Genotype patterns and characteristics of PRNP in the Korean population

Creutzfeldt-Jakob disease (CJD), included in the human transmissible spongiform encephalopathies (TSE), is widely known to be caused by an abnormal accumulation of misfolding prion protein in the brain. Human prion protein gene (PRNP) is mapped in chromosome 20p13 and many single nucleotide polymorphisms (SNPs) in PRNP have been discovered. However, the functionality of SNPs in PRNP is yet unclear, though several SNPs have been known as important mutation related with susceptibility human prion diseases. Our aim is to identify specific genotype patterns and characteristics in the PRNP genomic region and to understand susceptibility among Korean discriminated prion disease patients, suspected CJD patients and the KARE data group. Here, we have researched genotypes and SNPs allele frequencies in PRNP in discriminated prion disease patients group (n = 22), suspected prion diseases patients group (n = 163) and the Korea Association REsource (KARE) data group (n = 296) in Korea. The sequencing regions were promoter region, exon1 and exon2 with their junction parts among 481 samples. A total of 25 SNPs were shown in this study. Nucleotide frequencies of all SNPs are exceedingly tended to bias toward dominant homozygote types except in rs2756271. Genotype frequencies at codon 129 and 219 coding region were similar with previous studies in Korea and Japan. Pathogenic mutations such as 102P/L, 200E/K and 203V/I were observed in discriminated CJD patients group, and 180V/I and 232M/R were shown in suspected prion disease patients group and the KARE data group. A total of 10 SNPs were newly identified, six in the promoter region, one in exon 2 and three in the 3′ UTR. The strong and unique linkage disequilibrium (D' = 0.94, r² = 0.89) was observed between rs57633656 and rs1800014 which is located in codon 219 coding region. We expect that these data can be provided to determine specific susceptibility and a protective factor of prion diseases not only in Koreans but also in East Asians.     

Molecular genetic studies of Creutzfeldt-Jakob disease

Genetic study of over 200 cases of Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru have brought a reliable body of evidence that the familial forms of CJD and all known cases of GSS and FFI are linked to germline mutations in the coding region of the PRNP gene on chromosome 20, either point substitutions or expansion of the number of repeat units. No pathogenic mutations have so far been found in sporadic or infectious forms of CJD, although there are features of genetic predisposition in iatrogenic CJD and kuru. In FFI and familial CJD, clinically and pathologically distinct syndromes that are both linked to the 178^Asp→Asn substitution, phenotypic expression is dependent on a polymorphism at codon 129. Synthetic peptides homologous to several regions of PrP spontaneously form insoluble amyloid fibrils with unique morphological characteristics and polymerization tendencies. Peptides homologous to mutated regions of PrP exhibit enhanced fibrilogenic properties and, if mixed with the wild-type peptide, produce even more abundant and larger fibrous aggregates. A similar process in vivo may lead to amyloid accumulation and disease, and transmission of “baby fibrils” may induce disease in other hosts.     

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.     

Toward the molecular basis of inherited prion diseases: NMR structure of the human prion protein with V210I mutation

The development of transmissible spongiform encephalopathies (TSEs) is associated with the conversion of the cellular prion protein (PrP^C) into a misfolded, pathogenic isoform (PrP^Sc). Spontaneous generation of PrP^Sc in inherited forms of disease is caused by mutations in gene coding for PrP (PRNP). In this work, we describe the NMR solution-state structure of the truncated recombinant human PrP (HuPrP) carrying the pathological V210I mutation linked to genetic Creutzfeldt-Jakob disease. The three-dimensional structure of V210I mutant consists of an unstructured N-terminal part (residues 90-124) and a well-defined C-terminal domain (residues 125-228). The C-terminal domain contains three α-helices (residues 144-156, 170-194 and 200-228) and a short antiparallel β-sheet (residues 129-130 and 162-163). Comparison with the structure of the wild-type HuPrP revealed that although two structures share similar global architecture, mutation introduces some local structural differences. The observed variations are mostly clustered in the α₂-α₃ inter-helical interface and in the β₂-α₂ loop region. Introduction of bulkier Ile at position 210 induces reorientations of several residues that are part of hydrophobic core, thus influencing α₂-α₃ inter-helical interactions. Another important structural feature involves the alteration of conformation of the β₂-α₂ loop region and the subsequent exposure of hydrophobic cluster to solvent, which facilitates intermolecular interactions involved in spontaneous generation of PrP^Sc. The NMR structure of V210I mutant offers new clues about the earliest events of the pathogenic conversion process that could be used for the development of antiprion drugs.     

