Prion Disease
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Gene within a gene
High prevalence of pathogenic mutations in patients with early-onset dementia
Amyotrophy revisited
Scrapie: in vitro conversion of nine natural variants of PrP
Comparative monoclonal and polyclonal antibodies
Paraffin-embedded tissue blot detects prp(sc) early on
Cambridge Prion Consortium




Gene within a gene

20 Jan 00 webmaster research
Context: human chromosome 20p12.3 upstream of prion-doppel genes.
High lod scores for genes for corneal blindness (CHED-2) and brain iron deposition (HSS) 
  
Contig: 150kb AL121891 unfinished human contig in 13 pieces

Map Markers:
KIAA0552           17521 .. 17794   Hs.90232
WI-13669           18000 .. 18098  polyA tail of KIAA0552 
AFMa049yd1         69005 .. 69275
D20S1109            1402 ..  1519
D20S181           147913 .. 148054  last short 1064 bp contig 
  
Issues: 
... do neighboring genes shed light on function of prion gene? 
... is there a concentration of genes with neurological roles?
... why is this stretch of chromosome so gene-rich?

Bottom line: 3 complete genes expressed in brain within 79kb, apparent gene within gene
6+ genes in 150 k = high density (GenScan + EST + blastp support: 2 of 3)
KIAA0550
vlong
KIAA0860
bicarbonate anion exchange
intermediate filament
oncogene
This region of chromosome 20 -- even though sequencing was not finished -- was analyzed for genes because two serious diseases corresponding to unknown genes mapped close to microsatellite markers contained within the contig. The goal of the annotation was to find candidate genes in which defects could plausibly affect brain iron metabolism or corneal epithelial blindness. This involves annotation of the proteins corresponding to identified genes to the extent that their function is not already known.

Gene density on this arm of chromosome 20 is generally low, about 10-12 per million base pairs. However, this contig contained 6 genes with varying degrees of expression support within 150kb, a local gene density of 40 per million. Two adjacent genes had been previously identified (but not mapped) in studies of long expressed mRNAs in brain: KIAA0552 and KIAA0860 of 673 and 541 amino acids, respectively. Recall KIAA0168, which has a similar experimental history, is the immediate downstream neighbor of the prion-doppel genes.

The KIAA0860 gene was most unusual in that a third gene with a very long single coding exon (765 amino acids, temporarily called vlong) was intercalated on the same sense strand between the leading 5' UTR exon of KIAA0860 and its coding exons. [see annotation graphic]. While examples of intercalated genes were found on chr 22, they occurred within introns on the minus strand.

It is tempting to dismiss this as simply alternative splicing, that vlong is merely the first coding exon of the KIAA0860 gene and that the short form happened to have been cloned in the original study. However, none of the two dozen applicable EST sequences supports this scenario: none join exon 1 of KIAA0860 to vlong, none join any portion of vlong to distal CDS of KIAA0860, whereas the only EST extending to 5' UTR is spliced exactly the same as the experimentally recovered clone.

Further, GenScan identified a close-in promoter and initiator methionine for vlong. ESTs support a short 5' UTR following the promoter. A long uninterrupted coding sequence of 2295 bp terminates in a stop codon. Subsequent EST-supported 3' UTR contains a classical poly A signal a standard distance (20 bp) prior to a poly A site at which all downstream ESTs originate. A strong mouse Blastp homologue terminates at the same position.

In other words, vlong has all the properties a self-contained gene and is already 3x the length of an average human protein. Nothing is known about in vivo roles of these proteins; little is inferrable from homology to other species; protein properties determined ab initio were not especially informative. Significant protein matches in mouse and rabbit argue for a reasonably conserved function, 85% identity at those divergence distances.

The isolated upstream KIAA0860 exon is mis-identified by GenScan as part of an additional hypothetical protein on the minus strand. GenScan naturally could not make the connection to more distal parts of the KIAA0860 gene as could ESTs. [Other pieces of spurious protein have no support as more of this or any other gene as seen from tblastn(est), blastp(nrp), tblastn (nrn), tblastn (htgs).]

Tissues of expression, conveniently tabulated at the SAGE site, include adrenal gland, brain, colon, eye, heart, kidney, nose, ovary, pancreas, prostate, testis, uterus, and lung.

