NR AMQI
AU Windl,O.; Giese,A.; Schulz-Schaeffer,W.J.; Zerr,I.; Skworc,K.; Arendt,S.; Oberdieck,C.; Bodemer,M.; Poser,S.; Kretzschmar,H.A.
TI Molecular genetics of human prion diseases in Germany
QU Human Genetics 1999 Sep; 105(3): 244-52
PT journal article
AB Human prion diseases may be acquired as infectious diseases, they may be inherited in an autosomal dominant fashion or occur sporadically. Mutations and polymorphisms in the sequence of the coding region of the prion protein gene (PRNP) have been established as an important factor in all of these three types of prion diseases. Therefore, a total of 578 patients with suspect prion diseases referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit over a period of 4.5 years have been examined for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a missense mutation previously reported as pathogenic. Amongst these, the aspartate to asparagine change at codon 178 (D178N) was the most common mutation. All of these cases carried the D178N mutation in coupling with methionine at codon 129, resulting in the typical fatal familial insomnia (FFI) genotype. Most cases with pathogenic mutations were not found in the group of clinically "probable" cases according to established clinical criteria, supporting the notion that inherited prion diseases often exhibit atypical features. Two novel missense mutations (T188R and P238S) and several silent polymorphisms were found, demonstrating the quality of our screening procedure based on a modified version of the single-stranded conformational polymorphism technique. In "definite" CJD cases with no pathogenic mutation, the patients clinically classified as "probable" were mostly homozygous for methionine at the common polymorphism at codon 129, whereas there was a marked over-representation of patients homozygous for valine amongst those clinically classified as "possible". This large study on suspect cases of human prion diseases in Germany clearly shows that PRNP genetics is essential for a comprehensive analysis of prion diseases.
VT
Introduction
Human prion diseases occur as either sporadic diseases, in around 90% of the cases, or inherited disorders in around 10%. In addition, a small number of cases are caused by transmission from infectious human and presumably bovine material (Collinge et al. 1996; Will et al. 1996; Bruce et al. 1997; Prusiner 1997). Classically, three human prion diseases are distinguished according to the clinicopathological presentation, i.e. Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI). The majority of cases is represented by the CJD-type of prion disease, which is mainly sporadic and is inherited only in a small percentage of cases. One of the first reported CJD cases was a familial case. Hence, for a long time, CJD was considered a neurodegenerative and inherited disorder (Jakob 1921; Meggendorfer 1930; Kretzschmar et al. 1995a). The infectious nature of CJD was only shown in 1968 (Gibbs et al. 1968). In contrast to CJD, the rare GSS and FFI types of prion diseases seem to be exclusively inherited disorders. All inherited human prion diseases have been found to segregate with mutations in the coding region of the human prion protein gene (PRNP, Fig. 1a) (Mastrianni et al. 1996; Nitrini et al. 1997; Samaia et al. 1997; Windl and Kretzschmar 1999). Two types of mutations in PRNP have been found. First, point mutations, which lead to amino acid exchanges in the central and the C-terminal part of the prion protein (PrP). Second, insertions of multiples of a 24-bp unit that increase the number of octapeptide repeats within the N-terminal region of PrP. This normally carries five tandem repeats of the octapeptide motif, though the first repeat in the authentic human PrP is actually a nonapetide. Several of these mutations are associated with distinct clinicopathological phenotypes. For instance, P102L is associated with GSS, E200K with CJD and D178N with FFI (Goldgaber et al. 1989; Hsiao et al. 1989; Medori et al. 1992). In this last case, the presence of a methionine at the polymorphic codon 129 is required in coupling with the asparagine (N) at codon 178, to present the distinct features of FFI. In coupling with valine at codon 129, D178N is instead associated with CJD (Goldfarb et al. 1992).
---------------------------
Fig. 1. Known pathogenic mutations in the coding region of PRNP. The coding region of PRNP is depicted as a bar and the
various point mutations as well as the different insert mutations are drawn in scale. The single letter code for amino acids is
used and the stop codon is abbreviated by an asterisk in Y145*
---------------------------
The emergence of bovine spongiform encephalopathy (BSE) in the mid-1980s (Wilesmith et al. 1988) has caused great concern in the public. A new variant of CJD (nvCJD), to date mainly observed in the U.K. (Will et al. 1996), may be caused by infection of humans with BSE agent (Collinge et al. 1996; Bruce et al. 1997). A prerequisite for the detection of such a new variant of human prion disease was a surveillance program set up in the U.K. in 1990 and subsequently in many other European countries. As part of this surveillance, a genetic analysis of the coding region of PRNP is mandatory in suspect cases for several reasons. (1) It is necessary for the identification of inherited cases of prion diseases and vice versa defines the cases without mutation in PRNP as sporadic or acquired by infection. This is particularly important as many of the affected families are unaware of a genetic prediposition to dementing disease and can therefore be diagnosed only on the basis of a PRNP analysis. (2) Atypical forms of prion diseases are often caused by mutations in PRNP, and many of them are only detectable by genetic screening of cases clinically classified as not CJD. (3) Novel missense mutations and polymorphisms may define novel subtypes of prion diseases by displaying distinct clinical or pathological phenotypes. The analysis of more cases of known but extremely rare missense mutations and polymorphisms will provide more detailed information on the association between particular PRNP genotypes and their clinicopathological phenotype. (4) It is important to type for the common polymorphism at codon 129, because several variants of sporadic CJD with distinct clinical and pathological phenotypes are largely determined on the one hand by this polymorphism and on the other by two types of protease-resistant PrP. Both factors seem to be independent of each other and give rise to six different disease phenotypes of sporadic CJD (Parchi et al. 1996, 1998). (5) The diagnosis of nvCJD demands the analysis of PRNP to exclude a pathogenic mutation, which might mimic nvCJD, and also to determine the amino acid residue at the polymorphic codon 129 (Will et al. 1996). (6) Finally, the analysis of PRNP allows differentiation from other familial diseases with dementia, in particular the familial type of Alzheimer's disease.