Structural differences between allelic variants of the ovine prion protein revealed by molecular dynamics simulations

We have modeled ovine prion protein (residues 119-233) based on NMR structures of PrP from other mammalian species. Modeling of the C-terminal domain of ovine PrP predicts three helices: helix-1 (residues 147-155), flanked by two short beta-strands; helix-2 (residues 176-197), and helix-3 (residues 203-229). Molecular dynamics simulations on this model of ovine PrP have determined structural differences between allelic variants. At neutral pH, limited root mean-squared (RMS) fluctuations were seen in the region of helix-1; between beta-strand-2 and residue 171, and the loop connecting helix-2 and helix-3. At low pH, these RMS fluctuations increased and showed allelic variation. The extent of RMS fluctuation between beta-strand 2 and residue 171 was ARR > ARQ > VRQ. This order was reversed for the loop region connecting helix-2 and helix-3. Although all three variants have the potential to display an extended helix at the C-terminal region of helix-1, the major influence of the VRQ allele was to restrict the conformations of the Asn162 and Arg139 side-chains. Variations observed in the simulations in the vicinity of helix-1 correlated with reactivity of C-terminal specific anti-PrP monoclonal antibodies with peripheral blood cells from scrapie-susceptible and -resistant genotypes of sheep: cells from VRQ homozygous sheep showed uniform reactivity, while cells from ARQ and ARR homozygous sheep showed variable binding. Our data show that molecular dynamics simulations can be used to determine structural differences between allelic variants of ovine PrP. The binding of anti-PrP monoclonal antibodies to ovine blood cells may validate these structural predictions.     

Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases

Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP^Sc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP^Sc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP^Sc in a prion-infected cell line after treatment with bafilomycin A₁. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.     

Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases

Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP^Sc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP^Sc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP^Sc in a prion-infected cell line after treatment with bafilomycin A₁. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.     

Linkage disequilibrium in the North American Holstein population

Linkage disequilibrium was estimated using 7119 single nucleotide polymorphism markers across the genome and 200 animals from the North American Holstein cattle population. The analysis of maternally inherited haplotypes revealed strong linkage disequilibrium ( r ²   >   0.8) in genomic regions of ∼50 kb or less. While linkage disequilibrium decays as a function of genomic distance, genomic regions within genes showed greater linkage disequilibrium and greater variation in linkage disequilibrium compared with intergenic regions. Identification of haplotype blocks could characterize the most common haplotypes. Although maximum haplotype block size was over 1 Mb, mean block size was 26–113 kb by various definitions, which was larger than that observed in humans (∼10 kb). Effective population size of the dairy cattle population was estimated from linkage disequilibrium between single nucleotide polymorphism marker pairs in various haplotype ranges. Rapid reduction of effective population size of dairy cattle was inferred from linkage disequilibrium in recent generations. This result implies a loss of genetic diversity because of the high rate of inbreeding and high selection intensity in dairy cattle. The pattern observed in this study indicated linkage disequilibrium in the current dairy cattle population could be exploited to refine mapping resolution. Changes in effective population size during past generations imply a necessity of plans to maintain polymorphism in the Holstein population.     

The first report of polymorphisms and genetic characteristics of the prion protein gene (PRNP) in horses

ABSTRACT

Prion diseases have a wide host range, but prion-infected cases have never been reported in horses. Genetic polymorphisms that can directly impact the structural stability of horse prion protein have not been investigated thus far. In addition, we noticed that previous studies focusing on horse-specific amino acids and secondary structure predictions of prion protein were performed for limited parts of the protein. In this study, we found genetic polymorphisms in the horse prion protein gene (PRNP) in 201 Thoroughbred horses. The identified polymorphism was assessed to determine whether this polymorphism impedes stability of protein using PolyPhen-2, PROVEAN and PANTHER. In addition, we evaluated horse-specific amino acids in horse and mouse prion proteins using same methods. We found only one single nucleotide polymorphism (SNP) in the horse prion protein, and three annotation tools predicted that the SNP is benign. In addition, horse-specific amino acids showed different effects on horse and mouse prion proteins, respectively.

