TY - JOUR
AU - Lund, Eva Løbner
AB - Abstract This is the first report of presumed sporadic Creutzfeldt-Jakob disease (sCJD) and Gerstmann-Sträussler-Scheinker disease (GSS) with the prion protein gene c.305C>T mutation (p.P102L) occurring in one family. The father and son were affected with GSS and the mother had a rapidly progressive form of CJD. Diagnosis of genetic, variant, and iatrogenic CJD was ruled out based on the mother’s clinical history, genetic tests, and biochemical investigations, all of which supported the diagnosis of sCJD. However, given the low incidence of sCJD and GSS, their co-occurrence in one family is extraordinary and challenging. Thus, a hypothesis for the transmission of infectious prion proteins (PrPSc) via microchimerism was proposed and investigated. DNA from 15 different brain regions and plasma samples of the CJD patient was subjected to PCR and shallow sequencing for detection of a male sex-determining chromosome Y (chr. Y). However, no trace of chr. Y was found. A long CJD incubation period or presumed small concentrations of chr. Y may explain the obtained results. Further studies of CJD and GSS animal models with controlled genetic and proteomic features are needed to determine whether maternal CJD triggered via microchimerism by a GSS fetus might present a new PrPSc transmission route. Chromosome Y, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, Microchimerism, PRNP, Prions, Transmission INTRODUCTION Creutzfeldt-Jakob disease (CJD) is a rare, transmissible neurodegenerative disease caused by misfolding of the host-encoded prion protein (PrPC) into its protease-resistant forms (PrPSc), which are called prions (1). Human prion diseases occur in sporadic (80%–95%), genetic (10%–15%), and acquired (<1%) forms (2). Prior to the discovery of the prion protein gene (PRNP), genetic prion diseases were classified according to their clinicopathological features as genetic Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease (GSS), or fatal familial insomnia (3). Modern classification of genetic prion diseases is more detailed because missense and nonsense mutations and octapeptide-repeat-insertions and -deletions in the PRNP are well defined (4). The most common pathogenic variant in PRNP in GSS cases is a missense mutation, where a single nucleotide substitution (c.305C>T) leads to a replacement of the amino acid proline (P) with leucine (L) at codon position 102 (p.P102L) (3). The P102L mutation is transmitted autosomal dominantly with complete penetrance, but cases without a family history of neurodegenerative diseases also occur (5). Moreover, despite the lack of evidence for GSS transmission via blood in humans, GSS transmission has been demonstrated in experiments with apes and rodents that have been inoculated with human GSS brain tissue homogenates or blood (6–9). Iatrogenic CJD (iCJD) is the least prevalent form of CJD, but horizontal human-to-human PrPSc transmission has been shown via several methods, including contaminated surgical instruments, dura mater and ocular tissue grafts, blood transfusion, treatment with natural growth hormones, and gonadotropin obtained from PrPSc carriers (10, 11). Interestingly, there are few reported cases of seemingly etiologically distinct prion diseases in one family (12–15). Therefore, it is likely that other, yet unidentified, horizontal PrPSc transmission routes exist. Here, we present the first case of presumed sporadic CJD (sCJD) in a woman who was married to a man with GSS and who had a son with a confirmed P102L mutation causing GSS. To explain this unusual combination of rare prion diseases occurring in one family, we have evaluated the possibility of known transmission routes and investigated a hypothesis of a new PrPSc transmission route via acquired microchimerism. Microchimerism is a naturally occurring bidirectional exchange of cells between the fetus and mother (16). The fetus’ cells can circulate in the mother’s blood or reside in various organs, including the brain, and they can remain there for decades (16–20). There is evidence that fetal cells in the mother’s body differentiate into multiple cell types, including neurons, to contribute to maternal somatic maintenance (21, 22). Because fetal cells can reside in the mother’s brain and differentiate into neurons, we hypothesize that if a mother is carrying a fetus with a PRNP P102L mutation, she can be exposed to cells that could cause the development of prion disease. In this situation, P102L-derived PrPSc in fetal cells would be seeded into the