As part of the German surveillance program for human prion diseases, we examined all suspect cases referred to us after informed consent; these were screened for previously described and novel mutations in the coding region of PRNP.
Subjects and methods
Subjects
Blood samples were collected from suspect cases of CJD referred while still living to the National Surveillance Unit after informed consent of the patients or their relatives. Five hundred and two cases were seen by a neurologist of the surveillance team and were classified as "probable", "possible" or "other" according to published criteria (Masters et al. 1979; Budka et al. 1995). The remaining 76 cases were referred without clinical classification as suspect prion disease ("not classified"). The diagnosis "definite" CJD was made post-mortem using neuropathological criteria (Kretzschmar et al. 1996).
DNA extraction
Genomic DNA was extracted in most cases from blood using the QIAamp Blood Kit (Qiagen, Hilden, Germany). In some cases, the genomic DNA was extracted from fresh-frozen brain tissue or formalin-fixed and paraffin-embedded brain tissue using the QIAamp Tissue Kit (Qiagen). All extraction procedures were performed according to the manufacturer's recommendations.
Mutation detection
The entire coding region of PRNP was amplified by means of the polymerase chain reaction (PCR) as described previously (Windl et al. 1996). The PCR product was inspected on a 1% agarose gel to detect potential insertion mutations and deletions. The screening for prospective point mutations was performed by the single-strand conformational polymorphism (SSCP) technique (Orita et al. 1989). For this purpose, the PCR product spanning the coding region of PRNP was eluted from the agarose gel using the QIAEX II procedure (Qiagen) and re-amplified in four overlapping fragments with the following primers in four separate reactions:
Fragment 1. 5'-CTGACATTCTCCTCTTC-3' and 5'-CGGTTGCCTCCAGGGCT-3'
Fragment 2. 5'-CCTGGAGGATGGAACAC-3' and 5'-GTAGCCGCCAAGGCCCC-3'
Fragment 3. 5'-TGGCACCCACAGTCAGT-3' and 5'-TTCTCCCCCTTGGTGGT-3'
Fragment 4. 5'-CGTGAAAACATGCACCG-3' and 5'-CCTCAAGCTGGAAAAAG-3'
All of these primers were modified at their 5'-ends to allow non-radioactive detection of the PCR products. The primers of fragments 1 and 2 carried the fluorescent dye IRD-41 (LI-COR, Lincoln, Neb.), whereas digoxigenin (DIG) was attached to the primers of fragments 3 and 4. The PCR conditions for these nested reactions, the sample preparation and the SSCP gels were as previously published (Windl et al. 1996), except that radioactive isotopes were omitted. The detection of the labelled products was different for (A) fragments 1 and 2 and (B) fragments 3 and 4 due to the different nature of the labels. (A) The fluorescent products were visualised digitally during electrophoresis on an automated sequencing system (Model 4000L; LI-COR). (B) The DIG-labelled products were transferred to a nylon membrane (Hybond-N+, Amersham Buchler, Braunschweig, Germany) by contact blotting and visualised with the help of an alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim, Mannheim, Germany) and nitroblue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate, according to the manufacturer's recommendations.
Mutation analysis
The exact nature of the changes detected using the SSCP technique were determined by means of (1) digestion with the appropriate restriction endonuclease, (2) direct sequencing of the PCR product or (3) sequencing following cloning of parts of the PCR product.
1.The PCR product of all patients was analysed by means of NspI digestion to determine the codon 129 of PRNP. The restriction endonucleases DdeI, PvuII, Eco0109I, Tth111I and BsmAI were used to analyse codons 102, 117, 124, 178 and 200, respectively, whenever the SSCP analysis suggested a change at one of these codons in PRNP. In the cases of heterozygosity at codon 129 (methionine and valine) and the pathogenic D178N mutation on one of the alleles, the amino acid at codon 129 on the allele with the mutation was determined by the simultaneous digestion of the complete coding region of PRNP with MaeII and Tth111I. The digestion products were analysed on a 2% agarose gel. The enzymes were supplied by Amersham Buchler, Boehringer Mannheim, Promega (Madison, Wis.) and New England Biolabs (Beverly, Mass.) and were used according to the manufacturer's recommendations.
In the case of the silent polymorphism at codon 117, the coding region of PRNP was analysed a second time, as it is known for those patients that the allele carrying the polymorphic codon (GCG instead of GCA) is amplified preferentially and the other allele is possibly missed by the analysis (Palmer et al. 1996). This is due to an additional polymorphism within the sequence of the standard primer 5' to the coding region (e.g. primer A in Windl et al. 1996) co-segregating with the silent polymorphism at codon 117. Therefore, a different primer on the 5' side of the coding region was used (5'-TCCGGTACCGCAGAGCAGTCATTATGGCGAAC-3'), which overlaps with the start codon of the open reading frame. This primer together with the standard primer 3' to the coding region (primer B in Windl et al. 1996) was used for the amplification of the coding region of PRNP. The SSCP analysis (fragments 2, 3 and 4) and the NspI digest were repeated with this second product.