Sequencing error does not seem likely -- matches to previously determined mRNAs and ESTswere essentially perfect over thousands nucleotides, suggesting very low error rates. However, some sequencing vector is still present (beta-lactamase from E. coli) in contigs not used here. Three contigs were assembled and oriented using genes spread over more than a single contig but this was determined by various spanning ESTs and CDS colinearity. It remains possible that larger contigs were improperly assembled.

In other cases of intercalated genes, it is not hard to see how regulation of gene expression could work. An intron-intercalated unrelated gene on the minus strand does not result in unwanted coding sequence from read-through of the enveloping gene. The doppel gene is sometimes expressed through chimeric transcripts that begin with promoter and 5' UTR of the prion gene and skip the entire prion coding exon. This makes sense for proteins resulting from a tandem duplication that presumbably have related or coupled function.

KIAA0860 and vlong are similar to prion-doppel in that both are on the same strand and gene-skipping transcripts are established. KIAA0860, like doppel, may have additional promoter elements in conventional position. While KIAA0860 and vlong have no detectable homology, it is conceivable that their functions are related. No use of KIAA0860 promoter by vlong has been established, though naturally ESTs this far upstream are sparse.

Authentic KIAA0552 5257 CDS 1409..3430 673aa
MAKLETLPVRADPGRDPLLAFAPRPSELGPPDPRLAMGSVGSGVAHAQEFAMKSVGTRTGGGGSQGSFPGPRGSGSGASR ERPGRYPSEDKGLANSLYLNGELRGSDHTDVCGNVVGSSGGSSSSGGSDKAPPQYREPSHPPKLLATSGKLDQCSEPLVR PSAFKPVVPKNFHSMQNLCPPQTNGTPEGRQGPGGLKGGLDKSRTMTPAGGSGSGLSDSGRNSLTSLPTYSSSYSQHLAP LSASTSHINRIGTASYGSGSGGSSGGGSGYQDLGTSDSGRASSKSGSSSSMGRPGHLGSGEggggglpfaacsppSPSAL IQELEERLWEKEQEVAALRRSLEQSEAAVAQVLEERQKAWERELAELRQGCSGKLQQVARRAQRAQQGLQLQVLRLQQDK KQLQEEAARLMRQREELEDKVAACQKEQADFLPRIEETKWEVCQKAGEISLLKQQLKDSQADVSQKLSEIVGLRSQLREG RASLREKEEQLLSLRDSFSSKQASLELGEGELPAACLKPALTPVDPAEPQDALATCESDEAKMRRQAGVAAAASLVSVDG EAEAGGESGTRALRREVGRLQAELAAERRARERQGASFAEERRVWLEEKEKVIEYQKQLQLSYVEMYQRNQQLERRLRER GAAGGASTPTPQHGEEKKAWTPSRLERIESTEI

Authentic KIAA0860 4313 cds 155..1780 541 aa
MVINLCLPQFRPRIHCNKISADGYEVENLISEDLTKRSHGFRTEYFIKPPVYVTVSFPFNVEICRINIDLTAGGGQNVTG LEMYTSASSSRVSWNTPQCRTLGPAEPSVPDKEAFTLVGKVLLKNQSQVVFSHRGFKARPPFGAMEATLPSPAVVAQELW NKGALSLSHVAHLRICITHVTGGGIPCIKRLEVWGQPAKTCSQEVIDSILLVTSENLPQDVALQAPALPMESDCDPGDQP ESQQAPSSLQKLAEIIQDVPEEFLDPITLEIMPCPMLLPSGKVIDQSTLEKCNRSEATWGRVPSDPFTGVAFTPHSQPLP HPSLKARIDHFLLQHSIPGCHLLGRAQTALAVIPSSIVLPSQKRKIEQAEHVPDSNFGVNASCFSATSPLVLPTTSEHTA KKMKATNEPSLTHMDCSTGPLSHEQKLSQSLEIALASTLGSMPSFTARLTRGQLQHLGTRGSNTSWRPGTGSEQPGSILG PECASCKRVFSPYFKKEPVYQLPCGHLLCRPCLGEKQRSLPMTCTACQRPVASQDVLRVHF

