Introduction of Laboratory Diagnosis and Examination of Creutzfeldt-Jakob Disease

Abstract

Creutzfeldt-Jakob disease (CJD) is an infectious prion disease caused by the accumulation of misfolded prion proteins in the central nervous system. It is associated cognitive impairment and ataxia, and results in death within several months after the onset. CJD is subdivided into sporadic, genetic, and acquired types according to the phenotype, epidemiological features, and pathogenesis. Laboratory diagnosis of CJD offers the advantage of being autopsy-free and is essential for accurate diagnosis of the disease along with assessment of clinical symptoms and electroencephalography. In addition, since the incidence of CJD is increasing worldwide, including in the Republic of Korea, the importance of laboratory diagnosis of the disease is also increasing. The 14-3-3 protein analysis, real-time quaking-induced conversion (RT-QuIC), prion protein (PRNP) gene mutation analysis are mainly used to diagnose CJD. Since each of these diagnostic tests has different interpretations and limitations, combined analysis is essential for the accurate diagnosis and classification of CJD. For efficient disease management, a diagnosis accompanied by simultaneous multilateral analysis using various laboratory diagnostics is indispensable in patients with suspected CJD.

Key messages

① What is known previously?

Creutzfeldt-Jakob disease (CJD) is a prion disease in human, resulting in cognitive impairment and ataxia. It is an infectious neurodegenerative disease with a 100% fatality rate.

② What new information is presented?

For a definite diagnosis of CJD, histological analysis of brain tissue obtained through autopsy is essential; however, autopsy is rarely performed because of domestic circumstances. Therefore, the importance of an autopsy-free laboratory diagnosis has been highlighted. 14-3-3 protein analysis, real-time quaking-induced conversion (RT-QuIC), and prion protein (PRNP) gene mutation analysis are mainly used to diagnose CJD.

③ What are implications?

Since various diagnostic tests for CJD have different interpretations and limitations, combined analysis is essential for accurate diagnosis and classification of CJD. For efficient disease management, a diagnosis accompanied by simultaneous multilateral analysis using various laboratory diagnostics is indispensable in patients with suspected CJD.

Introduction

Creutzfeldt-Jakob disease (CJD) is one of the human transmissible spongiform encephalopathies or a prion (pronounced “pree-ON”) disease, and is one of the fatal neurodegenerative diseases [1]. A prion is an infectious disease protein particle, a term derived from two words “protein” and “infection,” and was coined by Prusiner [2], M.D. in 1982 while identifying proteins involved in disease. In general, prion is not introduced from the outside of the human body; instead, it normally exists in the human body (i.e., cellular prion protein [PrP^C]). In case of humans, PrP^C is a protein derived from chromosome 20, and the gene encoding this protein is called prion protein (PRNP) gene [3]. Although the exact function of the normal prion protein has not been clearly understood, prion diseases such as CJD are caused by the accumulation of an abnormal prion protein (scrapie prion protein [PrP^sc]) in the central nervous system by the misfolding of PrP^C (Figure 1) [4,5]. Although the mechanism that causes these structural changes is not yet known, the accumulation of PrP^sc causes vacuole formation in nerve cells and spongiform changes, leading to fatal neurodegenerative diseases that result in death without exception [1,3]. Additionally, PrP^sc requires a distinct disinfection method different from the generally used disinfection method, which is difficult to manage; therefore, it has a risk of spreading to the surroundings when exposed to the environment [4].

Figure 1. Schemeic diagram of misfolding of prion disease.

The pathogenic form of prion protein (PrP^sc) is the causative agent of the disease which acts as a seed in the cyclic conversion of normal cellular prion (PrP^C) into the misfolded pathogenic isoform. These newly formed PrP^sc further acts as a seed to this conversion mechanism to form PrP^sc oligomers and fibrils. PrP^sc=scrapie prion protein; PrP^C=cellular prion protein. Revised from the article of Khadka et al. (Cell Tissue Res 2022. [Epub] https://doi.org/10.1007/s00441-022-03621-0) [5].