2.For direct sequencing of the complete coding region of PRNP, we followed a protocol developed by B. Fartmann (MWG, Ebersberg, Germany). The purified PCR product was re-amplified using the primers 895Wta (5'-TGTAAAACGACGGCCAGTTCTCCTCTTCATTTTGCAGAG-3') and 896Wta (5'-CAGGAAACAGCTATGACCCCTCAAGCTGGAAAAAGATTAG-3'), which carried sequences at their 5'-ends homologous to the standard sequencing primers in pBluescript-derived vectors. The PCR conditions were identical to the amplification of PRNP from genomic DNA, except that only 20 cycles were performed. The re-amplified product was purified again and cycle sequenced using Thermo Sequenase (Amersham Buchler) according to the manufacturer's recommendations and 5'-IRD-41 labelled primers uni(-21) (5'-TGTAAAACGACGGCCAGT-3') or rev(-29) (5'-CAGGAAACAGCTATGACC-3'). The labelled products were separated by denaturing electrophoresis on a 4.3% long-ranger gel (AT Biochem, Malvern, Pa.) and monitored with the help of an automated system (Model 4000L; LI-COR).
3.For sequence analysis of PRNP of patients with insertion mutations, a part of the PRNP ORF comprising most of the N-terminal half of the protein was cloned. The PRNP ORF was cut with the restriction endonucleases MspI and PstI and cloned into pBluescriptIIKS(-) (Stratagene, La Jolla, Calif.), that was itself cut with the enzymes ClaI and PstI to create compatible ends. The inserted part of PRNP was sequenced employing Thermo Sequenase and the primers uni(-21) or rev(-29) as outlined in (B).
Results
Detection of mutations in PRNP
Due to the large number of suspect CJD cases in Germany and the considerable number of known mutations (Fig. 1), we decided to employ a screening procedure that allows for rapid detection of mutations in the coding region of PRNP. We developed a non-radioactive version of the SSCP procedure and, therefore, had to modify our original protocol in several aspects (Windl et al. 1996). The main feature consists of two different non-radioactive labels used for different fragments of PRNP. This turned out to be the most sensitive way to detect mutations in this gene. The reliability of our method is demonstrated by (1) the safe detection of known mutations in our patient sample and (2) the detection of several novel missense mutations and polymorphisms in the coding region of PRNP as outlined below.
Incidence of known pathogenic mutations in Germany
Within the 578 cases of suspect prion diseases, we found 40 cases with a previously described pathogenic mutation (Table 1). Most commonly (13 of 40 cases, 32.5%), we found the aspartate to asparagine change at codon 178 (D178N), which was first described by Goldfarb and colleagues in a Finnish CJD kindred (Goldfarb et al. 1991c). All of the 13 subjects of our study carried the mutation on the allele with methionine at codon 129 and, therefore, were of the FFI genotype (Goldfarb et al. 1992; Medori et al. 1992). The glutamate to lysine change at codon 200 (E200K) was detected in 8 of 40 cases (20%). This mutation was described as the most common cause of familial CJD (Goldgaber et al. 1989; Hsiao et al. 1989; Goldfarb et al. 1991b). The proline to leucine change at codon 102 (P102L) was found in seven cases (17.5%). The P102L mutation is known to be the major cause of GSS worldwide (Hsiao et al. 1989; Young et al. 1995). The valine to isoleucine change at codon 210 (V210I) was detected in six cases of our group (15% of all cases with pathogenic mutations). This mutation is associated with familial CJD. Finally, five cases with insertional mutations were discovered in our patient sample (12.5%), which will be described in more detail below, and one case was found (2.5%) with a threonine to alanine change at codon 183 of PRNP. These 40 patients with previously described pathogenic mutations belong to 34 families (Table 1).
---------------------------
Table 1. Type of mutations. NA not applicable
Mutation Cases Families Cases/family Type of insert
Inserts 5 (12.5%) 4 (12%) 2 5 x 24 bp
1 9 x 24 bp
1(2x) 5 x 24 bp
P102L 7 (17.5%) 6 (17.5%) 2 NA
1(5x) NA
D178N 13 (32.5%) 10 (29.5%) 2 NA
2 NA
2 NA
1(7x) NA
T183A 1 (2.5%) 1 (3%) 1 NA
E200K 8 (20%) 7 (20.5%) 2 NA
1(6x) NA
V210I 6 (15%) 6 (17.5%) 1(6x) NA
Total 40 (100%) 34 (100%)
---------------------------
The incidence of inherited cases within all "definite" prion cases is dependent on the denominator. To date, 184 patients of the 578 patients genetically examined have been classified as "definite" sporadic CJD, i.e. are neuropathologically confirmed and carry no mutation in PRNP. The addition of this number and the 40 genetically confirmed cases leads to 224 cases of confirmed prion diseases; the incidence of genetic cases is therefore 17.8%. If one adds to the denominator those clinically "probable" cases that have not been confirmed to date due to the lack of autopsy or because they are still alive (n=69), the incidence is 13.6% (40 of 293). Another attempt to get a realistic measure for this incidence is to limit the genetic cases to those 27 that were seen neuropathologically, i.e. had died and their material was available. If this number is divided by the sum of the 184 "definite" sporadic CJD cases, which by definition have been neuropathologically examined, and the 27 genetic cases, the incidence of genetic cases is 12.8% (27 of 211).