Predicted vlong 765 aa. Transmembrane 176-196; tblastn(est) matches to mouse, rabbit, chicken; htgs in drosophila; corneal epithelial in rabbit; perfect ESTs AW182833, AI768494 473.
MAATLKSLKLVRYRAFCSPSAFGAVRSVSYWNVSSTQHGGQDPPEHISLCHSAKKVKNICSTFSSRRILTTSSAHPGLEF SKTSSSKASTLQLGSPRATGVDEEDVEVFDSFENMRVFLQLRPEYRVHSYNASETSQLLSVSEGELILHKVRVNQNNLQA QVIVDYLCKLSSLPAEQHPVLLGSTSFALLCQLSVKKIQLFDTQDLINVLKAFVILGIPHSHSMLDVYETKCCHQVWEMN MDQLLLVADLWRYLGRKVPRFLNIFSSYLNLHWKDLSLSQLVHLIYVIGENRQVSQDLMQKLESLILKYIDLINLEEVGT ICLGFFKSSTNLSEFVMRKIGDLACANIQHLSSRSLVNIVKMFRFTHVDHINFMKQIGEIAPQRIPSLGVQGVMHLTLYC SALRFLNEGVMNAVAASLPPRVAHCRSKDVAKILWSFGTLNYKPPNAEEFYSSLISEIHRKMPEFNQYPEHLPTCLLGLA FLEYFPVELIDFALSPGFVRLAQERTKFDLLKELYTLDGTVGIECPDYRGNRLSTHLQQEGSELLWYLAEKDMNSKPEFL ETVFLLETMLGGPQYVKHHMILPHTRSSDLEVQLDVNLKPLPFNREATPAENVAKLRLEHVGVSLTDDLMNKLLKGKARG HFQGKTESEPGQQPMELENKAAVPLGGFLCNVADKSGAMEMAGLCPAACMQTPRMKLAVQFTNRNQYCYGSRDLLGLHNM KRRQLARLGYRVVELSYWEWLPLLKRTRLEKLAFLHEKVFTSAL-

best mouse match to vlong:165 terminal
PMPFNSEATPTEDGAQLRFKQVGVSLTDDLMNQLLKGKAKRYFQGQIELETGQPPMELRKKTTVPLVNSGCNGTDRLGDG MVGLCPLAHMQPPLVKLAIQFTNKNQYCYGSRALLGLHNMKRRQLVQIGYRVVELPHWEWLPLLKRTRLEKLAYLHEKVFTSAL

best rabbit match to vlong: C82541 rabbit corneal endothelial. 453aa 114/139 (82%) identical
NQYPEHLLTCLLGLAFSEYFPVEFINFALSPEFVRLAQERSKFELSKELYTLDGTVGIECPDYKGSRLSSHFQQEVSEKLWNL ARKDMTSKPEFLEALFSLETMLGGPQYIRHHMILPHTRSSDLEXQLDVYMKPLPFN

			13476 to ATG
   1	 554	554	5' UTR
 555	 711	157	MIR
 712	1099	388	more 5' UTR
1100	1131	32	simple repeat
1132	1398	267	end of 5' UTR
1399	1857	459	end exon 1 KIAA0552
1858	2201	344	intron 1
2202	2646	445	exon 2
2647	2764	118	glitch = intron 2
2765	4186	1422	to TGA exon 3
4187	5993	1807	to poly A signal
5994	6013	20	to site
		1398	5' UTR
		1827	3' UTR

   1	  113	113	loner exon 1
  114	10556	10443	inter-intron
10557	10596	40	promoter vlong
10597	10809	213	end of 5' UTR
10810	13104	2295	end of TGA
13105	13327	223	to poly A
13328	13361	34	to 3' end of vlong
13362	14901	1540	to glitch
14902	35006	20105	to 5' exon 2
35007	35047	41	to ATG KIAA0860
35048	35101	54	end exon 2
35102	35828	727	end of intron 2
35829	37021	1193	end of exon 3
37022	42938	5917	end of intron 3
42939	43108	170	end of exon 4
43109	48098	4990	end of intron 4
48099	48307	209	exon 5 to TGA
48099	49458	1360	to SINE
49459	49747	289	end SINE
49748	50003	256	to + SINE
50004	50299	296	end SINE
50300	50817	518	to poly A
50818	50837	20	to end exon 5
		  95	exon 2
		2948	exon 4

RepeatMasker:   79936 bp   
GC level:       49.33 %
bases masked    35349 bp (44.22 %) 

SINEs:              98       25153 bp    31.47 %
      ALUs          94       24615 bp    30.79 %
      MIRs           4         538 bp     0.67 %

