where possible. All patients were born a significant period before the mortuary practice of transumption ceased and their estimated incubation periods in some cases exceeded 50 years. The principal clinical features of kuru in the studied patients showed the same progressive cerebellar syndrome that had been previously described. Two patients showed marked cognitive impairment well before preterminal stages, in contrast to earlier clinical descriptions. In these patients, the mean clinical duration of 17 months was longer than the overall average in kuru but similar to that previously reported for the same age group, and this may relate to the effects of both patient age and PRNP codon 129 genotype. Importantly, no evidence for lymphoreticular colonization with prions, seen uniformly in vCJD, was observed in a patient with kuru at tonsil biopsy.

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.

Review. The origin of the prion agent of kuru: molecular and biological strain typing.

Kuru is an acquired human prion disease that primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. The central clinical feature of kuru is progressive cerebellar ataxia and, in sharp contrast to most cases of sporadic Creutzfeldt-Jakob disease (CJD), dementia is a less prominent and usually late clinical feature. In this regard, kuru is more similar to variant CJD, which also has similar prodromal symptoms of sensory disturbance and joint pains in the legs and psychiatric and behavioural changes. Since a significant part of the clinicopathological diversity seen in human prion disease is likely to relate to the propagation of distinct human prion strains, we have compared the transmission properties of kuru prions with those isolated from patients with sporadic, iatrogenic and variant CJD in both transgenic and wild-type mice. These data have established that kuru prions have prion strain properties equivalent to those of classical (sporadic and iatrogenic) CJD prions but distinct from variant CJD prions. Here, we review these findings and discuss how peripheral routes of infection and other factors may be critical modifiers of the kuru phenotype.