The PrP^sc phenotype, epidemiological characteristics, and pathogenesis of CJD found in humans are remarkably diverse; hence, based on this information, CJD is classified into sporadic, genetic, and acquired types [1]. Spontaneously occurring sporadic CJD (sCJD) is the most commonly identified human prion disease, accounting for approximately 85% of total CJD cases [1-3]. Familial CJD (fCJD) is a representative genetic CJD, and is mainly caused by PRNP gene mutation, accounting for approximately 10–15% of the total CJD cases [1,6]. Iatrogenic CJD (iCJD) and variant CJD (vCJD) are acquired types, caused by external factors, accounting for approximately 1% of the total cases [1]. The identified main external factors for iCJD are external administration of human pituitary growth hormone, dura mater transplantation, and use of contaminated equipment during surgical procedures such as ophthalmic procedures. In case of vCJD, which occurs mainly in the United Kingdom, consumption of beef infected with bovine spongiform encephalopathy is known to be the main external factor [1,4,7].

The average survival time of sCJD is approximately 6 months, and more than 90% of patients die within 1 year from the onset of symptoms. The incidence rate is highest in people in the age range of 70s, and the incidence rate is significantly low in the age ranges of 20s to 40s or 80s and older. The main symptoms include rapidly progressingz cognitive disorders such as dementia, ataxia, spatial perception disorders, and emotional disorders such as depression [7,8]. Clinical symptoms of fCJD and iCJD are similar to sCJD, but the age of onset varies from 50 to 60 years of age for fCJD, and 9.6 to 37.7 years of age for iCJD depending on the type of surgery. Most of the patients die within 6 months after the onset of symptoms in the case of fCJD, whereas, 12–28 months after the symptoms for iCJD [7]. vCJD is characterized by psychiatric symptoms, such as depression and anxiety, and abnormal sensory symptoms in the limbs even in the early stage of the disease. The mean age of onset of vCJD is relatively low at the age of 26, and most of the patients die within 14 months after the onset of vCJD-related symptoms [4,7].

Currently, the approved test method for CJD is to process specimens obtained through a brain biopsy or autopsy and measure the protease-resistant prion protein to confirm the diagnosis as PrP^sc [9]. However, since this method has a practical limitation in confirming CJD due to lower autopsy rates, CJD is currently diagnosed and treated through estimation using the clinical findings by specialists and laboratory-based diagnostic results. In the Republic of Korea (ROK), a case judgment committee composed of CJD experts determines whether or not CJD has occurred by integrating the patient's clinical symptoms, past medical history, and electroencephalogram (EEG)/magnetic resonance imaging (MRI) test results.

Early diagnosis of CJD is particularly important because it can minimize the risk of accidentally acquired transmission of prion into the human body, such as through the use of contaminated surgical instruments. In the present article, various laboratory tests will be introduced, which are mainly used for rapid diagnosis of CJD, to better understand them.

Methods

Through a literature review and publications from the Korea Disease Control and Prevention Agency, the National CJD Research & Surveillance Unit, and the Centers for Disease Control and Prevention in the United States, the laboratory diagnostic test methods for CJD were researched, organized, and explained.

Results

1. The 14-3-3 Protein Analysis

The 14-3-3 protein was named ‘14-3-3’ because it was initially found at the position 3.3 in the result of electrophoresis of sample 14 in the process of isolating protein from bovine brain tissue in 1967, and it is a protein of 30 kDa size that is expressed in various tissues, especially brain tissue [10]. The 14-3-3 protein performs numerous biological functions (e.g., differentiation and survival of neurons, neurite outgrowth, and ion channel regulation), and is known to have increased expression in CJD patients; therefore, it is used as an important biomarker for diagnosis [10]. In particular, for the presumptive diagnosis of sCJD, Western blot is adopted and used for detecting 14-3-3 protein [9,11]. The sensitivity of the 14-3-3 protein detection test is 79% for sCJD, 82% for fCJD, and 100% for iCJD, respectively; but, the sensitivity for vCJD is relatively low at approximately 50% [12]. This test method has a limitation of the possibility of false positives because the level of 14-3-3 protein expression is increased not only in CJD but also in other brain diseases such as stroke, status epilepticus, and inflammatory encephalopathy. In such cases, additional detection of tau protein, another biomarker for confirming nerve damage, should also be considered as a supplement to the 14-3-3 protein detection for CJD diagnosis [11].

