Current Molecular Medicine 2004, 4, 375-384 375 Genetic Studies in Relation to Kuru: An Overview

L.G. Goldfarb*, L. Cervenakova and D.C. Gajdusek

National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA American Red Cross J .H . Holland Laboratory , Rockville, MD 20855, USA Centre National de la Recharche Scientifique, Institut Alfred Fessard, Gif-sur-Yvette, France Abstract: Kuru is a subacute neurodegenerative disease presenting with limb ataxia, dysarthria, and a shivering tremor. The disease progress to complete motor and mental incapacity and death within 6 to 24 months. Neuropathologically, a typical pattern of neuronal loss, astrocytic and microglial proliferation, characteristic “kuru-type” amyloid plaques, and PrP deposits in the cerebral cortex and cerebellum are observed. Kuru is the prototype of a group of human transmissible spongiform encephalopathies (TSEs), or “prion” diseases, that include hereditary, sporadic and infectious forms. The latest member of this group, the variant Creutzfeldt-Jakob disease (vCJD), linked to transmission of bovine spongiform encephalopathy (BSE) to humans, shows features similar to kuru. Kuru has emerged at the beginning of the 1900s in a small indigenous population of New-Guinean Eastern Highlands, reached epidemic proportions in the mid-1950s and disappeared progressively in the latter half of the century to complete absence at the end of the 1990s. Early studies made infection, the first etiologic assumption, seem unlikely and led to a hypothesis that kuru might be a genetically determined or genetically mediated illness. After transmissibility of kuru had been discovered and all major epidemiologic phenomena adequately explained by the spread of an infectious agent with long incubation period through the practice of cannibalism, the pattern of occurrence still continued to suggest a role for genetic predisposition. Recent studies indicate that individuals homozygous for Methionine at a polymorphic position 129 of the prion protein were preferentially affected during the kuru epidemic. The carriers of the alternative 129Met/Val and 129Val/Val genotypes had a longer incubation period and thus developed disease at a later age and at a later stage of the epidemic. Observations made during the kuru epidemic are helpful in the understanding of the current vCJD outbreak, and vice versa clinical and experimental data accumulated in studies of other TSE disorders contribute to better understanding of the documented kuru phenomena.

INTRODUCTION to isolate an infectious agent continued [3]. Systematic research on kuru started in March Simultaneously, genetic patterns of disease 1957 when D. Carleton Gajdusek and Vincent Zigas distribution were also under study. The investigators studied 36 kuru cases and identified at least 300 were impressed by the fact that the illness was deaths from kuru that had occurred in the preceding restricted to Fore natives and their immediate 5 years [1]. These investigators first suspected that neighbors with whom Fore regularly intermarried, and kuru was a viral meningo-encephalitis leading to a that all known cases occurred in genetically related subacute or chronic disease with a pattern individuals [4]. reminiscent of Economo’s encephalitis and directly During a 1959 visit to D.C. Gajdusek’s Laboratory referred to it as a syndrome similar to paralysis at the National Institutes of Health in Bethesda, agitans [1, 2]. The association between kuru and veterinary neuropathologist James R. M. Innes noted cannibalism was considered during the first months similarities between neuropathology findings in kuru of studies, but rejected as unlikely when cases were and scrapie of sheep, a slow virus infection in which encountered in young children who were not susceptibility is genetically determined. Reflecting on believed to have engaged in the ritual cannibalistic published reports and an exhibit on kuru studies that consumption of deceased relatives [3]. Failure to find was set up by D.C. Gajdusek at the Wellcome any febrile response, cerebrospinal fluid pleocytosis, Medical Museum in London [5, 6], William J. Hadlow increased cellularity or perivascular cuffing in the wrote a letter to Lancet that suggested a possible neuropathologic images led to exclusion of an acute relationship between kuru and scrapie and infection as the cause of disease, although attempts recommended testing transmissibility of kuru in laboratory primates [7]. These observations stimulated enormously the ongoing attempts to find *Address correspondence to this author at the National Institutes of a microbial etiological agent since scrapie was the Health, Bldg 10, Room 4B37, 10 Center Dr., MSC 1361, Bethesda, only known example of non-inflammatory fatal CNS Maryland 20892-1361, USA; Tel: 301 402 1480; Fax: 301 496 6341; E- disease that was nonetheless shown to be mail: [email protected] transmissible. With the help of Carlton Herman and J.