Clinical classifications of patients with known pathogenic mutations
A total of 502 of the 578 cases of suspect CJD examined were classified clinically according to the criteria of Masters and colleagues (Masters et al. 1979; Budka et al. 1995). One hundred and eighty-one were found to be "probable" CJD, 187 were classified as "possible" cases and 134 as "other" disease. Within the 502 clinically classified cases, we found 31 with a known pathogenic mutation (Table 2). Most of these (18 cases) were found in the patient sample of "possible" cases; fewer patients with known mutations were identified within the groups of "probable" and "other" subjects (seven or six cases, respectively). Therefore, the frequency of known pathogenic mutations within the "possible" cases was close to 10% (9.7%), whereas in the "probable" or "other" cases, it was 3.9% or 4.5%, respectively. Furthermore, the types of mutations found varied considerably among the three patient groups. Most patients with V210I (five of six) and some patients with E200K (two of eight) showed a clinical presentation almost indistinguishable from sporadic CJD and were classified as "probable" CJD. However, patients with D178N (four of nine clinically classified cases) and insertion mutations (two of four clinically classified cases) tended to be misdiagnosed and classified as non-CJD ("other"). The highest diversity of mutations was detectable within the group of "possible" cases.
---------------------------
Table 2. Clinical classification of patients with known pathogenic mutations
Clinical Cases (n) Type of mutations Codon 129
classification Total With mutation
"Probable" 181 7 (3.9%) Five with V210I M/M (4); M/V (1)
Two with E200K M/M (2)
"Possible" 187 18 (9.6%) Two with insert: 2 with 5x24 V/V (2)
Four with P102L M/M (3); M/V (1)
Five with D178N M/M (5)
Six with E200K M/M (2); M/V (3); V/V (1)
One with V210I M/M (1)
"Other" 134 6 (4.5%) Two with insert: 1 with 524, M/V
1 with 924 M/M
Four with D178N M/M (2); M/V (2)
"Not classified" 76 9 (11.8%) One with insert: 1 with 524 M/V (1)
Three with P102L M/M (3)
Four with D178N M/M (4)
One with T183A M/M (1)
---------------------------
No systematic investigation is possible in the group of patients who were not classified by the surveillance unit. The high incidence of pathogenic mutations in this group (9 of 76, 11.8%) is very probably due to a bias towards diseases with a familial history but, prior to typing, unknown aetiology. The types of mutations found within this group were again quite diverse. It is of interest that no case within this group of patients contained an E200K or V210I mutation. Cases with these mutations are obviously very well diagnosed using the criteria for sporadic CJD.
Novel insertion mutations
Of the five cases with an insertion mutation in the repeat region of PRNP, one patient had an insert of 216 bp (924 bp) (Krasemann et al. 1995). The other four cases had an insert of 120 bp, but the exact nucleotide sequence of the inserted fragment was different from the three published families carrying a 120-bp insert (Goldfarb et al. 1991a; Cochran et al. 1996; Goldfarb et al. 1996). The exact sequence of repeats in two cases was R1, R2, R2, R3g, R3g, R3g, R2, R2, R3, R4 and in the other two cases R1, R2, R2, R3, R2, R2, R2a, R2, R2, R4 (nomenclature according to Goldfarb et al. 1991a). The mutated alleles carried methionine at codon 129 in the first two cases and valine at this polymorphic position in the remaining two cases. A detailed description of the clinicopathological features of these cases will be published elsewhere (K.S. et al., unpublished observations; O.W. et al., unpublished observations).
Novel missense mutations and silent polymorphisms in PRNP
Two novel missense mutations were found during our screening program (Fig. 2). In one case, a threonine (ACG) to arginine (AGG) change at codon 188 (T188R) was present; in the other case, a proline (CCA) to serine (TCA) change at codon 23 was identified (P238S). However, both patients are alive and no brain material is available for neuropathological examination, there is thus no proof at present that these patients suffer from a prion disease.
---------------------------
Fig. 2. Novel missense mutations and silent polymorphisms in the coding region of PRNP. The missense mutations are above and the silent polymorphisms are below the bar depicting the coding region of PRNP. In addition to the amino acid exchange, the nucleotide sequence is given in brackets for all novel variations. The position of known pathogenic mutations is indicated by vertical lines or an empty box (insertion mutations)
---------------------------
In addition to these missense mutations, we found four novel silent polymorphisms within the coding region of PRNP and one nucleotide change 4 bp downstream of the coding region (Fig. 2). One patient harboured a T to C exchange at the third position of codon 68 (P68P), one a G to A exchange at the third position of codon 212 (Q212Q), one an A to G exchange at the third position of codon 228 (R228R), one a G to A exchange at the third position of codon 230 (S230S) and one an A to T exchange at nucleotide number +766 (nucleotide +1 is the first base of the ATG initiation codon as defined by Kretzschmar et al. 1986).
Known polymorphisms have been detected in our sample with the following frequencies. The A to G exchange at the third position of codon 117 (A117A) was present in 31 subjects (5.4% of all patients examined), the C to G exchange at the third position of codon 124 (G124G) was present in three patients (0.5%) and the deletion of one of the octapeptides in the N-terminal repeat region was detected in five subjects (0.9%).