2. PRNP Gene Mutation Analysis

The PRNP gene encoding the prion protein consists of 253 amino acids [6]. Mutations such as point mutations or octapeptide repeats insertion/deletion (OPRI/OPRD) in the PRNP gene have been reported to cause genetic prion diseases such as fCJD, Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia (FFI) [12]. These genetic prion diseases are inherited in an autosomal dominant manner and are expressed and occur in multiple family members [3]. In the PRNP gene mutation analysis, DNA is extracted from the blood of suspected CJD patients, the PRNP gene is amplified through polymerase chain reaction, and then the presence or absence of mutation is identified through sequencing.

1) Single nucleotide polymorphism analysis for amino acid substitution

Although the exact mechanism has not been elucidated, mutations in the PRNP gene cause the expression of abnormal prion proteins, and more than 50 single nucleotide polymorphisms (SNPs) have been reported [12]. Among several SNPs in the PRNP gene, fCJD arises from SNPs of G114V, R148H, D178N-129V, V180I, T183A, T188K, E196K, E200K, V203I, R208H, V210I, E211Q, M232R, and P238S, among them, E200K is the most representative SNP (Figure 2) [12].

Figure 2. PRNP gene mutation causing fCJD.

^a)D178N is associated with familial CJD (fCJD) depending on the codon 129 polymorphism (valine [V]). PRNP=prion protein; OPRD=octapeptide repeats deletion; CHO=Asn-linked glycosylation site; GPI=glycosylphosphati dylinositol; S=serine; R=arginine; OPRI=octapeptide repeats insertion; G=glycine; H=histidine; D=aspartic acid; N=asparagine; I=isoleucine; T=threonine; A=alanine; K=lysine; E=glutamic acid; Q=glutamine; M=methionine; P=proline. Revised from the article of Jeong and Kim. (J Korean Med Sci 2014;29:623-32) [12].

2) Polymorphic gene analysis

The PRNP gene has sites with various polymorphisms, the most representative SNP variations are the polymorphisms by methionine (M) and valine (V) in the PRNP codon 129, i.e., homozygous (M/M, V/V) or heterozygous (M/V). The PRNP codon 129 polymorphism is a dangerous genetic factor in human prion diseases, and its ratio is known to vary by country and race/ethnicity [12]. In Europe, only approximately 49% of the general population were M/M homozygous, but 80% of sCJD patients and 100% of vCJD patients were M/M homozygous; meanwhile, in the ROK, 94% of the general population were M/M homozygous, but 100% of sCJD patients were identified as M/M homozygous type [12]. In addition, the polymorphism of codon 129 has different clinical features depending on the SNP of codon 178 associated with the substitution of aspartic acid (D) to asparagine (N): with the 178 codon SNP, FFI is associated with M/M homozygous of codon 129, whereas, fCJD is associated with M/V heterozygous of codon 129 [7,12].

3. Real-time Quaking-induced Conversion for Detecting Abnormal Prions

1) Real-time quaking-induced conversion

Real-time quaking-induced conversion (RT-QuIC) directly detects PrP^sc in cerebrospinal fluid (CSF), and it shows significantly higher sensitivity and specificity for CJD diagnosis than other test methods. In this method, recombinant prion protein (rPrP) is used as a seed, and then CSF containing PrP^sc is added to induce aggregation. After that, thioflavin-T (ThT) included in the reaction reagent reacts with the aggregated PrP^sc to change the ThT fluorescence spectrum, and the reaction is measured in real-time to confirm the presence of PrP^sc (Figure 3) [13,14]. RT-QuIC is an innovative and breakthrough test method because it can detect even an exceedingly small amount (1 fg) of PrP^sc, and it can be used with various samples such as brain homogenate, CSF, plasma, and olfactory mucosa, and has an automated reaction system, along with high sensitivity and specificity [14,15]. RT-QuIC has been added as a test method for diagnosing sCJD since 2018 [11].