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Anthony Morris, D.C. Gajdusek started intracerebral two groups, North and South. The administrative inoculation of many species of animals including boundary coincides with a minor cultural and monkeys, and later chimpanzees, anticipating linguistic division [15, 17]. unusually long incubation periods. In 1966, In the early years of the Administration frequent Gajdusek, Gibbs and Alpers reported transmission of fighting that claimed many lives was suppressed kuru to chimpanzee [8]. allowing the population to freely cultivate sweet Kuru, the prototype human TSE, was until potato in their gardens and breed pigs and chickens recently the only example of oral transmission without fear of attack [17, 18]. Unrelated to kuru, occurring in humans. Although the incidence of kuru among other measures directed at regulating the has markedly declined, further kuru studies are local’s social life and making it healthier, the Christian important not only from a historical perspective. The missionaries and the new Administration discouraged latest TSE member, vCJD, linked to consumption of the cannibalistic rituals [19], and cannibalism contaminated beef from animals incubating BSE [9] completely ceased between 1957 and 1962 [3]. has phenotypic similarity to kuru [10], therefore these During anthropological investigations conducted in disorders are expected to share basic disease 1951-1952 and 1952-1953, the presence of kuru mechanisms. The third infectious form of TSE, was noted among the Fore, Iate and Usurufa people iatrogenic Creutzfeldt-Jakob disease (iCJD), is a [18]. Fore always believed that kuru was a kind of disease associated with the use in modern medicine sorcery (among 34 other variants of sorcery) [18]. To of human tissues and extracts such as cadaveric induce kuru, the sorcerer had to obtain some object dura matter, corneal transplants, or growth hormone intimately associated with the victim, clothing, extracted from human pituitaries that may be excreta, hair or discarded food. He then packaged contaminated with the CJD agent [11]. the object in bark and leaves making a bundle, and Genetic studies of TSEs during the past decade half-buried it in a swamp. The sorcerer would return resulted in discoveries indicating that each hereditary daily to shake the bundle until his victim developed form of TSE, familial CJD, Gerstmann-Sträussler- “shaking disease” [2, 18]. Scheinker disease (GSS) and familial fatal insomnia Cannibalism was practiced in most Eastern (FFI) is associated with mutations in the PRNP gene Highlands populations before the Australian coding for the prion protein [12]. In addition, strong administrative control took over [18, 20, 21]. It evidence was presented for the existence of consisted primarily of the ceremonial consumption of genetically determined susceptibility to infectious and close-of-kin as an act of respect. Meat and viscera sporadic forms. Methionine/valine (Met/Val) coding were taken from the corpse and cooked in a steam variation at codon 129 of the PRNP gene has been oven wrapped in leaves or stuffed into bamboo extensively studied. Accumulated evidence has cylinders. The whole body was eaten. The shown that although this substitution does not by cannibalistic mourning rituals were attended and itself cause disease, it dramatically influences the performed almost exclusively by women and their phenotype produced by other PRNP mutations [13] young children, usually the closest relatives of the and is responsible for susceptibility/resistance to deceased, preferentially exposing them to the kuru, iatrogenic and sporadic forms of CJD [11, 14]. infectious agents. Men ate only the meat. Infants were contaminated by their mothers through rubbing their skin and eyes, and cleaning noses [3]. THE POPULATION OF THE KURU-AFFECTED REGION Kuru incidence grew dramatically in the 1940s and 1950s to reach a level of more than 200 new The Eastern Highlands Province of Papua New cases per year in 1957-1959 [22, 23]. More than Guinea is located at the beginning of the Highlands 80% of kuru cases ever recorded, of a total of about valleys at an average height of 1600m. It occupies 3,000, occurred in the Fore cultural and linguistic the valleys of Lamari, Asaro, Wahgi, Tua, Dunatina group, consisting of about 12,000 people. The and Ramu rivers. Common feature of this province is mortality rate of kuru in some Fore villages reached the steep rugged mountains covered with dense rain 35 per 1,000 [20]. In addition to Fore, neighboring forest graduating to sub-alpine vegetation. The total Keiagaana, Gimi, Yate, Kanite and Usurufa cultural population of the Eastern Highlands is 376,000 and linguistic groups, and parts of 3 other groups people divided in 37 linguistic groups, from 150 to were also affected by kuru, but to a lower degree [4]. 60,000 speakers per group [15]. Fore is one of the Intermarriage across the linguistic group borders and cultural and linguistic groups, a Stone Age culture migrations have played a major role in determining that up to the late 1950s practiced endo- the pattern of kuru incidence and distribution [4]. cannibalistic consumption of dead kinsmen as a rite Since the outlawing of cannibalism in the late 1950s, of mourning [16]. The first Lutheran evangelist there has been a gradual and steady decline in the mission was established in the region in 1949 and a occurrence of kuru, but a substantial disease Police patrol post in 1951. Okapa District incidence still existed in the 1960s and the 1970s, Administration of the Australian Territory of Papua and single-digit numbers of cases were recorded in and New Guinea was established in 1954. For the early 1990s [24]. The incidence of kuru in