LINEs:              16        3715 bp     4.65 %
      LINE1         11        2686 bp     3.36 %
      LINE2          2         293 bp     0.37 %

LTR elements:       10        3848 bp     4.81 %
      MaLRs          4         875 bp     1.09 %
      Retrov.        5        2510 bp     3.14 %

DNA elements:       10        1862 bp     2.33 %
      MER1_type      6        1245 bp     1.56 %
      MER2_type      1         238 bp     0.30 %
 
Simple repeats:     10         341 bp     0.43 %
Low complexity:      8         443 bp     0.55 %
GENSCAN_predicted|404_aa: anion exchange protein AE-2 chloride bicarbonate
XSTEEIIALFISITFVLDAVKGTVKIFWKYYYGHYLDDYHTKRTSSLVSLSGLGASLNASLHTALNASFLASPTELPSATHSGQATAVLSLLI MLGTLWLGYTLYQFKKSPYLHPCVREILSDCALPIAVLAFSLISSHGFREIEMSKFRYNPSESPFAMAQIQSLSLRAVSGAMGLGFLLSMLFF IEQNLVAALVNAPENRLVKGTAYHWDLLLLAIINTGLSLFGLPWIHAAYPHSPLHVRALALVEERVENGHIYDTIVNVKETRLTSLGASVLVG LSLLLLPVPLQWIPKPVLYGLFLYIALTSLDGNQLVQRVALLLKEQTAYPPTHYIRRVPQRKIHYFTGLQVLQLLLLCAFGMSSLPYMKMIFP LIMIAMIPIRYILLPRIIEAKYLDVMDAEHRP

Anion Exchange Protein AE-2 chloride bicarbonate closest nematode Identities = 186/402 (46%), Positives = 268/402 (66%); occupies most of 48464 contig #7; lacks strong human match. Fair number of EST matches,eg AI041607 100% putative oncogene. Best match: U2317 anion exchange protein [C elegans], identities 186/402 (46%) 605 1005 401aa of 1024
STEEIIALFISITFVLDAVKGTVKIFWKYYYGHYLDDYHTKRTSSLVSLSGLGASLNASLHTALNASFLASPTELPSATHSGQATAVLSLLIM LGTLWLGYTLYQFKKSPYLHPCVREILSDCALPIAVLAFSLISSHGFREIEMSKFRYNPSESPFAMAQIQSLSLRAVSGAMGLGFLLSMLFFI EQNLVAALVNAPENRLVKGTAYHWDLLLLAIINTGLSLFGLPWIHAAYPHSPLHVRALALVEERVENGHIYDTIVNVKETRLTSLGASVLVGL SLLLLPVPLQWIPKPVLYGLFLYIALTSLDGNQLVQRVALLLKEQTAYPPTHYIRRVPQRKIHYFTGLQVLQLLLLCAFGMSSLPYMKMIFPL IMIAMIPIRYILLPRIIEAKYLDVMDAEH

GENSCAN_predicted|353_aa intermediate filament same contig as anion and oncogene, plus strand.Identities = 91/119 (76%). ESTs so-so
MVAPVWYLVAAALLVGFILFLTRSRGRAASAGQEPLHNEELAGAGRVAQPGPLEPEEPRAGGRPRRRRDLGSRLQAQRRAQRVAWAEADENEE EAVILAQEEEGVEKPAETHLSGKIGAKKLRKLEEKQARKAQLCLGGGHTLAGGEGCAPMKTSLQAEEAEREERKRLESQREAEWKKEEERLRL EEEQKEEEERKAREEQAQREHEEYLKLKEAFVVEEEGVGETMTEEQSQSFLTEFINYIKKPEERSPVLALTFGGSLEQSKVVLLEDLASQVGL RTQDTINRIQDLLAEGTITGVIDDRGKFIYITPEELAAVANFIRQRGRVSIAELAQASNSLIAWGRESPAQAPA

GENSCAN_predicted|153_aa oncogene 1 hit.131/134 (97%) AF026816 of length 157 so possibly authentic would find first 23 aa; minus.strand
MAASLVGKKIVFVTGNAKKLEEVQGPVLVEDTCLCFNALGGLPGPYIKWFLEKLKPEGLHQLLAGFEDKSAYALCTFALSTGDPSQPVRLFR GRTSGRIVAPRGCQDFGWDPCFQPDGYEQTYAEMPKAEKNAVSHRFRALLELQEYFGSLAA