mother’s brain, where they would go on to trigger the misfolding of the maternal normal prion proteins (PrPC). MATERIALS AND METHODS Patients and Samples The family pedigree is shown in Figure 1, where the CJD patient’s husband is denoted as the GSS proband. Clinical information of the CJD patient (III-6), the son with GSS (IV-1), the GSS proband (III-5), and the proband’s paternal uncle (II-6) is summarized in Table 1 and described in detail in the Supplementary Material. TABLE 1. Summary of Clinical and Paraclinical Information . CJD Patient/Wife/Mother (III-6) . GSS Patient/Son (IV-1) . GSS Proband/Husband/Father (III-5) . GSS Proband’s Paternal Uncle (II-6) . Samples DNA from blood and plasma; half fresh frozen and half formic acid and formalin-fixed paraffin embedded brain; CSF DNA from blood sample Formalin fixed and formalin-fixed paraffin-embedded brain – Age at disease onset 76 48 55 74 Age at death 76 – 63 79 Disease duration 6 months > 4 years 8 years 5 years Disease onset symptoms Progressing imbalance, gait disturbance and confusion; cognitive decline; aphasia; double vision; hallucinations. Memory impairment; personality changes; increased aggressiveness; hypomania and lack of insight; interrupted horizontal eye pursuit movements; slow initiation of saccades; difficulty in performing Luria test, even with cueing and brisk reflexes. Insidious and progressing gait disturbance; imbalance; tremor and cramped handwriting. Gait disturbance; pain in the legs. Progressing symptoms With the initial symptoms getting worse, the patient also had delusions and became disoriented, developed severe myoclonus and mutism; eventually, the patient became rigid and deteriorated to a vegetative state. Significant global cognitive impairment with amnesia, anomia, acalculia, apraxia, and reduced concentration. Difficulties swallowing; double vision; urinary urgency; short-term memory impairment; dysarthria; upward gaze palsy and horizontal nystagmus; hypomimic facial expressions; postural tremor in the upper limbs; upper and lower limb ataxia; diminished tendon reflexes in the arms and lost in the legs; Babinski’s reflex diminished bilaterally. Lower back pain; paresis of the legs; arm and leg numbness; suicidal thoughts; swallowing difficulties; urinary incontinence; dysarthria; disorientation; upward gaze palsy and jerky eye movements with a horizontally gaze-evoked nystagmus; amyotrophic arms and legs; universal loss of tendon reflexes; extensor plantar reflexes. MRI DWI signal abnormalities in the right caudate nucleus. – – Normal. PET/MR FDG Whole-body scan was normal; brain scan revealed generally reduced metabolic activity. Parietotemporally moderately reduced (symmetrically distributed) metabolic activity with involvement of mesial parietal structures; only slight bifrontal affection. – – 11C-PIB PET – No amyloid binding in the gray matter. – – CT Slight cortical and central atrophy. – – Slight cortical and central atrophy; diffuse leukoariosis. EEG Encephalopatic; background slowing pattern and delta activity in frontal area; no CJD characteristic changes. – – – CSF RT-QuIC positive PrPSc amplification detected; amyloid beta 1-42 was decreased; pTau was normal; Tau, 14-3-3 protein and NSE were elevated. 14-3-3 protein and oligoclonal bands were not detected; amyloid beta 1-42, NSE, and pTau parameters were normal; Tau and neurofilament light polypeptides were elevated. – Mononuclear pleocytosis; raised spinal protein. DNA analysis No known pathogenic variants in PRNP, homozygous for V at codon 129 (129VV); silent mutation at codon 117 (A117A). P102L mutation in PRNP—mutated allele codes for M at codon 129 (P102L-129M), heterozygous at codon 129 (129MV), silent mutation at codon 117 (A117A). P102L mutation in PRNP; the information of other variants in PRNP was not available, and new analysis could not be performed. No mutations found in the genes for SCA type 1, 2, 3, 6, 7, 17; HD; and DRPLA. – . CJD Patient/Wife/Mother (III-6) . GSS Patient/Son (IV-1) . GSS Proband/Husband/Father (III-5) . GSS Proband’s Paternal Uncle (II-6) . Samples DNA from blood and plasma; half fresh frozen and half formic acid and formalin-fixed paraffin embedded brain; CSF DNA from blood sample Formalin fixed and formalin-fixed paraffin-embedded brain – Age at disease onset 76 48 55 74 Age at death 76 – 63 79 Disease duration 6 months > 4 years 8 years 5 years Disease onset symptoms Progressing imbalance, gait disturbance and confusion; cognitive decline; aphasia; double vision; hallucinations. Memory impairment; personality