administrative purposes, the Fore was divided into children and adolescents decreased first: by 1985 Genetic Studies in Relation to Kuru: An Overview Current Molecular Medicine, 2004, Vol. 4, No. 4 377 the disease was no longer seen in persons under 35 small semi-isolated communities whose isolation has years of age [21]. No one born after 1960 has ever been maintained by both the physical environment developed kuru [3, 24]. and the social rules. In such small communities there Kuru epidemic has dramatically distorted the was maximal opportunity for the operation of random population structure in the region. By 1960, the genetic drift [4]. (D) The age and gender distributions average adult female-to-male ratio in the South Fore indicated a higher susceptibility of certain population population was 1:1.67, while in less affected Gimi, groups [4]. (E) Kuru sometimes developed in Keiagaana and Kanite groups it was 1:1.19 to persons who had left kuru region, but there were no 1:1.14, and it was 1:1 in the essentially unaffected secondary kuru cases in their new surroundings. (F) Kamano group [25]. In some villages of South Fore Early clinical and laboratory findings were not the ratio was 1:2 to 1:3. The disparity increased with suggestive of an infectious disease: afebrile clinical age: the female-to-male ratio in young South Fore course, lack of pleocytosis in the cerebrospinal fluid, children was 1:1.04, but reached 1:3.41 in the 41 to no elevation of erythrocyte sedimentation rate, and 60 age group [25]. The female population deficit in no inflammatory changes in the brain [1, 4]. Large the South Fore was calculated at 1,676 persons, kuru pedigrees were compiled and analyzed [29-31]. while the total number of registered kuru cases was A genetic theory based on disease distribution 561. This indicates a long and intense past history of proposed that kuru is controlled by a single kuru in the South Fore [4, 17]. When kuru occurred autosomal gene that if mutated behaves as an in communities neighboring the Fore region, it was autosomal recessive trait in young children, but is usually observed in a Fore woman who had moved autosomal dominant in older females [30, 31]. But to marry a non-Fore man. Her illness typically this purely genetic explanation for a disease that presented many years after marriage and appeared was highly prevalent and highly lethal and had no sporadic. Although less frequent, some women from apparent concomitant heterozygote advantage was low-prevalence kuru regions would develop kuru not totally convincing [4]. after marrying into a highly affected Fore village [2]. After transmissibility of kuru was discovered [8] Six of 78 wives interviewed in a single Fore village and most epidemiologic phenomena explained by came from other cultural and linguistic groups, the spread of an infectious agent with long including three from kuru-free regions [26]. incubation period through the practice of A widely discussed point in the population studies cannibalism, the patterns of kuru distribution was when, where and how the kuru epidemic has continued to suggest a role for genetic started. Ethnographists R.M. Berndt and C. Berndt predisposition [15]. In order to obtain evidence and working in the Northern part of the kuru region in determine the role of genetics in kuru etiology and 1951-1953, saw kuru patients and regarded the pathogenesis, a study of erythrocyte antigens and disease psychosomatic in origin. They date the serum proteins in representative groups of the region earliest remembered cases of kuru in this community populations was conducted to determine whether approximately 50 years back, around 1901-1903 kuru susceptibility is associated with polymorphic [18]. First cases of kuru identified by name were markers known at the time [32-34]. Group-specific known since 1927 [26]. Kuru is believed to originate component (Gc) is an extremely polymorphic alpha-2- in two Keiagaana hamlets, Uwami and Amufi, and to globulin system widely used in population studies; subsequently spread to North Fore villages and more than 120 variant alleles have been identified. perhaps further through many visiting relatives who The gene coding for this protein is located on would come to see the strange disease and watch chromosome 4q12. The rare Australian Gc variant Aborigine (GcAb) has a single-nucleotide alteration in the patient die [27]. The initial source is unknown. Gc the second position of codon 429 resulting in a The most likely possibility is that an individual who protein with electrophoretically distinguishable died of sporadic CJD was consumed by kinsmen in pattern [35]. In a search for genetic markers the traditional ritual cannibalism fashion [3, 20, 28, associated with kuru, it was shown that the GcAb 29]. allele occurs with extremely high frequency in the kuru region, the highest being in the North and GENETIC STUDIES South Fore (10 to 24%), Keiagaana (24%), Gimi (10 to 21%), and Auyana (16%) groups. The distant Early data on clinical features, neuropathology second highest frequency of the GcAb allele was and epidemiology led the investigators to a observed in Australian aborigines (5-8%) [35]. It has hypothesis that kuru is a genetically determined or also been established that kuru patients possess the genetically mediated illness, based on the following GcAb allele more often than the general population facts. (A) The disease was restricted to Fore natives [33, 36]. Overall, 38.6% of kuru patients from kuru- and their immediate neighbors with whom Fore affected areas carried at least one GcAb allele. regularly intermarried [1, 2]. (B) Approximately three Homozygosity for the GcAb allele was proposed as quarters of patients were related to someone who the hereditary factor regulating susceptibility to kuru; had succumbed to kuru in the recent past, and the the calculated risk of contracting kuru for a GcAb-Ab majority had lost a mother, sibling or offspring [2]. (C) genotype carrier was 6.14:1 [36]. The results The Eastern Highlands population consisted of many describing the prevalence of the GcAb-Ab genotype in 378 Current Molecular Medicine, 2004, Vol. 4, No. 4 Goldfarb et al. kuru patients are difficult to interpret. Additional controls in the Fore villages at the same period of studies are needed to explain this association. 