High prevalence of pathogenic mutations in patients with early-onset dementia

Am. J. Hum. Genet., 66:110-117, 2000
Ulrich Finckh, Tomas Müller-Thomsen, .. Christoph Hock, Roger M. Nitsch, andAndreas Gal
Comment (webmaster): A small patient group (36) defined by early onset of dementia (EOD)and no significant early neurological sign turned out to be remarkably productive for both Alzheimer and CJD mutations. The authors make a persuasive case for routine PCR of the 4 relevent genes under these circumstances. Two novel CJD mutations were found, including a potentially very signficant stop mutation at position 160. Two known CJD mutations were also found, the common P102L and a second T183A kindred, as well as the commonest repeat deletion polymorphism, R34. These data further support severe under-diagnosis of CJD, with 1 in 9 of early-onset Alzheimer actually being familial CJD.

The T188K is interesting in that T188R was already known. This is a CpG mutational hotspot with different outcomes. A P105T family has also surfaced in Australia that is not; P105L was already known. Two types of mutation are also known at 117 and 212 but one each is neutral. The total number of prion gene mutations has reached 31, with 21 causative (2 stop), 7 silent, and 3 neutral.

It is fair to say that reported mutational sites in the prion gene have not reached saturation: 11 new ones have been reported in the last year from Europe alone. However, certain sites have numerous distinct kindreds (though only E200K has been properly investigated by microsatellites) and distinct mutations at 105 and 188 raise the whether the protein is especially intolerant to change at these sites. The distribution of causative amino acid change along the polypeptide is far from uniform.

The second stop mutation at position 160 reinforces the idea from position 145 that alpha helix to beta sheet transitioning has no relevence to abnormal conformer formation, in fact does not occur, and indeed was a misinterpetation fraom the get-go of the circular dichroism spectrum attributable to improper deconvolution of cross-beta structure. While no autopsy material is available yet, the cases suggest that abridged mini-prions might simplify study of in vitro conformational change and infectivity.

P105 is especially intriguing because threonine and leucine are fairly conservative substitutions relative to hydrophobicity and size, suggesting that the special rigidity of an imino acid is important at this position (as well as P102). This effectively refutes the notion from nmr studies that this region is always unstructured, supporting instead the idea that when copper is bound that the region has a conventional folded protein structure. The slow rate of evolutionary change of this region is also completely contradictory to the proposed nmr structure.

We are left wondering how it is possible that larger series of patients elsewhere are failing to turn up this density of prion gene mutation. It seems that either the patient group used by Finckh et al was a very good choice or that sequencing methods are better.

Highlights:
Clinical differential diagnosis of early-onset dementia (EOD) includes familial Alzheimer disease (FAD) and hereditary prion disease. In both disease entities, postmortem brain histopathological examination is essential for unambiguous diagnosis. Mutations in the genes encoding the presenilins (PS1 and PS2) and amyloid precursor protein (APP) are associated with FAD, whereas mutations in the prion protein (PrP) gene are associated with prion disease.

To investigate the proportion of EOD attributable to known genes, we prospectively (i.e., antemortem) screened these four genes for mutations by sequencing genomic PCR products from patients with EOD before age 60 years.

Family history for dementia was positive (PFH) in 16 patients, negative (NFH) in 17 patients, and unknown (UFH) in 3 patients. In 12 patients, we found five novel mutations (in PrP, Q160X and T188K) and five previously reported mutations (in PrP, P102L and T183A) that are all considered to be disease causing. Of these 12 patients, 9 had PFH. This indicates a detection rate of 56% (9/16) in patients with PFH. We found two mutations in two of the three UFH patients, and only one mutation (PrP T188K) in 1 of the 17 patients with NFH. We conclude that because of the lack of specific antemortem diagnostic markers for FAD and hereditary prion disease, all four genes should be included in a molecular diagnostic program in patients with EOD who had PFH.

To determine the spectrum of mutations and the relative contribution of the previously mentioned four genes, we performed a prospective molecular diagnostic program in an ethnically heterogeneous group of patients with EOD and no significant early neurological signs. Such a study may help to establish criteria for molecular diagnostics in dementia.

