No evidence for prion protein gene locus multiplication in Creutzfeldt-Jakob disease

Neuroscience Letters 472 (2010) 16–18

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No evidence for prion protein gene locus multiplication in Creutzfeldt-Jakob disease Steven J. Collins a,c,∗ , Maaike Schuur b,d , Alison Boyd a,c , Victoria Lewis a,c,1 , Genevieve M. Klug a,c , Amelia McGlade a,c , Andrew van Oosterhout b , Guido Breedveld e , Ben A. Oostra e , Colin Masters a,c , Cornelia M. Van Duijn b,∗∗ a

Australian National CJD Registry, Department of Pathology, The University of Melbourne, Parkville 3010, Australia Department of Epidemiology, Erasmus MC University Medical Center, Rotterdam, The Netherlands c Mental Health Research Institute of Victoria, The University of Melbourne, Parkville 3010, Australia d Department of Neurology, Erasmus MC University Medical Center, Rotterdam, The Netherlands e Department of Clinical Genetics, Erasmus MC University Medical Center, Rotterdam, The Netherlands b

a r t i c l e

i n f o

Article history: Received 7 December 2009 Accepted 19 January 2010 Keywords: Creutzfeldt-Jakob disease Dementia Prion disease Prion protein gene multiplication

a b s t r a c t Precedent of causative multiplication of key gene loci exists in familial forms of both Alzheimer’s and Parkinson’s diseases. Genetic Creutzfeldt-Jakob disease (CJD) is often clinically indistinguishable from sporadic disease and inexplicably, a negative family history of a similar disorder occurs in around 50–90% of patients harboring the most common, disease-associated, prion protein gene (PRNP) mutations. We undertook semi-quantitative analysis of the PRNP copy number in 112 CJD patients using quantitative polymerase chain reaction. All included cases satisfied classification criteria for probable or definite sporadic CJD, ascertained as part of longstanding, prospective, national surveillance activities. No examples of additional copies of the PRNP locus as an explanation for their disease was found in any of the 112 sporadic CJD patients. Hence, contrasting with more common, age-related neurodegenerative diseases, the genetic aetiology in human prion disease continues to appear entirely restricted to small scale mutations within a single gene, with no evidence of multiplication of this validated candidate gene locus as a cause. © 2010 Elsevier Ireland Ltd. All rights reserved.

Creutzfeldt-Jakob disease (CJD) constitutes the most common human phenotype of the rare, transmissible neurodegenerative disorders known as prion diseases, with this disorder characterized by rapidly progressive dementia with a median survival of only 4–5 months [4]. In contrast to variant CJD, a zoonosis related to bovine spongiform encephalopathy, most CJD (approximately 85%) occurs without explanation (sporadic), with detectable mutations in the prion protein gene (PRNP) accounting for only 10–15% of cases (genetic) and the very small remainder due to inadvertent health care related transmissions (iatrogenic) [4]. Although unrecognized or covert horizontal transmission related to surgery or other medical procedures is presumed to explain some apparently sporadic

∗ Corresponding author at: Australian National CJD Registry, Level 5 The Medical Building, Department of Pathology, The University of Melbourne, Parkville, Victoria 3010, Australia. Tel.: +61 3 8344 1949; fax: +61 3 9349 5105. ∗∗ Corresponding author. Tel.: +31 10 7043391; fax: +31 10 7044657. E-mail addresses: [email protected] (S.J. Collins), [email protected] (C.M. Van Duijn). 1 Present address: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, LIGHT Laboratories, Clarendon Way, University of Leeds, Leeds LS2 9JT, United Kingdom. 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.01.043

CJD [3], this contentious mechanism is quite unlikely to be the basis for all such cases. Analogous to the much more common neurodegenerative disorders Alzheimer’s disease (AD) and Parkinson’s disease (PD), which are also patho-aetiologically linked to the accumulation of neurotoxic protein species within the CNS [9,12] sporadic CJD evinces a notable increase in incidence with age [7]. Diverse evidence suggests a decline in proteasomal function occurs with advancing age [1], with the likely compromise in protein clearance thought to be one pathogenic factor contributing to the progressive build-up of certain abnormal polypeptides, such as A␤ and ␣-synuclein so commonly observed in the brains of older individuals. Further illustrating age-related effects on protein homeostasis and the capacity of neurons to maintain a critical balance between protein synthesis and the maintenance of quality control are examples of overexpression such as Down’s syndrome due to trisomy of chromosome 21 and carriers of duplications of the Amyloid Precursor Protein (APP) gene. The presence of an extra copy of the APP gene is almost invariably associated with the early development of AD brain pathology [11,13]. Similarly, the PARK 4 autosomal dominant form of genetic PD is explained by either duplication [5] or triplication [10] of the ␣-synuclein gene (SNCA) locus, which results in