1957-1959; these individuals did not develop kuru The role of genetically determined susceptibility through the end of the epidemic, according to the could not be further evaluated until the human gene Kuru Registry. Forty-eight persons represented coding for prion protein, PRNP, was cloned and neighboring non-Fore villages where kuru had never characterized [37, 38]. Multiple mutations in the occurred or the incidence was 100 times lower than PRNP gene have been linked to various phenotypes that among the Fore. In addition, 26 Fore subjects of hereditary TSE [12]. Currently, 26 pathogenic were tested in the post-epidemic time, during 1988- missense mutations, nine variants of insertional 1993. The frequency of Met/Val genotypes in the mutations, a deletion, and seven DNA kuru group was compared with each of the three polymorphisms have been identified in the PRNP control groups. Codon 129 genotype distribution in gene. An incidental finding indicating that two kuru kuru patients at the height of the epidemic was patients were homozygous at codon 129 of the found to significantly deviate from Fore survivors or PRNP gene was reported [39] and subsequently non-Fore controls. Based on representative confirmed [40], prompting further research on statistics, a conclusion was made that individuals susceptibility/resistance to kuru and other infectious homozygous for the 129Met allele had an earlier age forms of TSE. The methionine/valine (Met/Val) of disease onset and a shorter incubation time, while variation at position 129 was later characterized as a the 129Met/Val and 129Val/Val carriers developed neutral polymorphism [41], but accumulated kuru at a later age, after a longer incubation period evidence has shown that the homozygous carriers of of 20 or more years, and many survived the epidemic the 129Met or 129Val alleles were overrepresented [43, 45]. A group of individuals who lived through the among patients with sporadic and iatrogenic CJD. kuru epidemic and never developed kuru was found The iCJD patients with CNS route of infection were to deviate from Hardy-Weinberg equilibrium due to mainly homozygous for the 129Met allele [11], while the almost complete absence of the 129Met/Met homozygosity for the 129Val allele was more genotype. The 129Met/Met genotype depletion in frequent in iCJD with peripheral route of infection this group of survivors was significant as compared [11, 42]. The Met/Met:Met/Val:Val/Val genotype to the healthy population of neighboring non-Fore frequencies determined in the background villages sampled during the same 1957-1959 time populations vary from 0.32/0.43/0.24 in New period. Depletion of the 129Met/Met genotype was Guineans to 0.37/0.51/0.12 in the British to associated with the loss of the 129Met/Met 0.92/0.08/0 in the Japanese [42-44]. individuals who had increased susceptibility to kuru and became early victims of the epidemic [43]. Studies of genetic mechanisms underlying susceptibility to kuru were conducted in the 1990s To further investigate this hypothesis, genotype with specimens collected at several points of the frequencies in Fore male patients who (unlike the kuru epidemic. DNA was recovered from frozen brain females) had a single age-related peak of exposure tissue, brain suspensions previously used in to kuru, predominantly between 1 and 10 years of transmission studies, blood clots, serum samples and age, were separately analysed [43]. Fifty-three other tissues stored in tissue bank archives [43, 45]. percent of young Fore males who developed kuru in The Kuru Registry book provided precious medical 1957-1959 had the 129Met/Met genotype, while the information about individuals who did and did not survivors who were exposed to kuru at the same age develop kuru throughout the epidemic. Eighty kuru and at the same time, completely lacked patients identified and examined in Fore villages in the129Met/Met genotype. The results of this analysis 1957 through 1959, at the time of the highest kuru demonstrate that among young Fore males just incidence, and 13 patients diagnosed during 1964- entering the age of risk and presumably having a 1988 were studied. Germ-line mutations, known or normal initial genotype distribution, the 129Met/Met unknown, were not identified in five kuru patients in individuals were preferentially affected with kuru [43]. whom the entire PRNP coding region has been This clearly indicates early involvement of the sequenced, despite of the fact that a parent (mother 129Met/Met carriers during the initial phases of the in most cases) of each of these patients have died epidemic. Having "used up" the more susceptible of kuru [40, 43]. In view of the fact that all five kuru 129Met/Met genotype, the kuru epidemic eventually patients studied by sequencing were homozygous began absorbing the less susceptible 129Met/Val for Val at codon 129, additional kuru patients and and 129Val/Val genotypes prevailing in the region. et population controls were screened for the codon 129 Indeed, two kuru cases recently studied by Lantos al et al sequence by using restriction analysis of the PCR- . [10], 3 of 5 cases studied by McLean . [47] amplified PRNP coding