Genomic DNA was prepared from peripheral blood leukocytes of 36 unrelated German, Austrian, Italian, Hungarian, and Swiss patients with EOD and onset before age 60 years, and 3 unrelated German patients with late-onset dementia after age 65 years. None of the patients had significant neurological signs in the early disease stage, and all had a clinical diagnosis of dementia of unknown type or possible or probable AD. Sixteen of the EOD patients had positive family history (PFH) of EOD, whereas the 3 late-onset patients had PFH of late-onset dementia. PFH was assumed if at least one first-degree relative with dementia was reported ... Twenty-four patients were ascertained by AD research centers (22 in Hamburg, Germany and 2 in Brescia, Italy).

In 14 patients, we found a total of 12 different mutations in PSEN1, PSEN2, APP, and PRNP. Of the 12 mutations, 5 were previously undescribed and 7 were known.

PrP del r34 . In addition to the PS2 T122P mutation, patient 6 carried a heterozygous 24-bp in-frame deletion in PRNP (del r34). This deletion removes part of the last two of the five proline-rich octapeptide repeat elements. In patient 6, del r34 was in cis with the M129-encoding allelic variant of PrP 129M/V. As a result of the high sequence similarity of the repeat elements, it is impossible to determine the precise location of the deletion that may be identical to previously reported PRNP deletions. Both the patient and her father were heterozygous for M129V. The patient's father, who was not demented at age 81 years and did not carry the PS2 mutation, also carried M129 in cis with del r34. Therefore, del r34 is likely a nonpathogenic polymorphism. This conclusion is supported by a report on cosegregation of dementia with PS1 H163R but not with a 24-bp PRNP deletion in two siblings with mixed neuropathological features of AD and CJD (El Hachimi et al. 1996).

PrP P102L. This mutation in PRNP codon 102 was found in two siblings, both with onset at age 40 years. Both siblings shared the GSS haplotype P102L in cis with M129, and were heterozygous for M/V at codon 129. At age 43 years, the affected brother of the index patient had a diagnostic antemortem brain biopsy of the right prefrontal cortex. Histopathologically (staining techniques: HE, trichrom. Masson, PAS, Bielschowsky) were found some atrophic neurons, multiple small and larger-sized amorphic amyloid areas with only few glial reactions surrounding them, and neurofibrillary aggregates in some single neurons. The white matter was described as spongious, with some areas of discrete reactive glial proliferation. All of these together were interpreted as signs of primary cortical atrophy of Alzheimer type. The patient died at age 54 years; no autopsy was done. Analysis of the pedigree revealed several relatives with ataxia and largely variable clinical phenotype characteristic for GSS.

PrP Q160X. In an Austrian family, we identified the novel nonsense mutation PrP Q160stop. This mutation, in cis with PrP M129, was found both in the index patient (patient 12), with onset of dementia at age 32 years and a severe and rapidly progressing course, and in his elder brother, with onset at age 48 years and a mild course. The wild-type allele of the younger brother encodes M129 and that of the elder brother encodes V129.

Repeated EEGs from patient 12 done at at ages 35, 36, 37, and 38 years were slightly abnormal, with diffuse theta increment and intermittent theta groups but without hints of seizures. Both cranial CT at age 35 years and magnetic-resonance imaging (MRI) at age 37 years showed severe ventricular and sulcal enlargement. Cranial MRI of the elder brother, done at age 55 years, showed almost normal findings with only slight cerebellar atrophy.

So far, after disease durations of 6 and 8 years, respectively, no neurological sign of CJD or ataxia has been observed in the two brothers. Their father also had dementia with onset at age 48 years and a severe course resembling that of the index patient. The father died with pneumonia at age 60 years. Pathology reports from 1980 noted a reduced brain weight of 1,200 g, diffuse cortical atrophy, extensive enlargement of ventricles, little arteriosclerosis of brain vessels, and no stenosis of the carotid arteries. No microscopic analyses were done.

PrP T183A. Patient 13 represents the second case with this mutation first described in a Brazilian family with EOD and histopathologically confirmed spongiform encephalopathy (Nitrini et al. 1997). The clinical course in patient 13 was observed over a period of 4 years. Because there were no neurological signs of CJD, the clinical diagnosis of possible AD was made. The patient died at age 44 years, and autopsy confirmed hereditary prion disease by demonstration of spongiform encephalopathy (U. Mann, unpublished data).