S.J. Collins et al. / Neuroscience Letters 472 (2010) 16–18

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Table 1 Summary of cases undergoing semi-quantitative analysis of the PRNP locus. Country

Definite sCJD

Probable sCJD

Male

Female

Total

Age range, yearsa (mean)

sCJD aged <61 years

Australia Netherlands Combined

48 41 89

23 23

19 26 67

29 38 45

48 64 112

34–87 (65.4)

35

a

Ages are at time of death.

the invariable development of disorders either recapitulating PD or dementia with Lewy bodies, with gene dosage directly correlating with younger age of onset and greater neuropathological severity. Current diagnostic criteria for sporadic CJD require the absence of mutations in the PRNP or exclusion of a positive family history of prion disease in first degree relatives. Analogous to phenotypes observed in some PARK 4 kindreds in comparison to idiopathic PD [5], genetic CJD is often clinically indistinguishable from sporadic disease. Of further importance, the penetrance of many PRNP mutations appears to be low, as a negative family history of a similar disorder occurs in around 50–90% of patients harboring the most common PRNP mutations associated with the CJD phenotype [6]. To date, only point mutations and polynucleotide insertions and deletions restricted to the open reading frame of PRNP are recognized as causally related to genetic CJD [4]; no gene locus multiplications have been reported, which contrasts with the findings in PD and AD. Acknowledging the aforementioned, and the fact that conventional genetic analytical techniques are insensitive for detecting disease-associated large scale chromosomal rearrangements [10], we undertook the present study to semi-quantitatively assess whether multiplication of the PRNP locus was an occasional explanation for disease in a cohort of classified sporadic CJD patients, especially in younger age at onset cases. Many of the Australian and Dutch patients included in the present study have been reported on previously [2,7]. All included cases satisfied classification criteria for probable or definite sporadic CJD, ascertained as part of longstanding, prospective, national surveillance activities with case definitions and surveillance methods described previously [2,7]. In brief, definite cases were pathologically confirmed, while probable cases had alternative diagnoses excluded and manifested rapidly progressive dementia (of less than 2 years duration), accompanied by any two of myoclonus, visual or cerebellar dysfunction, extrapyramidal or pyramidal signs or akinetic mutism, with an EEG showing typical periodic discharges or the presence of 14-3-3 proteins in the CSF. Permission for inclusion in research studies was obtained for all patients. For Dutch cases, genomic DNA was extracted from whole blood samples according to a standard non-phenol:chloroform method [8]. For Australian cases, genomic DNA was extracted using a phenol:choloroform extraction method. Briefly, approximately 50 mg frozen brain tissue was homogenised in 450 ␮l DNA extraction buffer (100 mM NaCl, 10 mM Tris–Cl pH 8.0, 25 mM EDTA pH 8.0, 0.5% (w/v) SDS), by passing through 18 g, then 20 g needles. Proteins were digested overnight at 37 ◦ C by addition of proteinase K (final concentration 1 mg/ml). An equal volume of phenol:chloroform (phenol:chloroform:isoamyl alcohol, 25:24:1, Pierce) was added, samples mixed gently, and centrifuged for 5 min, maximum speed in a bench top microfuge (approximately 13,000 rpm). The aqueous (top) layer was removed to a fresh tube, and the phenol:chloroform extraction was repeated twice more, or until there was no white precipitate at the interface between the organic and aqueous layers. The extraction was then repeated, substituting the phenol:chloroform with chloroform. DNA was precipitated from the final aqueous layer by the addition of 2.5 times the volume of ice cold 100% ethanol, which was pelleted by centrifugation at maximum speed in a bench top microfuge at 4 ◦ C. The DNA pel-