region. and a single case studied by Hainfellner [48], as well as 5 of 8 our latest kuru cases studied in 1988-1993, A population-based study in kuru-affected areas were homozygous for the 129Val allele. Many of the of New Guinea was carried out by comparing the 129Met/Val and 129Val/Val genotype carriers frequency of the 129Met/Met genotype in kuru survived the epidemic altogether demonstrating a patients and adequate controls [43]. For this lower susceptibility to kuru. Lack of the 129Met/Met purpose, DNA samples were obtained from 21 individuals in the post-epidemic population control control individuals who were selected as paired may be explained by the massive loss of this Genetic Studies in Relation to Kuru: An Overview Current Molecular Medicine, 2004, Vol. 4, No. 4 379 genotype to kuru in a small population [43]. may be seen in the presence of minimal neuronal Apparently, the decline of kuru incidence in Fore change and extend into subcortical white matter [46, population was determined by two independent 47, 48]. Mild myelin degeneration was described in events: discontinuation of exposure and exhaustion the majority of kuru cases, involving predominantly of the susceptible genotype. spinocerebellar and corticospinal tracts [46, 52]. A strong “balancing selection” imposed by the Highly characteristic (“kuru-type”) amyloid plaques are kuru epidemic on the Fore population resulting in present in large numbers in the granular layer of the overrepresentation of 129Met/Val heterozygotes was cerebellum and occasionally in the molecular layer recently confirmed in another study [49]. Surprisingly, [46, 47]. The unicentric kuru plaques are about 30 a similar balancing selection at the PRNP locus was microns in diameter and composed of a dense discovered in some other world populations and central core and delicate fibrillary radiating spikes interpreted as evidence of selection pressure from surrounding the core. Abundant synaptic and possible epidemics of prion diseases in prehistoric perineuronal types of PrP deposits were revealed humans [49]. No other hypotheses explaining the with the use of immunohistochemistry. PrP 129Met/Val genotype survival advantages have yet depositions are localized predominantly in the been evaluated. Haplotype analysis based on cerebellum, but numerous PrP plaques and intense characterization of 56 polymorphic sites within a 25 synaptic-type deposits are also present throughout kb region spanning the PRNP gene failed to identify all gray matter areas of the brain [47, 48]. The haplotypes specifically linked to vCJD [50]. deposits are consistently present in regions showing spongiform change and astrogliosis. No significant inflammatory changes have been observed. GENOTYPE-PHENOTYPE RELATIONSHIPS Neuropathological descriptions of kuru patients Kuru is a rapidly progressive fatal ataxia. studied in the 1950s as compared with cases Disequilibrium, clumsiness and tremor are the initial collected and studied in the 1970s present signs of illness affecting the lower extremities and significant differences. The former show vacuolation resulting in unstable gait before progressing to of neurons and status spongiosus in limited parts of involve trunk and arms. Speech deteriorates as the the cortex and basal ganglia and are present in only disease advances [3, 29]. Tremors are of about one-half of studied patients [46, 53]. In action/intention type known to be other clinical signs patients who developed kuru in later stages of the of cerebellar dysfunction [51]. Dystonic movements epidemic, more diffuse and intense spongiform and myoclonus-type jerks are present in the trunk, change was found in the frontal, central, parietal and limbs, neck and jaw. There is no muscle weakness, cingulated cortices, and additional sites of prominent fasciculation or atrophy. Emotional instability with spongiform change were observed in the cerebellum, inappropriate outbursts of laughter or crying is noted striatum, mesencephalon and pontine nuclei [47, in some patients, others become depressed and 48]. These neuropathologic differences between the withdrawn, or paranoid and aggressive. Mental cases studied in the early and late stages of the slowing is apparent, but severe dementia may not be kuru epidemic consisting of a more diffuse and evident until later stages of illness [3]. Terminally, intense spongiform change in all studied patients urinary and fecal incontinence develop, and may depend not solely on technological progress, dysphagia leads to thirst and starvation. Total but also the genetic make-up. Descriptions of kuru duration of illness is usually six to nine months, with patients who were studied in the 1950s and a range from four to 24 months. Routine biochemical subsequently genetically typed indicate that the and hematological investigations are normal, as is presence of amyloid plaques was predominantly the cerebrospinal fluid. seen in patients carrying at least one Methionine allele [45]. In contrast, kuru patients studied in the Dominant kuru neuropathology is represented by 1960s and the 1970s were predominantly of the sub-acute degeneration with a pattern of neuronal 129Val/Val genotype [47, 48]. The results of loss, spongiform change, astrocytic and microglial neuropathological investigations in other TSEs, proliferation, amyloid plaques and PrP deposits in particularly sporadic CJD [54], FFI and hereditary the cerebral cortex and cerebellum. Loss of neurons CJD with the D178N mutation [55, 56], confirm that is most severe in the central and parietal cortex, most severe spongiform degeneration in the cerebral cerebellum, striatum, mesencephalon, cortex occurs in the carriers of at least one 129Val hypothalamus, and pontine nuclei [46], leading to