Abbreviations: PRNP: prion protein gene; SNP: single nucleotide polymorphism; CJD: Creutzfeldt-Jakob disease; CWD: chronic wasting disease; TME: transmissible mink encephalopathy; FSE: feline spongiform encephalopathy; MD: molecular dynamics; ER: endoplasmic reticulum; GPI: glycosylphosphatidylinositol; NMR: nuclear magnetic resonance; ORF: open reading frame; GWAS: genome-wide association study; NAPA: non-adaptive prion amplification; HMM: hidden Markov model; NCBI: National Center for Biotechnology Information     

单核苷酸多态概述

单核苷酸多态ＳＮＰ是遍布于基因组中的一种ＤＮＡ序列变化类型,人类基因组中平均约每一千碱基中有一个。单核苷酸多态是一种双等位型多态,群体中出现的频率大于１％或２％者视为多态,低于１％或２％的则视为突变。由于其具有高信息量、高密度又便于自动化操作的特点,单核苷酸多态在遗传性疾病基因的克隆和药物的设计与开发方面具有广阔的应用前景。本文对单核苷酸的概念、特点、应用前景,及其研究应用的一些问题作一综述。     

Association between the PRNP 1368 polymorphism and the occurrence of sporadic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disorder. The etiology of sporadic form of CJD remains unsolved. In addition to the codon 129 polymorphism, polymorphisms in the non-coding region of PRNP are considered as important factors in sCJD development. To assess a possible association between PRNP 1368 SNP and sCJD, we compared the genotype, allele and haplotype frequencies of the 1368 SNP among 46 sCJD patients of Dutch origin with the respective frequencies in healthy controls. We detected a significant association between sCJD and 1368T/T genotype. A significant difference was also observed in 1368 alleles’ distribution. In the haplotype analysis, haplotype 1368C-129G was associated with decreased risk of sCJD in Dutch population. Our findings support the hypothesis that genetic variations in the regulatory region of the PRNP gene may influence the pathogenesis of sCJD.     

APOA1 gene polymorphisms in the South Asian immigrant population in the United States

BACKGROUND:

Coronary artery disease (CAD) is a leading cause of death in the United States. South Asian immigrants (SAIs) from the Indian subcontinent living in the US are disproportionately at higher risk of CAD than other immigrant populations. Unique genetic factors may predispose SAIs to increased risk of developing CAD when adopting a Western lifestyle including a higher-fat diet, more sedentary behavior and additional gene-environment interactions. SAIs are known to have low levels of the protective high density lipoprotein (HDL) and an altered function for Apo-lipoprotein A-1 (ApoA1), the main protein component of HDL cholesterol. One gene that may be genetically distinctive in this population is APOA1 which codes for ApoA-1 protein, a potentially important contributing factor in the development of CAD.

MATERIALS AND METHODS:

DNA sequencing was performed to determine the status of the seven single-nucleotide polymorphisms (SNPs) in the APOA1 gene from 94 unrelated SAI adults. Genotypes, allelic frequencies, and intragenic linkage disequilibrium of the APOA1 SNPs were calculated.

RESULTS:

Several polymorphisms and patterns were common among persons of south Asian ethnicity. Frequencies for SNPs T655C, T756C and T1001C were found to be different than those reported in European Caucasian individuals. Linkage disequilibrium was found to be present between most (13 of 15) SNP pairings indicating common inheritance patterns.

CONCLUSIONS:

SAIs showed variability in the sequence of the APOA1 gene and linkage disequilibrium for most SNPS. This pattern of APOA1 SNPs may contribute to decreased levels of HDL cholesterol reported in SAIs, leading to an increased risk for developing CAD in this population.     

Association between the PRNP 1368 polymorphism and the occurrence of sporadic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare transmissible neurodegenerative disorder. The etiology of sporadic form of CJD remains unsolved. In addition to the codon 129 polymorphism, polymorphisms in the non-coding region of PRNP are considered as important factors in sCJD development. To assess a possible association between PRNP 1368 SNP and sCJD, we compared the genotype, allele and haplotype frequencies of the 1368 SNP among 46 sCJD patients of Dutch origin with the respective frequencies in healthy controls. We detected a significant association between sCJD and 1368T/T genotype. A significant difference was also observed in 1368 alleles’ distribution. In the haplotype analysis, haplotype 1368C-129G was associated with decreased risk of sCJD in Dutch population. Our findings support the hypothesis that genetic variations in the regulatory region of the PRNP gene may influence the pathogenesis of sCJD.     