let was then washed with 70% ethanol, allowed to air dry, and re-suspended in an appropriate volume of sterile Milli-Q H2 O. PRNP gene locus copy number variation was analysed by qPCR (Hydrolysis Probes). For the PRNP locus, we used a FAM labeled Applied Biosystems TaqMan probe (Hs01940892) located in exon 2 of the PRNP gene (NM 000311) and the RNase P Control Reagents Kit (VIC labeled, Applied Biosystems) as reference locus. Both probes (final concentration 1×) were tested in one single reaction, total volume 20 ␮l, containing 1× qPCR MasterMix Plus w/o UNG (Eurogentec) and 15 ng of genomic DNA using an Applied Biosystems 7300 Real Time PCR System. The amplification protocol was as follows: initial denaturation for 10 min at 95 ◦ C followed by 40 cycles of denaturation at 95 ◦ C for 15 s and annealing, extension and data collection at 60 ◦ C for 60 s. Relative quantification was performed using the RQ-Study Application from the Sequence Detection (SDS) software V1.3.1 (Applied Biosystems). The linear amplification phase for analysis of PRNP was generally observed between 25 and 27 cycles. A total of 112 sporadic CJD patients (89 definite, 23 probable) underwent PRNP locus quantification by qPCR; in three additional cases amplification was attempted but was unsuccessful (two due to degraded DNA; one for unclear reasons). Table 1 summarizes the demographic features of analysed cases. The ages of the 112 patients at time of death ranged from 34 to 87 years (mean 65.4 years), with 67 females (59.8%). Thirty-five individuals (31.3%) were 60 years of age or younger at their time of death, with 12 (10.7%) less than 56 years. None of the 112 successfully, semi-quantitatively, assessed sporadic CJD cases showed evidence of a definite increase in PRNP locus copy number with the relative quantification range 0.76–1.39 (normal: 0.7–1.3); two patients each had copy ratios just above the normal range on a single occasion, which were within the reference range on repeat testing. Despite precedent in other age-related neurodegenerative diseases, albeit with more overt genetic associations, our study of 112 sporadic CJD patients failed to disclose any examples of additional copies of the PRNP locus as an explanation for their disease. Although investigation of familial CJD occurring in the absence of PRNP mutations would perhaps, a priori, be considered more likely to uncover locus copy number multiplication, such pedigrees were not available. We speculated that given the insensitivity of conventional analytical techniques to gene locus multiplication and through extrapolated analogy to the unexplained but very frequent occurrence of a negative family history despite probands harboring causative PRNP mutations, it was reasonable to investigate a sizeable cohort of apparently sporadic CJD patients. Even though the number of cases included in our study is perhaps comparatively small, this finding certainly militates against undetected duplication or triplication of this genetic locus constituting a relatively common cause and possibly excludes this type of genetic basis as even a rare cause of apparently sporadic disease. From previous reports of PARK 4 kindreds, age of disease onset correlates with gene dosage, although for SNCA duplications ages at onset were only modestly or equivocally reduced in comparison to idiopathic PD [5] with the most dramatic effects observed in relation to triplication of SNCA [10]. With nearly one third of our sporadic CJD cohort below the mean age at death reported for this disease (around 65–70 years) [2], we believe our studied group

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included optimally aged patients to detect this type of underlying genetic disorder. In conclusion, contrasting with the more common, age-related neurodegenerative diseases AD and PD, the genetic aetiology in human prion disease continues to appear entirely restricted to small scale mutations within a single gene, the PRNP, with no evidence of multiplication of this validated candidate gene locus as a cause. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The Australian National Creutzfeldt-Jakob disease Registry (ANCJDR) is funded by the Commonwealth Department of Health and Ageing. This work was supported in part by an NH&MRC Program Grant (#400202). SJC is supported by an NH&MRC Practitioner Fellowship (#400183) and by an NH&MRC Project Grant (#454546). The Dutch CJD Surveillance is funded by the Dutch Ministry of Health, Welfare and Sports. The ANCJDR and the Dutch Surveillance Center thank the families of all the sporadic CJD patients and their managing clinicians for their support. The methodology undertaken in this study complies with the ethical standards and statutes of the participating countries. References [1] G. Carrard, A.L. Bulteau, I. Petropoulos, B. Friguet, Impairment of proteasome structure and function in aging, The International Journal of Biochemistry & Cell Biology 34 (2002) 1461–1474. [2] S. Collins, A. Boyd, J.S. Lee, V. Lewis, A. Fletcher, C.A. McLean, M. Law, J. Kaldor, M.J. Smith, C.L. Masters, Creutzfeldt-Jakob disease in Australia 1970–1999, Neurology 59 (2002) 1365–1371.

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