a allele, while it is mild in the carriers of the 129Met/Met relatively constant pattern of spongiform change [47, genotype. Genotype-dependent pathologic 48]. In the cerebellum, there is severe loss and expression was also observed in transmission degeneration of both Purkinje and granule cells [52]. experiments of BSE and vCJD to transgenic mice Specific features of neuronal degeneration include carrying human Val/Val as opposed to Met/Met loss of Nissl substance and single or multiple transgene: the characteristic neuropathological and cytoplasmic vacuoles of various sizes. Astrocytic molecular phenotype of human vCJD was proliferation and astrogliosis are widespread and reproduced in Met/Met but not Val/Val mice [57]. intense. They are more pronounced in grey matter areas, coinciding with severe neuronal damage, but The shortest kuru incubation period was encountered in young children who were massively 380 Current Molecular Medicine, 2004, Vol. 4, No. 4 Goldfarb et al. contaminated at the cannibalistic feasts. The squirrel, capuchin, woolly, marmoset) developed kuru youngest known kuru patient was diagnosed at age after incubation times of 10 to 51 months [60]. 4 and died at age 5 years, several years before his Summarily, 11 kuru cases were experimentally mother developed kuru [3]. The average and the transmitted [60]. Incubation period lasted 14 to 38 longest incubation time was estimated using three months. The infectivity titer in 1g of a kuru patient’s exceptionally well documented separate cannibalistic brain tissue was 103 -108. On consecutive passages, feasts in Fore villages [58]. Fourteen adults and the incubation time substantially reduced. eleven children participating in the feasts were Transmissibility of other human TSEs, including followed for 29 years after the events. All 14 adults sporadic, iatrogenic and hereditary CJD, GSS and have died of kuru with an average incubation time of FFI to apes and monkeys, Syrian hamsters and 11 years, whereas the children had an average several strains of mice, was also demonstrated [3, incubation time of 21 years and four have not 61-63]. Experimental model of kuru was established developed kuru 29 years later. The longest in the chimpanzees [64-66]. Chimpanzees incubation time in this series was 29 years [58]. The developed experimental kuru after intracerebral or longest known incubation time reported in the 1990s peripheral inoculation with the kuru patient brain was 40 years [24]. The calculated mean incubation material and in sub-passages from other period was assessed between 10.3 and 13.2 years chimpanzees. The major clinical symptoms of [59], close to the estimates of the incubation time in experimental kuru were ataxia, action and postural iCJD patients [11]. tremor, apathy and abnormal behavior. Experimentally induced kuru showed remarkably uniform neuropathology consisting of severe TRANSMISSIBILITY spongiosis predominantly in the cerebral cortex and Attempts at isolation of the infectious agent other gray matter structures of the brain [65, 66]. causing kuru started in the first months of kuru Marked proliferation and hypertrophy of astrocytes investigation in 1957. Blood, urine, CSF and brain was observed in the areas of massive neuronal loss. tissue were inoculated into small laboratory animals The white matter was not obviously thinned, but (mice, rats, hamsters, guinea pigs and rabbits) and a extensive breakdown of myelin was detected. variety of primary and continuous cell lines. These Damage to the cerebellum was not as severe as in experiments failed to identify a propagating agent kuru patients. These findings led to a conclusion that [3]. Similarities between the neuropathological the neuropathologic process in experimental kuru features of scrapie and kuru suggested that kuru like was essentially the same as in natural kuru, with scrapie may be transmissible [7]. Scrapie has been some differences in distribution that may have known as a “slow” infection characterized by depended on the site of intracerebral inoculation of incubation times in sheep experimental transmissions the infectious material [25]. lasting one to five years. With these considerations, a slow-virus program was set up at the National Institutes of Health using for experimental MOLECULAR PATHOGENESIS transmissions a wide variety of experimental animals The predominant current view of TSE and expecting unusually long incubation times [3]. pathogenesis is based on the “protein only” According to an established protocol, 10% hypothesis. According to this hypothesis, the critical suspension of freshly frozen kuru brain tissue was event in TSE pathogenesis is an irreversible inoculated intracerebrally and in some cases transformation of the normal host-encoded cellular intravenously into experimental monkeys and apes, protein PrP-c into an abnormal disease-associated including rhesus, cynomolgus, African green and isoform PrP-sc [67]. PrP-sc is the only molecule that squirrel monkeys and later chimpanzee, gibbon, has been intrinsically linked to infectivity. PrP-c is a baboon, spider, capuchin, marmoset, lemur, and glycoprotein attached to the plasma membrane by patas monkeys. The rational for using a wide variety glycosyl-phosphatydylinositol anchor. While the PrP-c of laboratory animals came from experience with is easily degraded by proteases, its pathogenic transmission of scrapie, suggesting that the disease counterpart PrP-sc shows substantial resistance to could be reproduced only in select breeds of sheep, proteolysis. Purified by non-denaturing procedures goats, mice and hamsters, but not guinea pigs, and examined by Fourier transform infrared rabbits, rhesus or cynomolgus monkeys. Hence, the spectroscopy and circular