Codon 129 polymorphism
The polymorphism at codon 129 [homozygosity for methionine (M/M), homozygosity for valine (V/V) or heterozygosity (M/V)] was determined in all patients. The distribution of the three genotypes in patients without a pathogenic mutation [n=538 (578 examined cases minus 40 with pathogenic mutation); thereof n=471 clinically classified and n=67 clinically "not classified"] was of particular interest, as the genotype at 129 is one major determinant of disease phenotype in sporadic CJD (Parchi et al. 1998). We focused on the patients being clinically classified (n=471) and a subset (n=176) who in addition were neuropathologically confirmed as "definite" CJD cases. Within the clinically classified cases (n=471), 174 were "probable" (meeting the clinical criteria for CJD by Masters et al. 1979), 169 were "possible" [same criteria as "probable", but without typical electroencephalogram (EEG) alterations] and 128 were classified as "other" disease (not meeting the criteria). The distributions of the genotype at codon 129 were the following in the three different groups of clinically classified cases: within the group of "probable" cases, M/M was found in 150 (86.2%), M/V in 16 (9.2%) and V/V in 8 (4.6%) patients; within the group of "possible" cases M/M occurred in 70 (41.4%), M/V in 52 (30.8%) and V/V in 47 (27.8%) patients; finally, in the group of "other" cases M/M was found in 53 (41.4%), M/V in 56 (43.8%) and V/V in 19 (14.8%). Compared with the normal controls or the "other disease" group, the homozygotes for methionine are extremely over-represented in the cases clinically classified as probable (86.2%), whereas the homozygotes for valine are over-represented in the possible cases (27.8%). As shown in Fig. 3, the "other disease" group (Fig. 3B) showed a genotype distribution virtually identical to the distribution in the normal German population (Fig. 3A, Schulz-Schaeffer et al. 1996). Only a part of the "probable" (105 of 174), the "possible" (65 of 169) and the "other" (6 of 128) cases could be confirmed by neuropathological examination of the brain to date ("definite" cases). The remaining patients are either still alive, have not been autopsied or were not confirmed as having CJD according to neuropathological criteria. The focus on the "definite" cases taking into account their original clinical classification showed that the homozygotes for methionine represent 90.5% of the originally "probable" cases (Fig. 3D) and homozygotes for valine 33.8% of the originally "possible" cases (Fig. 3E). Due to their small number the "definite" cases with the clinical classification "other" (n=6) cannot be evaluated for the distribution of the polymorphism at codon 129. Grouping the numbers of "definite" cases differently, with the genotype as leading parameter (Fig. 4), reveals a drastic effect concerning the homozygous patients for valine at codon 129. Twenty-two of twenty-six patients with V/V (85%) were originally classified as "possible", i.e. they met the clinical criteria of sporadic CJD apart from the typical EEG alterations.
---------------------------
Fig. 3. Distribution of the 129 genotype in (A) the normal German population (control; Schulz-Schaeffer et al. 1996), (B) cases clinically classified as "other", (C) all neuropathologically diagnosed cases ("definite") without a pathogenic mutation which were clinically classified, (D) the subgroup of "definite" cases clinically classified as "probable" and (E) the subgroup of "definite" cases clinically classified as "possible"
---------------------------
Fig. 4. The 129 genotype of "definite" CJD cases with their preceding clinical classification ("probable", "possible" CJD or
"other" disease). The height of columns corresponds to the number of cases
---------------------------
In conclusion, the correlation of codon 129 polymorphism in "definite" sporadic cases of human prion diseases and their preceding clinical classification allows two strong statements. (1) Approximately 90% of the clinically "probable" cases are homozygous for methionine and (2) approximately 85% of cases homozygous for valine were classified as "possible", i.e. do not show the typical changes on EEG.
Discussion
A total number of 578 suspect cases of human prion diseases in Germany referred to the national reference centre were screened for mutations and polymorphisms in the coding region of PRNP. We found 40 cases with a previously described pathogenic mutation within this group of patients. The most common mutation was the D178N mutation in coupling with methionine at codon 129, which is known to be the FFI genotype (Goldfarb et al. 1992; Medori et al. 1992). This high incidence of the D178N-129M genotype might be due to a founder effect in or somewhere close to Germany, but at the moment it is difficult to judge whether this finding is specific for the population of Germany, since studies of similar size are not published for other European countries. A French study on 57 cases of "definite", "probable" or "possible" cases of CJD did not detect a case with a D178N mutation, yet the authors found eight cases with the E200K mutation within their patient sample (Laplanche et al. 1994). Therefore, the major mutation in France seems to be the E200K mutation. The difference to the German study presented here might either represent true differences in the respective populations or be influenced by a different screening or identification system in the selection of the examined cases. In our study, a high proportion of cases clinically classified as "other" (n=134; Table 2) or not seen by a neurologist of the surveillance team ("not classified"; n=76) were included and, indeed, eight of the 13 cases with the FFI genotype were found in these two groups (Table 2). A British study of 120 cases of suspect CJD revealed nine cases with a known pathogenic mutation; in detail, three cases with an E200K mutation, two with a D178N mutation, two with a P102L mutation and two with an insertion mutation were found (Windl et al. 1996). There was no significant preponderance of any known mutation within this patient sample in the UK. It remains to be seen whether screening programs from other countries, which are similar in size and patient sample, will reveal whether there are regional differences in the frequency of certain mutations.
The clinical classification of the cases with mutations showed that the majority of cases with inherited prion disease are not classified as "probable" CJD. The typical clinical picture of an inherited prion disease is therefore unlike the typical picture of sporadic CJD and, in a strict sense, the inherited prion diseases are not ideally registered by a survey based on clinical criteria for sporadic CJD. Only two cases with the E200K mutation and most cases (five of six) with the V210I mutation met the criteria for clinically "probable" CJD. This is in agreement with published reports of these two mutations, whose clinicopathological appearance can be very similar to sporadic CJD (Goldfarb et al. 1991b; Pocchiari et al. 1993; Ripoll et al. 1993; Korczyn and Chapman 1996). On the other end of the spectrum of inherited prion diseases are those with a clinical classification as "other disease". Only patients with insertion mutations and the FFI mutation D178N(cis-129M) were in this category. This is not out of the ordinary, as the term FFI indicates a clinicopathological entity with significant differences to CJD; the insertion mutations are also known for a high degree of clinicopathological diversity, including atypical prion diseases (Collinge et al. 1992; Van Gool et al. 1995). Curiously, the constellation D178N(cis-129V) was not found in our patient sample but has been reported in Germany in one historic family (Meggendorfer 1930; Kretzschmar et al. 1995b).