Genome-wide population structure and evolutionary history of the Frizarta dairy sheep

In the present study, we used genomic data, generated with a medium density single nucleotide polymorphisms (SNP) array, to acquire more information on the population structure and evolutionary history of the synthetic Frizarta dairy sheep. First, two typical measures of linkage disequilibrium (LD) were estimated at various physical distances that were then used to make inferences on the effective population size at key past time points. Population structure was also assessed by both multidimensional scaling analysis and k-means clustering on the distance matrix obtained from the animals’ genomic relationships. The Wright’s fixation F_ST index was also employed to assess herds’ genetic homogeneity and to indirectly estimate past migration rates. The Wright’s fixation F_IS index and genomic inbreeding coefficients based on the genomic relationship matrix as well as on runs of homozygosity were also estimated. The Frizarta breed displays relatively low LD levels with r² and |Dʹ| equal to 0.18 and 0.50, respectively, at an average inter-marker distance of 31 kb. Linkage disequilibrium decayed rapidly by distance and persisted over just a few thousand base pairs. Rate of LD decay (β) varied widely among the 26 autosomes with larger values estimated for shorter chromosomes (e.g. β=0.057, for OAR6) and smaller values for longer ones (e.g. β=0.022, for OAR2). The inferred effective population size at the beginning of the breed’s formation was as high as 549, was then reduced to 463 in 1981 (end of the breed’s formation) and further declined to 187, one generation ago. Multidimensional scaling analysis and k-means clustering suggested a genetically homogenous population, F_ST estimates indicated relatively low genetic differentiation between herds, whereas a heat map of the animals’ genomic kinship relationships revealed a stratified population, at a herd level. Estimates of genomic inbreeding coefficients suggested that most recent parental relatedness may have been a major determinant of the current effective population size. A denser than the 50k SNP panel may be more beneficial when performing genome wide association studies in the breed.     

Exploring the zoonotic potential of animal prion diseases

Following the discovery of a causal link between bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt–Jakob disease (vCJD) in humans, several experimental approaches have been used to try to assess the potential risk of transmission of other animal transmissible spongiform encephalopathies (TSEs) to humans. Experimental challenge of non-human primates, humanised transgenic mice and cell-free conversion systems have all been used as models to explore the susceptibility of humans to animal TSEs. In this review we compare and contrast in vivo and in vitro evidence of the zoonotic risk to humans from sheep, cattle and deer prions, focusing primarily on chronic wasting disease and our own recent studies using protein misfolding cyclic amplification.     

A lack of association between adiponectin polymorphisms and coronary artery disease in a Chinese population

We investigated the association between two single nucleotide polymorphisms (SNPs) in the adiponectin gene (rs822395 and rs266729) and coronary artery disease (CAD) in a case-control study of 198 unrelated Chinese CAD patients (with ≥ 70% coronary stenosis or previous myocardial infarction) and 237 non-CAD controls. The ligase reaction was used to detect SNPs rs822395 and rs266729, and the allelic association of these SNPs with the occurrence and severity of CAD was assessed. There were no significant differences in the genotypic or allelic frequencies of the two SNPs between control and CAD individuals. In addition, there was no association between the two SNPs and the severity of CAD based on the number of diseased vessels. The frequencies of alleles C and G at rs266729 differed significantly between females in the CAD and control groups, but not between males. Female carriers of allele G at rs266729 had a higher risk of CAD compared with allele C carriers (OR = 1.30, 95% CI: 1.09-2.64, p = 0.02). These results indicate a gender-specific effect of the adiponectin gene rs266729 variant in modulating the risk of CAD in women.     

Linkage disequilibrium and signatures of selection on chromosomes 19 and 29 in beef and dairy cattle

The objective of this study was to quantify the extent of linkage disequilibrium (LD) on bovine chromosomes 19 and 29 and to study the pattern of selection signatures in beef and dairy breeds (Angus and Holstein) of Bos taurus. The extent of LD was estimated for 370 and 186 single nucleotide polymorphism markers on BTA19 and 29 respectively using the square of the correlation coefficient (r(2)) among alleles at pairs of loci. A comparison of the extent of LD found that the decline of LD followed a similar pattern in both breeds. We observed long-range LD and found that LD dissipates to background levels at a locus separation of about 20 Mb on both chromosomes. Along each chromosome, patterns of LD were variable in both breeds. We find that a minimum of 30 000 informative and evenly spaced markers would be required for whole-genome association studies in cattle. In addition, we have identified chromosomal regions that show some evidence of selection for economically important traits in Angus and Holstein cattle. The results of this study are of importance for the design and application of association studies.