dichroism, PrP-c shows a emphasis in this new series of investigations was on high alpha-helix content (42%) and little beta-sheet the use of as many species as possible. (3%), while the PrP-sc isoform contains 34% beta- sheet and 20% alpha-helix structures [68]. PrP-sc First positive results were obtained in 1966 in reduced by proteolysis to a core protein with three chimpanzees each inoculated with brain molecular weight of 27-30 kD [69] has 43% beta- material from a separate kuru patient. Chimpanzees sheets and no alpha-helix. The PrP27-30 core developed the disease after 18, 21 and 21 months protein exhibits remarkable resistance to physical of incubation [8]. The clinical features and and chemical agents. Turnover of PrP-sc is neuropathology of experimental kuru were similar to approximately 5 times slower than that of the wild human kuru. In addition to the chimpanzee, all type PrP-c, and with the disease progression it tends tested species of the New World monkeys (spider, Genetic Studies in Relation to Kuru: An Overview Current Molecular Medicine, 2004, Vol. 4, No. 4 381 to form insoluble fibrillar aggregates in cell cell cultures and transgenic mice expressing the compartments. PrP-sc and its toxic fragments VRQ versus ARR allelic variants [78, 79]. ARQ/ARQ gradually accumulate in neurons resulting in and AHQ/AHQ genotypes show the shortest neuronal death. After cell death, PrP-sc accumulates incubation time upon BSE transmission [80]. in the brain extracellular space forming amyloid Experiments with oral transmission of BSE to Cheviot plaques such as the plaques observed in kuru sheep also identified the ARR/ARR genotype as brains. PrP-sc alone or in association with another as resistant and ARQ/ARQ as susceptible [81]. yet unidentified molecule [70] has the capacity to After oral inoculation, PrP-sc is first identified in initiate the transformation of PrP-c into PrP-sc if Peyer’s patches, mesenteric lymph nodes and inoculated into a susceptible host. Evidence enteric nerves of hamsters infected with scrapie [82]. indicating that PrP-sc is the pathogenic protein One to three months later infection becomes responsible for the disease and its transmission to detectable in the spleen and non-mesenteric lymph other hosts comes from several experimental nodes, indicating that the infectious agent is findings: (A) accumulation of PrP-sc in the brain is spreading through the lymphatic system [83]. In observed in all forms of TSE, (B) procedures other studies, peripheral nerves were considered as destroying proteins inactivate infectivity, (C) PrP-sc a most efficient pathway of neuroinvasion [84]. copurifies with infectivity, and the concentration of Experimental oral infection in lemurs with a range of PrP-sc is directly proportional to the infectivity titer, observations from early stages of incubation to and (D) mice devoid of PrP gene are resistant to advanced disease indicated that the BSE agent is TSE [67]. Since PrP-c and PrP-sc are encoded by first taken up by the epithelial cells lining the lumen the same PRNP gene, they are antigenically of the digestive tract and spreads to lymphatic indistinguishable by the host, accounting for the tissues. It can later be traced along the nerve absence of immunologic and inflammatory changes pathways through ventral and dorsal root ganglia in the TSE. into the brain cortex where it accumulates during Analysis of kuru epidemic clearly indicates that advanced disease [85]. TSE can be transmitted orally. This has TSE isolates originating from different sources subsequently been confirmed in numerous have distinct biological characteristics. These isolates experiments. Of two squirrel monkeys orally fed with termed “strains” are distinguishable by their disease kuru brain tissue, one developed disease 36 months characteristics in experimentally infected animals, in after infection. Four squirrel monkeys were similarly particular the incubation periods and neuropathology fed with brain tissue of a patient with CJD or animals they produce in panels of inbred mice [86]. The with scrapie. All four developed spongiform strain “signature” is retained after experimental encephalopathy after 23 to 32 months [71]. Oral passage through a variety of intermediate species. transmission requires higher amounts of agent and is According to the “protein only” paradigm and generally an inefficient route of disease transmission. terminology, prion strains differ according to their Thus, transmission of scrapie to tga20 and wild-type host-specific amino acid sequence and the PrP-sc mice by i/c or i/p injection induced fatal disease in molecular conformation. The PrP-sc electrophoretic both species, whereas oral transmission of the same mobility pattern reflecting the size and abundance of RML scrapie agent resulted in disease in tga20 but the 3 major glycotypes is associated with its not the wild-type mice. Some of the wild-type mice conformation [87], and is therefore becoming a developed subclinical disease identified by infectivity useful tool for classification of TSE strains and and the presence of PrP-sc in the brain tissue [72]. establishing correlations with the disease Similar observations were made in hamsters phenotypes. PrP-sc extracted from the brain tissue subjected to dietary exposure to scrapie strain 263K and subjected to limited proteolysis with proteinase K [73]. results in truncated PrP27-30 molecule that is Data on genetically determined susceptibility to identified by western blotting. Three major PrP27-30 oral TSE transmission have become available from glycoforms, di-glycosylated, mono-glycosylated and experiments with scrapie and BSE in sheep. Analysis non-glycosylated, can be identified by molecular of the ovine PrP gene has shown