changes; increased aggressiveness; hypomania and lack of insight; interrupted horizontal eye pursuit movements; slow initiation of saccades; difficulty in performing Luria test, even with cueing and brisk reflexes. Insidious and progressing gait disturbance; imbalance; tremor and cramped handwriting. Gait disturbance; pain in the legs. Progressing symptoms With the initial symptoms getting worse, the patient also had delusions and became disoriented, developed severe myoclonus and mutism; eventually, the patient became rigid and deteriorated to a vegetative state. Significant global cognitive impairment with amnesia, anomia, acalculia, apraxia, and reduced concentration. Difficulties swallowing; double vision; urinary urgency; short-term memory impairment; dysarthria; upward gaze palsy and horizontal nystagmus; hypomimic facial expressions; postural tremor in the upper limbs; upper and lower limb ataxia; diminished tendon reflexes in the arms and lost in the legs; Babinski’s reflex diminished bilaterally. Lower back pain; paresis of the legs; arm and leg numbness; suicidal thoughts; swallowing difficulties; urinary incontinence; dysarthria; disorientation; upward gaze palsy and jerky eye movements with a horizontally gaze-evoked nystagmus; amyotrophic arms and legs; universal loss of tendon reflexes; extensor plantar reflexes. MRI DWI signal abnormalities in the right caudate nucleus. – – Normal. PET/MR FDG Whole-body scan was normal; brain scan revealed generally reduced metabolic activity. Parietotemporally moderately reduced (symmetrically distributed) metabolic activity with involvement of mesial parietal structures; only slight bifrontal affection. – – 11C-PIB PET – No amyloid binding in the gray matter. – – CT Slight cortical and central atrophy. – – Slight cortical and central atrophy; diffuse leukoariosis. EEG Encephalopatic; background slowing pattern and delta activity in frontal area; no CJD characteristic changes. – – – CSF RT-QuIC positive PrPSc amplification detected; amyloid beta 1-42 was decreased; pTau was normal; Tau, 14-3-3 protein and NSE were elevated. 14-3-3 protein and oligoclonal bands were not detected; amyloid beta 1-42, NSE, and pTau parameters were normal; Tau and neurofilament light polypeptides were elevated. – Mononuclear pleocytosis; raised spinal protein. DNA analysis No known pathogenic variants in PRNP, homozygous for V at codon 129 (129VV); silent mutation at codon 117 (A117A). P102L mutation in PRNP—mutated allele codes for M at codon 129 (P102L-129M), heterozygous at codon 129 (129MV), silent mutation at codon 117 (A117A). P102L mutation in PRNP; the information of other variants in PRNP was not available, and new analysis could not be performed. No mutations found in the genes for SCA type 1, 2, 3, 6, 7, 17; HD; and DRPLA. – MRI, magnetic resonance imaging; DWI, diffusion-weighted imaging; PET, positron-emission tomography; MR FDG, metabolic rate of fluorodeoxyglucose; 11C-PIB PET, positron emission tomography imaging with 11C-labelled Pittsburgh Compound-B ligand; CT, computed tomography; EEG, electroencephalography; CSF, cerebrospinal fluid; NSE, neuron-specific enolase; SCA, spinocerebellar ataxia; HD, Huntington disease; DRPLA, Dentato-Rubro-Pallido-Luysian atrophy. Open in new tab TABLE 1. Summary of Clinical and Paraclinical Information . CJD Patient/Wife/Mother (III-6) . GSS Patient/Son (IV-1) . GSS Proband/Husband/Father (III-5) . GSS Proband’s Paternal Uncle (II-6) . Samples DNA from blood and plasma; half fresh frozen and half formic acid and formalin-fixed paraffin embedded brain; CSF DNA from blood sample Formalin fixed and formalin-fixed paraffin-embedded brain – Age at disease onset 76 48 55 74 Age at death 76 – 63 79 Disease duration 6 months > 4 years 8 years 5 years Disease onset symptoms Progressing imbalance, gait disturbance and confusion; cognitive decline; aphasia; double vision; hallucinations. Memory impairment; personality changes; increased aggressiveness; hypomania and lack of insight; interrupted horizontal eye pursuit movements; slow initiation of saccades; difficulty in performing Luria test, even with cueing and brisk reflexes. Insidious and progressing gait disturbance; imbalance; tremor and cramped handwriting. Gait disturbance; pain in the legs. Progressing symptoms With the initial symptoms getting worse, the patient also had delusions and became disoriented, developed severe myoclonus and mutism; eventually, the patient became rigid and deteriorated to a vegetative state. Significant global cognitive impairment with