Figure 3. Schemic represntation of RT-QuIC.

The real-time quaking-induced conversion (RT-QuIC) reaction may be divided into the lag phase, the exponential phase and the plateau phase. During lag phase, the seed is allowed to contact the substrate and to trigger the conformational change. The monomers aggregates into oligomers and amyloid fiber in exponential phase, thioflavin-T (ThT) reacts with this fibers. Eventually, when all the substrate is incorporated into fibrils, a plateau phase is observed. Revised from the article of Candelise et al. (Acta Neuropathol Commun 2020;8:117) [14].

The reaction conditions of RT-QuIC have been changed gradually through numerous studies, and currently, RT-QuIC can be largely divided into first-generation and second-generation methods [13]. The first-generation RT-QuIC targeted the prion protein (23-231 amino acids [aa]) of rPrP [14,15], had a sensitivity of 73–100%, and a specificity of 98–100%, showing excellent discrimination against negative samples [13]. The second-generation RT-QuIC, developed through continuous research to ensure the accuracy and efficiency of the test method, shortens the reaction time by changing the reaction conditions using the N-terminal truncated rPrP (90-231aa), having improved sensitivity of 91–100%, and improved specificity of 98.5–100% [14,15].

2) Enhanced quaking-induced conversion

RT-QuIC is useful for diagnosing general CJD, but has a lower sensitivity of 25% for vCJD diagnosis [11]. To improve such drawback, a test method called “enhanced QuIC (e-QuIC)” was developed by adding an immunoprecipitation (IP) to the existing RT-QuIC test method [16]. e-QuIC detects PrP^sc protein using a PrP^sc-specific antibody after adding diluted brain homogenate of vCJD patients to plasma [16]. This test method could be considered as an advanced test method because it improved the low sensitivity of the existing RT-QuIC to vCJD, and detected PrP^sc in blood with many reaction inhibitory substances, at a much lower concentration than existing RT-QuIC; but it is difficult to standardize the test method and it reduced reproducibility [15].

Discussion

Prion protein, the causative agent of CJD, is a pathogen completely different from other infectious pathogens such as bacteria and viruses in terms of characteristics, route of transmission, and diagnostic methods. Moreover, since CJD has an exceptionally long incubation period ranging from several months to several years, and does not show any prodromal symptoms such as inflammatory response and disease-specific immune response, we may not be able to recognize CJD until related clinical symptoms appear. There is a 100% risk of death due to CJD because of no currently available treatments. In general, CJD is not an infectious disease that is transmitted through respiratory droplets or contact, but in case of iCJD or vCJD, they should be managed and controlled with caution because the risk of transmission and the cause of the outbreak of iCJD or vCJD might be related to social issues such as medical care and livestock farming.

Worldwide, 1 to 2 cases of CJD per 1,000,000 people are reported every year, and the CJD case numbers have tended to be gradually increasing [1,4,17-19]. Since 2011, the ROK has been conducting surveillance on all CJD patients. Although the number of cases of Korean domestic CJD patients is insignificant compared to major outbreaks in other countries, the number of reported cases per year has been between 29 and 65. In particular, compared to 2011, when total case surveillance began, the number of cases has increased to be more than double in 2021 [20].