that susceptibility weight. Originally, Parchi et al. [54] studied patients to scrapie is tightly linked to polymorphisms at with sporadic CJD and reported glycotypes 1 and 2 codons 136 (A/V), 154 (R/H), and 171 (Q/R) [74]. defined by the non-glycosylated glycoform variant Homozygosity for allele with A at position 136; R at with electrophoretic mobility corresponding to 154, and R at 171 (ARR/ARR) confers absolute molecular mass of either 21 or 19 kDa, and the resistance to subcutaneous inoculation of SSBP/1 PRNP genotype at codon 129. The difference in scrapie strain in Cheviot and Suffolk sheep; in molecular weight results from cleavage of PrP-sc at contrast, ARQ/ARQ genotype in Cheviot and different sites, which reflects distinct conformations VRQ/VRQ genotype in Suffolk sheep corresponds to [54]. Importantly, circular dichroism studies showed highest susceptibility [75, 76]. Strong association that Met-containing synthetic peptide has a greater between the VRQ allele and susceptibility to natural propensity to adopt a beta-sheet conformation and scrapie was demonstrated in many other breeds of aggregate into amyloid fibrils [88]. In a more sheep [77]. These susceptibility/resistance extended study that included hereditary TSE, kuru, relationships were fully confirmed in experiments in iCJD and vCJD cases, the classification of glycotypes 382 Current Molecular Medicine, 2004, Vol. 4, No. 4 Goldfarb et al. was modified to be based not only on the Recent genetic studies of vCJD demonstrated the differences in electrophoretic mobility and the PRNP absence of germ-line mutations in eight patients in genotype at codon 129, but also the ratio of three which the entire PRNP coding region was glycoforms [89]. The number of identifiable sequenced [96]. The PRNP codon 129Met/Met glycotypes was increased to six. Two studied kuru genotype has been a uniform finding in vCJD patients were classified as type 2 [89, 90]; vCJD patients, as it was in the early stages of kuru patients were classified as type 4 [89]. epidemic. If the analogy between kuru and vCJD can be extended to the increased, but not exclusive susceptibility of the 129Met/Met genotype, further RELEVANCE OF KURU STUDIES TO THE cases of vCJD may show longer incubation periods UNDERSTANDING OF THE VCJD OUTBREAK and occur in older individuals with alternative codon The situation in the Fore villages observed almost 129 genotypes, signaling a maturing evolution of the 50 years ago is in many respects similar to the vCJD epidemic. If the length of the incubation time in current outbreak of the newest infectious form of the carriers of the alternative genotypes is similar to TSE, vCJD. Convincing evidence has been what was observed in kuru (up to 40 years), the presented suggesting that vCJD is linked to vCJD epidemic would last longer and the overall consumption of contaminated meat products of number of vCJD cases be higher than currently animals incubating BSE [9, 92], and is therefore a estimated [97]. The phenotype in these genetically predominantly orally transmitted infection. PrP-sc alternative cases may be somewhat different from analysis has shown that the vCJD agent has strain the clinical and neuropathological features of the characteristics distinct from other types of human current cases [45]. TSE, but similar to characteristics of the BSE agent [93]. BSE was first identified in 1986 and spread throughout the U.K. to involve over 180,000 animals ABBREVIATIONS [92]. The disease resulted from changes in rendering TSE = Transmissible spongiform encephalopathy ovine and bovine carcasses for the production of CJD = Creutzfeldt-Jacob disease animal food pellets which permitted residual activity of the infectious agent. Although the use of vCJD = Variant Creutzfeldt-Jacob disease ruminant-derived cattle feed ceased in 1988, the iCJD = Iatrogenic Creutzfeldt-Jacob disease BSE epidemic reached a peak only in the late 1992 and subsided by 2001. In 1995 and 1996, GSS =Gerstmann-Sträussler-Scheinker disease approximately ten years after the beginning of the FFI = Fatal familial insomnia BSE epidemic, vCJD cases with distinctive clinical presentations and neuropathologic features were BSE = Bovine spongiform encephalopathy identified in British adolescents and young adults [9]. (“mad cow disease”) Further observations confirmed that this was a new PRNP = Human prion protein gene variant of CJD related to the BSE epidemic. More than 140 cases of vCJD have occurred in Great PrP = Prion protein Britain and single cases in several other countries, PrP-c = Host-encoded cellular PrP but there are many uncertainties about the future PrP-sc = Abnormal disease-associated PrP isoform course of the epidemic because the expected incubation period may be very long [94]. CNS = Central nervous system vCJD is characterized by a unique and consistent CSF = Cerebrospinal fluid phenotype of early behavioral and personality Gc = Group-specific component change, depression and memory loss. Ataxia is an early clinical feature in almost all patients. Dementia GcAb = Aborigine variant of Gc (GcAborigine) and myoclonus develop as the disease progress [9]. kDa = Kilodalton There are no periodic complexes on EEG. Average duration of illness is 14 months. The defining Amino acids: neuropathologic feature is a “kuru-type” amyloid Met=Methionine plaque composed of a central core and fibrillary periphery, surrounded by a rim of spongiform Val = Valine degeneration (“daisy” or “florid” plaques) [95]. A=Alanine Clusters of PrP-immunoreactive deposits not associated with spongiform change are widespread H=Histidine in the cerebral and cerebellar cortices, basal ganglia, Q=Glutamine thalamus and brainstem [95]. 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