The two novel missense mutations which were found during the screening process (T188R and P238S in one patient each) are not unequivocally associated with a prion disease at this moment. The lack of brain tissue which could be neuropathologically examined prevents any far-reaching conclusions. Both mutations result in exchanges of amino acids which are strictly conserved during mammalian evolution (Schätzl et al. 1995; Windl et al. 1995) and have not been noted previously. The five silent changes found in and around the coding region of PRNP in single patients are most probably without pathogenic consequence, but their tracing shows the power of the screening method. In principle, it is conceivable that a non-coding (silent) mutation may have an effect on RNA stability and, therefore, potential consequences on the translational efficiency which might hypothetically influence the susceptibility to a prion disease. While there is currently no indication that one of the known or one of the novel silent mutations alters the probability to get a prion disease, a recent finding in patients who carry the E200K mutation put the focus on the expression levels of wild-type versus mutated PRNP inasmuch as a downregulation of the transcriptional level of the wild-type allele during the disease process suggested an influence of the relative expression level of the mutated PRNP allele on the disease (Rosenmann et al. 1997).
We have determined the incidences of known polymorphisms, i.e. two silent nucleotide changes (A117A and G124G) and a deletion of one octapeptide repeat. The A117A polymorphism, which abolishes a PvuII site, was reported to occur in 20% of European Caucasians (Wu et al. 1987). We found this polymorphism in only 5.4% of the examined patients. Even knowing that our patient sample does not represent the normal population, the previously reported value seems very high compared with our finding, and it remains to be seen what the exact incidence of this silent mutation will be in the normal population.
The distribution of the polymorphism at codon 129 in CJD patients without a pathogenic mutation is significantly different from the normal population. This was reported originally by Palmer and colleagues (1991) and subsequently confirmed by several other studies in different European countries (Laplanche et al. 1994; Salvatore et al. 1994; Windl et al. 1996). On the basis of a much larger patient sample, we were able to specify the original findings more exactly by taking into consideration the clinical classification and the neuropathological diagnosis of "definite" CJD in many cases. Within "definite" CJD cases, which were clinically classified as "probable", the main genotype (90.5%) was homozygosity for methionine at codon 129, whereas homozygosity for valine (1.9%) and heterozygosity (7.6%) were extremely under-represented compared with the normal German population or the cases clinically classified as "other" (M/M 41.4%; M/V 43.8%; V/V 14.8%). The clinically typical CJD case according to established criteria (presenting with the typical altered EEG pattern) is therefore homozygous for methionine at codon 129. The situation is different in the group of clinically "possible" cases, which by definition do not show a typically altered EEG pattern. In those clinically "possible" cases, which were then neuropathologically confirmed as CJD, a marked over-representation of patients homozygous for valine at codon 129 (33.8%) was noticeable compared with the normal population. The inverse correlation is even more striking, as approximately 92% of confirmed CJD cases homozygous for valine were clinically not classified as "probable", i.e. did not show the typical changes in the EEG (periodic sharp-wave complexes; PSWC). Homozygosity for valine at codon 129 and PSWC in sporadic CJD seem to be almost mutually exclusive.
The extensive screening efforts of this and other groups in suspect cases of prion diseases has established the crucial role of the PRNP gene in the inherited and sporadic types of prion diseases. Inherited cases are no longer rare oddities, but distinct and numerically important disease entities. The current number of families with inherited prion diseases detected by this study (34 families, i.e. one family per approximately 2.5 million inhabitants in about 5 years) suggests that inherited prion diseases may be more commonly diagnosed than inherited Alzheimer's disease or other inherited dementias with the exception of Huntington's disease. The differential diagnosis of an inherited prion disease should therefore always be kept in mind when seeing patients presenting with an unusual psychiatric or neurological disorder.
Acknowledgements. We thank the physicians who made the site visits to the hospitals in the study: A. Szudra, S. Räcker, M. Otto, S. Grosche, M. Lantsch, W. Murach, K. Weidehaas, A. Otto, C. Riedemann, S. Kropp, S. Laske and A. Schröter. We thank all physicians and neuropathologists who reported suspect cases to the German CJD surveillance unit and provided data and material. We are indebted to D. Hause-Reithner and W. Dröse for expert technical assistance. Special thanks to B. Fartmann (MWG, Ebersberg, Germany) for his protocols and helpful advice concerning all applications on the LI-COR sequencer. This study was supported by the Bundesministerium für Gesundheit (Federal Ministry of Health).
