spinal muscular atrophy (Kugelberg-Welander syndrome) Journal of the . Kuru S, Sakai M, Konagaya M, Yoshida M, Hashizume Y, Saito K. Primary Sjögrens’s syndrome (pSS) is an autoimmune, chronic . (11), and the NCBI 16S rRNA reference sequence set (ftp:// ). .. Kuru B, McCullough MJ, Yilmaz S, Porter SR. Human prion diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann- Straussler-Scheinker syndrome (GSS), kuru, fatal familial.

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Conceived and designed the experiments: Sporadic Creutzfeldt-Jakob disease sCJD is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein PrP Sc. In both forms of CJD, heterozygosity at residue for methionine M or valine V in the prion protein gene may affect disease phenotype, onset and progression.

Therefore, factors other than PrP Sc allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD. In Creutzfeldt-Jakob disease CJDheterozygosity at residue for methionine or valine in normal prion protein may affect disease phenotype, onset and progression.

However, the relative contribution of each prion protein allotype to the infectious, disease associated form of prion protein PrP Sc is unknown. Here we report the novel observation that in heterozygous cases of sporadic CJD the PrP Sc allotype ratio is highly variable. This case-by-case variability is consistent with the origin of sporadic CJD being the spontaneous, sendrou random, misfolding of either host prion protein allotype into infectious PrP Sc.

By contrast, in heterozygous cases of iatrogenic Hz in the United Kingdom resulting from exposure to contaminated human growth hormone, the PrP Sc allotype ratio is much more homogeneous and consistent with exposure to infectious PrP Sc containing valine at residue Surprisingly, the PrP Sc allotype ratio did not correlate with disease onset or duration in either disease type.

Thus, factors other than PrP Sc allotype ratio likely influence the clinical progression of heterozygous cases of CJD. Moreover, our results suggest that the ratio of methionine to valine in PrP Sc may be a means of determining the origin of prion infection. Prion diseases are fatal neurodegenerative disorders affecting humans and various other mammals.

They are associated with the misfolding of monomeric prion protein PrP C into a pathological isoform termed PrP Sc that is partially protease resistant, aggregated, and infectious.

Although CJD occurs in sporadic, genetic, and iatrogenic forms, the most common form is sporadic CJD sCJDwhich occurs at approximately 1—2 cases per million people per year in any given population.

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CJD is associated with a wide diversity of clinicopathological features [ 145 ], but the factors that determine these different CJD phenotypes are still being elucidated. Sporadic CJD is classified by a neuropathological profile that appears to correlate with the biochemical properties of PrP Sc [ 4 — 7 ] as well as the sequence of the patient prion protein gene PRNP at codon These correspond well to the differing clinicopathological presentation found in patients in the six well-recognized sCJD phenotypic subtypes: Based on these criteria, as well as transmission studies in non-human primates [ 10 ] and transgenic mice expressing different human PRNP genotypes [ 1112 ], 5 major strains of sCJD have been proposed: Although not causing disease itself, the presence of M or V at residue in PrP C does affect many aspects of prion disease susceptibility and phenotype [ 14 — 913 ].

For example, in a familial form of human prion disease associated with an aspartic acid to asparagine mutation at residue DNa methionine in cis at position manifests as FFI, while a valine in cis at position manifests as CJD [ 14 ]. The codon genotype also affects susceptibility to CJD. Methionine and valine homozygosity at codon are overrepresented in sCJD patients when compared to the general population, whereas MV heterozygosity at codon is underrepresented [ 15 — 18 ].

A similar allelic distribution has been observed in human growth hormone associated cases of iCJD in France [ 19 ]. The predisposition towards homozygosity in CJD is even more pronounced in variant CJD, in which all probable and definite clinical cases reported to date have been MM homozygous [ 16 ].

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Thus, the epidemiological data suggest that the effect of homozygosity or heterozygosity at codon on CJD pathogenesis varies depending upon the type of CJD. This variability may be due to multiple factors including the relative abundance of different populations of PrP Sc.

For example, in the brain the amount of protease-sensitive PrP Sca conformationally distinct population of PrP Sc that is aggregated but susceptible to digestion with proteinase K kuuru 21 ], has been correlated with disease progression rate. This latter observation is particularly intriguing since the presence of two different PrP allotypes in the same brain can often lead, in a dose-dependent manner, to inefficient PrP Sc formation sendfomu increased disease incubation times [ 2627 ].