PrP T188K. We discovered this novel missense mutation in PRNP codon 188 in a 59-year-old patient (patient 14) with a NFH. Lleading signs of the disease were dysphasia along with dementia. In this patient, the disease is rapidly progressing and has reached a most severe dementia in less than 1 year.

Patients with dementia who had mutations in PSEN1, PSEN2, or APP most likely have FAD, whereas mutations in PRNP lead most likely to prion disease. In all three deceased index patients of our prospective study, molecular prediction was confirmed histopathologically at autopsy. An affected sibling of one of our index patients with the well-known GSS mutation, PrP P102L, had a biopsy-based histopathological diagnosis of AD in 1987, 2 years before the first report on a familial PrP mutation appeared.

In up to 10% of histopathologically confirmed CJD cases, AD-typical neuropathologic changes can also be seen (Hainfellner et al. 1998). So the limited amount of antemortem brain biopsy material available and the lack of PrP-specific immunocytochemistry may explain the interpretation of the findings as AD in this patient. The observation of 4 mutations in PRNP, among a total of 12 pathogenic mutations in patients with EOD, suggests an important overlap in clinical symptoms between FAD and hereditary prion disease. Our data support the idea that AD and prion disease may occur jointly in individual patients.

CCG  CTG   P102L    early
CAA  TAA   Q160X    early
ACA  GCA   T183A    early
ACG  AAG   T188K    negative

Cambridge Prion Consortium

web site for new prion research group 18 Jan 00
[Initial webpage: fuller research version promised soon.]:

The Cambridge Prion Consortium was established in December 1999. The consortium represents a union of scientists within the University of Cambridge that are working in the area of research encompassing the prion diseases, transmissible spongiform encephalopathies, prions (including those of yeast) or the normal cellular protein, the prion protein. The aim of the consortium is to enhance and advance prion research in Cambridge by interaction, collaboration and pooling of resources.

At present the consortium includes researchers from six departments who are currently working in the field or interested in the field. Thus the consortium has the talents of biochemists, immunologists, veterinarians, chemists, biotechnologists and experimental psychologists. If you work in Cambridge and have an interest in or already work on prions or TSEs please contact the organizers via the address below.

Department of Biochemistry:  Dr. David Brown

Institute of Biotechnology: Professor Chris Lowe, Dr. Denise Dear

Department of Chemistry: Professor Alan Fersht, Dr. Despina Galani

Department of Experimental Psychology:: Dr. Harry Baker, Dr. Ros Ridley

School of Veterinary Clinical Medicine: Professor Ian McConnell, Dr. Raymond Bujdoso

Department of Pathology: Professor Tony Minson, Dr. Stacey Efstathiou

Prion diseases or spongiform encephalopathies are disease of the central nervous system. These diseases are characterized by neurodegeneration which leads to death of brain cells. This loss of cells has fatal consequences. These disease include Creutzfeldt-Jakob disease, Bovine Spongiform Encephalopathy, scrapie and others.

Prions are proteins that transmit conformational modification between cells in a non-Mendellian manner (i.e.. DNA is not involved) such that the same protein expressed by the "infected" cell shows the conformational state of the infecting prion which is characterized as abnormal. Two proteins have been shown to have these effects and are both expressed by yeast. However, most people these days accept that the mammalian protein - the prion protein also has such a rogue isoform.

The prion protein is a normal protein expressed by brain cells and cells of the immune system. It is highly expressed in neurones. It is cell surface glycoprotein that binds copper and possible acts as an anti-oxidant protein in synapses and elsewhere on the cell surface.

In the not too distant future the consortium will host a series of lectures, seminars and journal clubs related to prions. This web page is a temporary version. A full site will soon be constructed providing detailed information and contacts of research on prion, prion proteins and prions diseases being carried out at the University of Cambridge.

Full information concerning the Cambridge Prion Consortium can be obtained from

Dr. David R. Brown 
Department of Biochemistry 
University of Cambridge Cambridge CB2 1QW United Kingdom 
Phone: +01223 339319 Fax: +01223 333345 email: 

Amyotrophy in prion diseases.

Arch Neurol 2000 Jan;57(1):33-8
Worrall BB, Rowland LP, Chin SS, Mastrianni JA
Amyotrophic lateral sclerosis was once thought to be caused by persistent viral infection, partly because some patients with transmissible Creutzfeldt-Jakob disease showed prominent amyotrophy. However, in the past 15 years there has been little interest in the amyotrophy in prion diseases, and the possible link to amyotrophic lateral sclerosis has been eschewed. We analyzed case reports of prion disease published after 1968 for evidence of amyotrophy.