References
Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, McCardie L, Chree A, Hope J, Birkett C, Cousens S, Fraser H, Bostock CJ (1997) Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 398:489-501
Budka H, Aguzzi A, Brown P, Brucher J-M, Bugiani O, Collinge J, Diringer H, Gullotta F, Haltia M, Hauw J-J, Ironside JW, Kretzschmar HA, Lantos PL, Masullo C, Pocchiari M, Schlote W, Tateishi J, Will RG (1995) Tissue handling in suspected Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases). Brain Pathol 5:319-322
Cochran EJ, Bennett DA, Cervenáková L, Kenney K, Bernard B, Foster NL, Benson DF, Goldfarb LG, Brown P (1996) Familial Creutzfeldt-Jakob disease with a five-repeat octapeptide insert mutation. Neurology 47:727-733
Collinge J, Brown J, Hardy J, Mullan M, Rossor MN, Baker H, Crow TJ, Lofthouse R, Poulter M, Ridley R, et al. (1992) Inherited prion disease with 144 base pair gene insertion. 2. Clinical and pathological features. Brain 115:687-710
Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF (1996) Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD. Nature 383:685-690
Gibbs CJ, Jr., Gajdusek DC, Asher DM, Alpers MP, Beck E, Daniel PM, Matthews WP (1968) Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161:388-389
Goldfarb LG, Brown P, McCombie WR, Goldgaber D, Swergold GD, Wills PR, Cervenáková L, Baron H, Gibbs CJ, Jr., Gajdusek DC (1991a) Transmissible familial Creutzfeldt-Jakob disease associated with five, seven, and eight extra octapeptide coding repeats in the PRNP gene. Proc Natl Acad Sci U S A 88:10926-10930
Goldfarb LG, Brown P, Mitrowa E, Cervenáková L, Goldin L, Korczyn AD, Chapman J, Galvez S, Cartier L, Rubenstein R, et al. (1991b) Creutzfeldt-Jakob disease associated with the PRNP codon 200 Lys mutation. An analysis of 45 families. Eur J Epidemiol 7:477-486
Goldfarb LG, Haltia M, Brown P, Nieto A, Kovanen J, McCombie WR, Trapp S, Gajdusek DC (1991c) New mutation in scrapie amyloid precursor gene (at codon 178) in Finnish Creutzfeldt-Jakob kindred. Lancet 337:425-425
Goldfarb LG, Petersen RB, Tabaton M, Brown P, Leblanc AC, Montagna P, Cortelli P, Julien J, Vital C, Pendelbury WW, Haltia M, Wills PR, Hauw JJ, McKeever PE, Monari L, Schrank B, Swergold GD, Gajdusek DC, Lugaresi E, Gambetti P (1992) Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 258:806-808
Goldfarb LG, Cervenáková L, Brown P, Gajdusek DC (1996) Genotype-phenotype correlations in familial spongiform encephalopathies asociated with insert mutations. In: Court L, Dodet B (eds) Transmissible subacute spongiform encephalopathies. Elsevier, Paris, pp 425-431
Goldgaber D, Goldfarb LG, Brown P, Asher DM, Brown WT, Lin S, Teener JW, Feinstone SM, Rubenstein R, Kascsak RJ, Boellaard JW, Gajdusek DC (1989) Mutations in familial Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker's syndrome. Exp Neurol 106:204-206
Hsiao K, Baker HF, Crow TJ, Poulter M, Owen F, Terwilliger JD, Westaway D, Ott J, Prusiner SB (1989) Linkage of a prion protein missense variant to Gerstmann-Sträussler syndrome. Nature 338:342-345
Jakob A (1921) Über eigenartige Erkrankungen des Zentralnervensystems mit bemerkenswertem anatomischem Befunde (spastische Pseudosklerose-Encephalomyelopathie mit disseminierten Degenerationsherden). Dtsch Z Nervenheilkd 70:132-146
Korczyn AD, Chapman J (1996) The varied manifestations of E200K Creutzfeldt-Jakob disease. In: Court L, Dodet B (eds) Transmissible subacute spongiform encephalopathies. Elsevier, Paris, pp 417-419
Krasemann S, Zerr I, Weber T, Poser S, Kretzschmar HA, Hunsmann G, Bodemer W (1995) Prion disease associated with a novel nine octapeptide repeat insertion in the PRNP gene. Mol Brain Res 34:173-176
Kretzschmar HA, Stowring LE, Westaway D, Stubblebine WH, Prusiner SB, DeArmond SJ (1986) Molecular cloning of a human prion protein cDNA. DNA 5:315-324
Kretzschmar HA, Bogumil T, Giese A, Windl O (1995a) Pathologie und Genetik der humanen spongiformen Enzephalopathien in Deutschland. Verh Dtsch Ges Pathol 79:567
Kretzschmar HA, Neumann M, Stavrou D (1995b) Codon 178 mutation of the human prion protein gene in a German family (Backer family): sequencing data from 72 year-old celloidin-embedded brain tissue. Acta Neuropathol (Berl) 89:96-98
Kretzschmar HA, Ironside JW, DeArmond SJ, Tateishi J (1996) Diagnostic criteria for sporadic Creutzfeldt-Jakob disease. Arch Neurol 53:913-920
Laplanche JL, Delasnerie-Lauprêtre N, Brandel JP, Chatelain J, Beaudry P, Alperovitch A, Launay J-M (1994) Molecular genetics of prion diseases in France. Neurology 44:2347-2351
Masters CL, Harris JO, Gajdusek DC, Gibbs CJ, Jr, Bernoulli C, Asher DM (1979) Creutzfeldt-Jakob disease: patterns of worldwide occurrence and the significance of familial and sporadic clustering. Ann Neurol 5:177-188
Mastrianni JA, Iannicola C, Myers RM, DeArmond S, Prusiner SB (1996) Mutation of the prion protein gene at codon 208 in familial Creutzfeldt-Jakob disease. Neurology 47:1305-1312