amnesia, anomia, acalculia, apraxia, and reduced concentration. Difficulties swallowing; double vision; urinary urgency; short-term memory impairment; dysarthria; upward gaze palsy and horizontal nystagmus; hypomimic facial expressions; postural tremor in the upper limbs; upper and lower limb ataxia; diminished tendon reflexes in the arms and lost in the legs; Babinski’s reflex diminished bilaterally. Lower back pain; paresis of the legs; arm and leg numbness; suicidal thoughts; swallowing difficulties; urinary incontinence; dysarthria; disorientation; upward gaze palsy and jerky eye movements with a horizontally gaze-evoked nystagmus; amyotrophic arms and legs; universal loss of tendon reflexes; extensor plantar reflexes. MRI DWI signal abnormalities in the right caudate nucleus. – – Normal. PET/MR FDG Whole-body scan was normal; brain scan revealed generally reduced metabolic activity. Parietotemporally moderately reduced (symmetrically distributed) metabolic activity with involvement of mesial parietal structures; only slight bifrontal affection. – – 11C-PIB PET – No amyloid binding in the gray matter. – – CT Slight cortical and central atrophy. – – Slight cortical and central atrophy; diffuse leukoariosis. EEG Encephalopatic; background slowing pattern and delta activity in frontal area; no CJD characteristic changes. – – – CSF RT-QuIC positive PrPSc amplification detected; amyloid beta 1-42 was decreased; pTau was normal; Tau, 14-3-3 protein and NSE were elevated. 14-3-3 protein and oligoclonal bands were not detected; amyloid beta 1-42, NSE, and pTau parameters were normal; Tau and neurofilament light polypeptides were elevated. – Mononuclear pleocytosis; raised spinal protein. DNA analysis No known pathogenic variants in PRNP, homozygous for V at codon 129 (129VV); silent mutation at codon 117 (A117A). P102L mutation in PRNP—mutated allele codes for M at codon 129 (P102L-129M), heterozygous at codon 129 (129MV), silent mutation at codon 117 (A117A). P102L mutation in PRNP; the information of other variants in PRNP was not available, and new analysis could not be performed. No mutations found in the genes for SCA type 1, 2, 3, 6, 7, 17; HD; and DRPLA. – . CJD Patient/Wife/Mother (III-6) . GSS Patient/Son (IV-1) . GSS Proband/Husband/Father (III-5) . GSS Proband’s Paternal Uncle (II-6) . Samples DNA from blood and plasma; half fresh frozen and half formic acid and formalin-fixed paraffin embedded brain; CSF DNA from blood sample Formalin fixed and formalin-fixed paraffin-embedded brain – Age at disease onset 76 48 55 74 Age at death 76 – 63 79 Disease duration 6 months > 4 years 8 years 5 years Disease onset symptoms Progressing imbalance, gait disturbance and confusion; cognitive decline; aphasia; double vision; hallucinations. Memory impairment; personality changes; increased aggressiveness; hypomania and lack of insight; interrupted horizontal eye pursuit movements; slow initiation of saccades; difficulty in performing Luria test, even with cueing and brisk reflexes. Insidious and progressing gait disturbance; imbalance; tremor and cramped handwriting. Gait disturbance; pain in the legs. Progressing symptoms With the initial symptoms getting worse, the patient also had delusions and became disoriented, developed severe myoclonus and mutism; eventually, the patient became rigid and deteriorated to a vegetative state. Significant global cognitive impairment with amnesia, anomia, acalculia, apraxia, and reduced concentration. Difficulties swallowing; double vision; urinary urgency; short-term memory impairment; dysarthria; upward gaze palsy and horizontal nystagmus; hypomimic facial expressions; postural tremor in the upper limbs; upper and lower limb ataxia; diminished tendon reflexes in the arms and lost in the legs; Babinski’s reflex diminished bilaterally. Lower back pain; paresis of the legs; arm and leg numbness; suicidal thoughts; swallowing difficulties; urinary incontinence; dysarthria; disorientation; upward gaze palsy and jerky eye movements with a horizontally gaze-evoked nystagmus; amyotrophic arms and legs; universal loss of tendon reflexes; extensor plantar reflexes. MRI DWI signal abnormalities in the right caudate nucleus. – – Normal. PET/MR FDG Whole-body scan was normal; brain scan revealed generally reduced metabolic activity. Parietotemporally moderately reduced (symmetrically distributed) metabolic activity with involvement of mesial parietal structures; only slight bifrontal affection. – – 11C-PIB PET – No amyloid binding in the gray matter. – – CT Slight cortical and central atrophy. – – Slight cortical and central atrophy; diffuse leukoariosis. EEG Encephalopatic; background slowing pattern and delta activity in frontal area; no CJD characteristic changes. – – – CSF RT-QuIC positive PrPSc amplification detected; amyloid beta 1-42 was decreased; pTau was normal; Tau, 14-3-3 protein and NSE were elevated. 