Thus, a ready explanation for the variability in disease onset and duration in heterozygous cases of CJD would be quantitative differences between PrP Sc allotypes containing M or V at residue Tandem mass spectrometry was then used to differentiate peptides containing M at residue from those containing V.


In previous MS-based analysis of purified hamster and mouse PrP Scwe found that one of the most commonly identified PrP peptides spanned residues — PrP [ 2829 ]. The same procedure was used on brain samples from several non-CJD neurological controls and the samples were compared. Many of these peptides spanned the region of PrP from residues — and showed variable levels of methionine oxidation S1 Table.

Importantly, the MV polymorphism at residue was distinguishable based upon the unique fragmentation patterns of peptides containing either M or V Fig 1a and 1b. Peaks were visualized using Scaffold v4.

Italicized methionine residues and in panel A and methionine in panel B are oxidized in this representative example. However, the relative abundance and thus potential extent of the contribution of each allotype to disease pathogenesis is unknown. Spectral counting, which is defined as the number of MS spectra identified for a protein, is a semi-quantitative technique that is often used as a practical method for estimating protein abundance [ 31 ].

It provides a robust label-free estimate for comparing the relative sensromu of proteins in or between sample groups [ 32 ]. At a molar ratio of Thus, spectral counting allowed us to estimate the relative abundance of M or V at residue in a heterozygous mixture of human PrP, albeit with a slight bias towards detection of the M versus the V PrP peptide. Patient brain samples from 14 cases of heterozygous sCJD were analyzed and their clinical and molecular data are presented in Table 1.

The prominent neuropathological features of each case are described in S2 Table and exemplified by images of PrP immunohistochemistry performed on cerebral cortex CC and, where relevant to this study, cerebellar cortex CbC samples Fig 2a. The pathological and molecular features correspond broadly to the histopathological types proposed by Parchi et al [ 5 ]. However, in certain cases, such as case 3, there was a discrepancy between molecular and histopathological findings see S2 Table suggesting an atypical case of MV1 sCJD.

Analysis of samples from one sCJD MV1 case specimen consistently showed a mobility slightly ahead of the Type 1 reference standard Fig 3acase 4. The blots further resolved the presence of a Type 2 doublet 2d in 6 out of the 9 MV2 subtype cases Table 1 and Fig 3a. The relative amounts of the two doublets varied between samples from equivalence case 10 CC to barely detectable amounts of the least abundant band case 7 CC. Original magnifications were X The PrP antibody used was the mouse monoclonal antibody KG9 [ 34 ].

Case number and brain region are indicated above each blot while PrP Sc type is indicated below. The most informative exposure for the test and reference standard samples from a series of timed exposures is shown. For each patient sample, the number of PrP spectra spanning residues PrP was determined. However, the amount of PrP Sc -M Fig 4 as well as the type of PrP Sc deposition Fig 2a differed significantly between brain regions in the two other cases, 9 and While only 4 sCJD cases were analyzed, the data suggest that within the same patient the amount of PrP Sc -M and PrP Sc -V can differ markedly between different regions of the brain with distinctive neuropathologies.

The case number is indicated under each pair of bars. In order to determine if differences in the amount of each allotype were associated with phenotypic variables, the relative abundance of PrP Sc -M was compared to the sCJD molecular subtype, age of onset, and duration of clinical disease.

Thus, despite significant differences between cases, the MV ratio of the PrP Sc allotypes did not appear to overtly influence disease onset or progression. In two cases, 9 and 14, differences in the PrP Sc allotype ratio between brain regions Fig 4 were associated with different neuropathologies Fig 2a.

However, when all of the MV sCJD cases were compared, there was no definitive correlation between the amount of PrP Sc -M and various neuropathological features such as the presence of amyloid plaques, pattern of PrP Sc deposition or type of spongiform change S2 Table. The prominent neuropathological features of each case are described in S2 Table and exemplified by images of PrP immunohistochemistry performed on cerebral cortex samples in Fig 2b. Western blot analysis of a tissue sample from the specimen used for MS using the method of Parchi et al.

With the caveat that only 2 cases of sCJD MV2K were available for analysis, these data are nonetheless consistent with the hypothesis that UK cases of human growth hormone associated iCJD are the result of exposure to the V2 strain of human prion.

Each point represents the results from a single brain region from a single patient. For some patients, both CC and CbC were available for analysis. Efficient conversion of PrP C to PrP Sc can be strongly dependent upon homology at a single amino acid residue in PrP C [ 35 ], including residue [ 3637 ], and differences in key amino acid residues can effect disease onset and progression [ 273839 ].