Medori R, Tritschler H-J, LeBlanc A, Villare F, Manetto V, Chen HY, Xue R, Leal S, Montagna P, Cortelli B, Tinuper P, Avoni P, Moghi M, Baruzzi A, Hauw JJ, Ott J, Lugaresi E, Gambetti P (1992) Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene. N Engl J Med 326:444-449
Meggendorfer F (1930) Klinische und genealogische Beobachtungen bei einem Fall von spastischer Pseudosklerose. Z Ges Neurol Psychiatry 128:337-341
Nitrini R, Rosemberg S, Passos-Bueno MR, Teixeira da Silva LS, Iughetti P, Papadopoulos M, Carrilho PM, Caramelli P, Albrecht S, Zatz M, LeBlanc A (1997) Familial spongiform encephalopathy associated with a novel prion protein gene mutation. Ann Neurol 42:138-146
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorhisms. Proc Natl Acad Sci U S A 86:2766-2770
Palmer MS, Dryden AJ, Hughes JT, Collinge J (1991) Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 352:340-342
Palmer MS, Van Leeven RH, Mahal SP, Campbell TA, Humphreys CB, Collinge J (1996) Sequence variation in intron of prion protein gene, crucial for complete diagnostic strategies. Hum Mutat 7:280-281
Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sima AAF, Trojanowsky JQ, Petersen RB, Gambetti P (1996) Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neurol 39:767-778
Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, Budka H, Julien J, Kopp N, Poser S, Rojiani AM, Streichenberger N, Vital C, Zerr I, Ghetti B, Kretzschmar HA, Gambetti P (1998) The molecular and clinico-pathologic spectrum of phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD). Neurology 50:A336
Pocchiari M, Salvatore M, Cutruzzolá F, Genuardi M, Allocatelli CT, Masullo C, Macchi G, Alemá G, Galgani S, Xi YG, Petraroli R, Silvestrini MC, Brunori M (1993) A new point mutation of the prion protein gene in Creutzfeldt-Jakob disease. Ann Neurol 34:802-807
Prusiner SB (1997) Prion diseases and the BSE crisis. Science 278:245-251
Ripoll L, Laplanche JL, Salzmann M, Jouvet A, Planques B, Dussaucy M, Chatelain J, Beaudry P, Launay JM (1993) A new point mutation in the prion protein gene at codon 210 in Creutzfeldt-Jakob disease. Neurology 43:1934-1937
Rosenmann H, Meiner Z, Kahana E, Halimi M, Lenetsky E, Abramsky O, Gabizon R (1997) Differential allelic expression of PrP mRNA in carriers of the E200K mutation. Neurology 49:851-856
Salvatore M, Genuardi M, Petraroli R, Masullo C, DAlessandro M, Pocchiari M (1994) Polymorphisms of the prion protein gene in Italian patients with Creutzfeldt-Jakob disease. Hum Genet 94:375-379
Samaia HB, Mari JD, Vallada HP, Moura RP, Simpson AJG, Brentani RR (1997) A prion-linked psychiatric disorder. Nature 390:241-241
Schätzl HM, Da Costa M, Taylor L, Cohen FE, Prusiner SB (1995) Prion protein gene variation among primates. J Mol Biol 245:362-374
Schulz-Schaeffer WJ, Giese A, Windl O, Kretzschmar HA (1996) Polymorphism at codon 129 of the prion protein gene determines cerebellar pathology in Creutzfeldt-Jakob disease. Clin Neuropathol 15:353-357
Van Gool WA, Hensels GW, Hoogerwaard EM, Wiezer JHA, Wesseling P, Bolhuis PA (1995) Hypokinesia and presenile dementia in a Dutch family with a novel insertion in the prion protein gene. Brain 118:1565-1571
Wilesmith JW, Wells GAH, Cranwell MP, Ryan JBM (1988) Bovine spongiform encephalopathy: epidemiologic studies. Vet Rec 123:638-644
Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, Smith PG (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921-925
Windl O, Kretzschmar HA (1999) Prion diseases. Contemp Neurol (in press)
Windl O, Dempster M, Estibeiro P, Lathe R (1995) A candidate marsupial PrP gene reveals two domains conserved in mammalian PrP proteins. Gene 159:181-186
Windl O, Dempster M, Estibeiro JP, Lathe R, De Silva R, Esmonde T, Will R, Springbett A, Campbell TA, Sidle KCL, Palmer MS, Collinge J (1996) Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic variations in the PRNP gene. Hum Genet 98:259-264
Wu Y, Brown WT, Robakis NK, Kobkin C, Devine-Gage E, Merz P, Wisniewski HM (1987) A PvuII RFLP detected in the human prion protein (PrP) gene. Nucleic Acids Res 15:3191-3191
Young K, Jones CK, Piccardo P, Lazzarini A, Golbe LI, Zimmerman TR, Dickson DW, McLachlan DC, George-Hyslop PSt, Lennox A, Perlman S, Vinters HV, Hodes ME, Dlouhy S, Ghetti B (1995) Gerstmann-Sträussler-Scheinker disease with mutation at codon 102 and methionine at codon 129 of PRNP in previously unreported patients. Neurology 45:1127-1134
MH DNA/chemistry/genetics; DNA Mutational Analysis; Genotype; Germany/epidemiology; Human; Incidence; Mutagenesis, Insertional; Mutation; Mutation, Missense; Polymorphism (Genetics); Prion Diseases/epidemiology/*genetics; Prions/*genetics; Support, Non-U.S. Gov't
AD Otto Windl, Armin Giese, Walter Schulz-Schaeffer, Katherina Skworc, Stefanie Arendt, Christina Oberdieck, Hans A. Kretzschmar, Institut für Neuropathologie, Universität Göttingen, Robert-Koch-Str. 40, D-37075 Göttingen, Germany; Inga Zerr, Monika Bodemer, Sigrid Poser, Neurologische Klinik, Göttingen, Germany
SP englisch
PO Deutschland
EA HTML-Version und pdf-Datei