Accordingly, the importance of managing and diagnosing CJD patients has also been increasing in the ROK. However, in reality, an autopsy to confirm CJD is quite difficult in the ROK, so a diagnosis of CJD patients mostly relies on presumptive diagnosis [5,20]. The presumptive diagnosis of CJD in the ROK is determined by the CJD case judgment committee composed of subject matter experts in the related field by integrating the patient’s clinical symptoms, EEG/MRI test, and laboratory test results; among this information, the laboratory test result is considered the most crucial factor because it is necessary for making the diagnostic decision as per the diagnostic criteria in Table 1. Although there are diverse types of CJD diagnostic tests, the meaning and interpretation of each test result are different, and the integrated results are essential for classifying CJD patients. Therefore, an accurate diagnosis can be made only when the data is analyzed from multi-level perspectives. In addition, since each test method has different limitations, supplementing it with the results of various other test methods is essential and required. In case of the 14-3-3 protein analysis, the destruction of brain tissue due to the accumulation of amyloid fibrils can be judged by the increase in the amount of 14-3-3 protein in the CSF, but the ability to differentiate it from other degenerative brain diseases is limited [11]. Therefore, it is important to cross-validate false positives by comparing the detection results of another biomarker such as tau protein, or the detection results of PrP^sc using RT-QuIC. In case of PRNP gene mutation analysis, although the heritability for CJD can be identified, it is not possible to directly determine whether or not CJD is present, so a comprehensive analysis should also be performed along with 14-3-3 protein detection results or RT-QuIC results. Considering these characteristics of various CJD diagnostic methods, medical institutions need to request various laboratory tests together for accurate diagnosis of CJD and efficient patient management, when CJD is suspected according to the patient’s clinical symptoms and EEG results.

Table 1.

Probable diagnostic criteria for CJD

Type	Probable diagnostic criteria	DC	Reference
sCJD	- A person corresponding any of the following ①, ②, ③ positive in [DC1] ①, ②, ④ positive in [DC1] ①, ② positive in [DC1], and 14-3-3 protein detection in CSF RT-QuIC positive in CSF or other tissues, and progressive neuropsychiatric disorder	[DC1] ①Rapidly progressive dementia (cognitive impairment) ②Two or more of the following Myoclonus Visual or cerebellar dysfunction Pyramidal or extrapyramidal features Akinetic mutism ③Typical EEG findings (periodic sharp wave complexes) ④Typical MRI findings (high signal in caudate/putamen or at least two cortical regions either on DWI or FLAIR)	[10-12,20]
fCJD	- A person corresponding any of the following Neuropsychiatric disorder and definite or probable CJD in 1st degree relative Progressive neuropsychiatric disorder and pathogenic PRNP gene mutation in [DC2]	[DC2] G114V, R148H, D178N-129V, V180I, T183A, T188K, E196K, E200K, V203I, R208H, V210I, E211Q, M232R, P238S, 96bpi, 120bpi, 144bpi, 168 bpi, 48bpdel
iCJD	- A person corresponding any of the following Progressive cerebellar syndrome in a recipient of human cadaveric derived pituitary hormone Probable sCJD with [DC3]	[DC3] ①Administration of reproductive gland stimulating hormone, transplantation of human origin pituitary growth hormone and dura matter ②Corneal transplantation from prion disease definite or probable patient ③Exposure from neurosurgical instruments used in prion disease definite or probable patient
vCJD	- A person corresponding any of the following ①, ②, ③A, ③B positive in [DC4] ①, ④ positive in [DC4]	[DC4] ①All positive following clinical history Progressive neuropsychiatric disorder 6 months or more of disease duration No other diagnosis to suspect No history of exposure to transmissible iatrogenic risk factors No evidence of fCJD ②Four or more of the following Psychiatric symptoms such as depression, anxiety, and apathy in early stage Persistent painful sensory symptoms Ataxia Muscular dysfunction Dementia ③EEG and MRI results No typical EEG findings in early stage or no EEG test results High signal of both pulvinar in MRI results ④Adaptable result for vCJD in biopsy of tonsil

CJD=Creutzfeldt-Jakob disease; DC=detailed criteria; sCJD=sporadic CJD; fCJD=familial CJD; iCJD=iatrogenic CJD; vCJD=variant CJD; CSF=cerebrospinal fluid; RT-QuIC=real-time quaking-induced conversion; PRNP=prion protein; EEG=electroencephalogram; MRI=magnetic resonance imaging; DWI=diffusion weighted imaging; FLAIR=fluid attenuation inverse recovery; i=inserstion; del=deletion.