We defined amyotrophy as clinically evident fasciculation buttressed by electromyographic results in some cases. We sought evidence of motor neuron degeneration at autopsy. Prion disease was proved by transmissibility, immunohistochemistry demonstration of protease-resistant prion protein, or finding a mutation in the prion protein gene.

Amyotrophy was noted in 27 patients: 13 with sporadic Creutzfeldt-Jakob disease, 2 with familial Creutzfeldt-Jakob disease, and 12 with Gerstmann-Straussler-Scheinker disease. Of the 27, 23 showed clinical fasciculation and 10 had electromyographic evidence of denervation. The spinal cord was examined in 8 patients: 6 showed loss of motor neurons, 1 showed vacuolation of motor neurons, and 1 reported no abnormalities. Another 23 patients had typical histopathological characteristics but lacked molecular or biochemical proof of prion disease. The total number of patients with amyotrophy and proven prion disease that we identified was 50. This case review supports the belief that amyotrophy is occasionally a prominent feature of Creutzfeldt-Jakob disease and underscores the importance of documenting lower motor neuron function and the crucial role of examining the spinal cord at autopsy in cases of prion disease.

Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP

J Virol 2000 Feb;74(3):1407-1414
Bossers A, de Vries R, Smits MA
. Polymorphisms in the prion protein (PrP) gene are associated with phenotypic expression differences of transmissible spongiform encephalopathies in animals and humans. In sheep, at least 10 different mutually exclusive polymorphisms are present in PrP. In this study, we determined the efficiency of the in vitro formation of protease-resistant PrP of nine sheep PrP allelic variants in order to gauge the relative susceptibility of sheep for scrapie. No detectable spontaneous protease-resistant PrP formation occurred under the cell-free conditions used. All nine host-encoded cellular PrP (PrP(C)) variants had distinct conversion efficiencies induced by PrP(Sc) isolated from sheep with three different homozygous PrP genotypes.

In general, PrP allelic variants with polymorphisms at either codon 136 (Ala to Val) or codon 141 (Leu to Phe) and phylogenetic wild-type sheep PrP(C) converted with highest efficiency to protease-resistant forms, which indicates a linkage with a high susceptibility of sheep for scrapie. PrP(C) variants with polymorphisms at codons 171 (Gln to Arg), 154 (Arg to His), and to a minor extent 112 (Met to Thr) converted with low efficiency to protease-resistant isoforms. This finding indicates a linkage of these alleles with a reduced susceptibility or resistance for scrapie. In addition, PrP(Sc) with the codon 171 (Gln-to-His) polymorphism is the first variant reported to induce higher conversion efficiencies with heterologous rather than homologous PrP variants. The results of this study strengthen our views on polymorphism barriers and have further implications for scrapie control programs by breeding strategies.

A Comparative Study of Immunohistochemical Methods for Detecting Abnormal Prion Protein with Monoclonal and Polyclonal Antibodies.

J Comp Pathol 2000 Jan;122(1):43-53
Hardt M, Baron T, Groschup MH
Transmissible spongiform encephalopathies are associated with the accumulation of abnormal prion protein (PrP(Sc)) in the central nervous system which can be detected immunohistochemically. Using a monoclonal antibody (L42) to an epitope on the first alpha-helix of ruminant PrP, we compared previously reported immunohistochemical antigen unmasking and "visualization" systems. In addition, a variety of polyclonal and monoclonal antibodies to other epitopes on ruminant PrP were assessed. Antigen unmasking by hydrated autoclaving and proteinase K treatments, and antigen detection with L42 and an avidin-biotin complex system, enabled intra- and extra-neuronal PrP(Sc)to be demonstrated in scrapie-affected sheep carrying three different PrP alleles, as well as in cases of bovine spongiform encephalopathy.

The Paraffin-Embedded Tissue Blot Detects PrP(Sc) Early in the Incubation Time in Prion Diseases.

Am J Pathol 2000 Jan;156(1):51-56
Schulz-Schaeffer WJ, Tschoke S,... Groschup MH, Kretzschmar HA
... there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals.... We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies.

The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.


 


 


 





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