However, we found no evidence that M or V at residue in PrP Sc correlated with disease onset or progression. This is consistent with some studies of familial human prion disease which also suggest that disease phenotype does not necessarily correlate with the presence of M or V at residue in mutant PrP C [ 2440 ].


It is possible that, in heterozygous cases of sCJD, the polymorphism at residue does not necessarily lead to protease-resistant PrP Sc conformations that are significantly different. Since PrP Sc conformation is thought to encode prion strain phenotypes [ 1 ], PrP Sc -M and PrP Sc -V might therefore contribute minimally to conformational differences affecting disease phenotype. Unknown host factors or other forms of abnormal PrP which may or may not be equivalently infectious, such as those with atypical protease-digestion profiles [ 23 ] or increased protease-sensitivity [ 21 ], may have a more significant influence on age of onset or duration of clinical disease than the presence of an M or V at residue in Type 1 or Type 2 PrP Sc.

The epidemiological associations of Type 1 PrP Sc with methionine homozygosity and Type 2 PrP Sc with valine homozygosity within sCJD patient cohorts could be used to argue that the presence of methionine at position of the prion protein predisposes it to misfold to a Type 1 conformation, whereas the presence of valine at the same position predisposes it to misfold to a Type 2 conformation. Data from transmission studies in transgenic mice [ 111241 ] and in vitro assays of PrP Sc formation [ 37 ] are consistent with this prediction.

It is possible, however, that there may be some correlation with phenotypic differences between brain regions in the same patient. In cases 9 and 14, where distinctive pathologies were observed in the cerebral cortex and cerebellar cortex Fig 2the PrP Sc allotype ratios were also significantly different Fig 4.

While these data are suggestive that PrP Sc allotype may correlate with regional differences in neuropathology, further analysis of multiple brain regions from a greater number of patients will be necessary to prove this correlation. An alternative explanation for the heterogeneity in the MV PrP Sc allotype ratio may lie in its proposed aetiology. The relatively equal representation of the different possible PrP Sc allotypes does not appear to be consistent with one PrP C molecule being more predisposed to misfold into PrP Sc than the other, as has been suggested by several in vitro studies [ 42 — 44 ].

In this context, the differences in relative abundance between the two allotypes could be interpreted as indicating which PrP C molecule initially misfolded into PrP Sc.

However, it is important to note that we have only analyzed a small portion of the brain from the end stage of a long and complex disease process.

It is more likely that there are multiple variables, both host and prion specific, that contribute to the relative abundance of each PrP Sc allotype at the end stage of disease.

A similar hypothesis was proposed by Hosszu et al.

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By contrast, there is evidence that there are differences in the stability of the downstream products of PrP C misfolding. Therefore, while similar free energy barriers for both PrP C -M and PrP C -V would suggest that they may be equally likely to misfold into PrP Scvariability in the thermodynamic stability of the PrP Sc end products may be more important in determining the final relative abundance of each PrP Sc allotype.

Based on the earlier cases, Brandel et al. Used in this way, mass spectrometry analysis of heterozygous PrP Sc could be the molecular equivalent of in vivo traceback studies that have used the transmission properties of iCJD into transgenic mice [ 4748 ] or non-human primates [ 10 ] to determine the prion strain originally responsible for infection in groups of patients. Trypsin was purchased from Promega.

Imperial Coomassie blue stain and iodoacetamide were purchased from Thermo-Fisher Scientific. For the purposes of this study, samples were pseudoanonymized using a Brain Bank reference number. Cerebellar and cerebral cortex tissue from two additional sCJD patients were obtained from the University of Verona, Italy. These tissues were obtained at autopsy and sent to the Neuropathology Unit at the University of Verona for statutory definite diagnosis of CJD.

The proteins were expressed as inclusion bodies using Escherichia coli Rosetta cells and then purified as described for recombinant hamster PrP [ 49 ]. Briefly, guanidine denatured PrP from bacterial inclusion bodies was clarified by centrifugation, bound to NiNTA resin Qiagenand then subjected to on-column refolding without reducing agents using a non-denaturing refolding buffer 10mM Tris, mM sodium phosphate, pH 8. After elution using refolding buffer adjusted to pH 5.

In addition, the molecular weight of purified recombinant PrP was verified by intact mass analysis using a Sciex QTrap system. Concentrations of purified rHuPrP-M and rHuPrP-V proteins were determined individually by absorbance at nm and then adjusted to contain molar ratios of 0: Final mixtures containing 0. Coomassie blue staining displayed a single strong band which was excised and subjected to in-gel trypsin digestion as described below.