14-3-3 protein and oligoclonal bands were not detected; amyloid beta 1-42, NSE, and pTau parameters were normal; Tau and neurofilament light polypeptides were elevated. – Mononuclear pleocytosis; raised spinal protein. DNA analysis No known pathogenic variants in PRNP, homozygous for V at codon 129 (129VV); silent mutation at codon 117 (A117A). P102L mutation in PRNP—mutated allele codes for M at codon 129 (P102L-129M), heterozygous at codon 129 (129MV), silent mutation at codon 117 (A117A). P102L mutation in PRNP; the information of other variants in PRNP was not available, and new analysis could not be performed. No mutations found in the genes for SCA type 1, 2, 3, 6, 7, 17; HD; and DRPLA. – MRI, magnetic resonance imaging; DWI, diffusion-weighted imaging; PET, positron-emission tomography; MR FDG, metabolic rate of fluorodeoxyglucose; 11C-PIB PET, positron emission tomography imaging with 11C-labelled Pittsburgh Compound-B ligand; CT, computed tomography; EEG, electroencephalography; CSF, cerebrospinal fluid; NSE, neuron-specific enolase; SCA, spinocerebellar ataxia; HD, Huntington disease; DRPLA, Dentato-Rubro-Pallido-Luysian atrophy. Open in new tab FIGURE 1. Open in new tabDownload slide The pedigree of the family. The GSS proband (III-5), indicated with an arrow, carried the P102L mutation in the PRNP causing GSS (horizontal pattern). The CJD patient (III-6, wife of the proband) is marked with a vertical pattern. The son (IV-1) shares the PRNP P102L mutation and is diagnosed with GSS (horizontal pattern). Many of the proband’s paternal family members had motor symptoms and dementia (dotted pattern) that might represent GSS cases. The CJD patient’s mother also had dementia (checked pattern). The family members whose mutation status is unknown are indicated with a question mark. FIGURE 1. Open in new tabDownload slide The pedigree of the family. The GSS proband (III-5), indicated with an arrow, carried the P102L mutation in the PRNP causing GSS (horizontal pattern). The CJD patient (III-6, wife of the proband) is marked with a vertical pattern. The son (IV-1) shares the PRNP P102L mutation and is diagnosed with GSS (horizontal pattern). Many of the proband’s paternal family members had motor symptoms and dementia (dotted pattern) that might represent GSS cases. The CJD patient’s mother also had dementia (checked pattern). The family members whose mutation status is unknown are indicated with a question mark. Informed consent was obtained from the patients’ families. The use of the samples was in accordance with the ethical standards of the Danish ethical committee (De Videnskabsetiske Komiteer Protokol nr.: H-16029969) and with the 1964 Helsinki Declaration and its later amendments. Half of the CJD patient’s brain was pretreated in formic acid, formalin-fixed, and paraffin-embedded (FFPE), and the other half was fresh frozen. For the search of chr. Y in the CJD patient’s brain, DNA was extracted from 15 different areas of the frozen half of the brain: frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate gyrus, hippocampus, amygdala, caudate nucleus, putamen, globus pallidus, thalamus, medulla, pons, cerebellum, and spinal cord. Cerebrospinal fluid (CSF) and blood samples were obtained while the patient was hospitalized and were made available for the study. The son is alive, and his blood sample was made available for the study. Half of the GSS proband’s brain was FFPE, and half of it was formic acid pretreated and formalin fixed. Immunohistochemistry FFPE brain tissues of the CJD patient and GSS proband were cut into 3-µm-thick sections and stained with hematoxylin and eosin (H&E). Immunohistochemistry included staining for glial fibrillary acid protein (1:6400, Dako, Glostrup, Denmark); microglia—CD68 (1:200, Dako); p62 (1:800, Santa Cruz Biotechnology, Dallas, TX); β-amyloid (1:200, Dako); Tau (1:400 Dako); α-synuclein (1:5500, Millipore, Burlington, MA); ubiquitin (1:400, Dako); and protease-K-resistant prion proteins binding 12F10 (1:1500, Dako) and KG9 (1:1000, DiaNova, Hamburg, Germany) antibodies. The conditions used for the 12F10 and KG9 staining are described below. 12F10 The sections were pretreated in a microwave oven with 2 mM hydrogen chloride at 121°C for 8–10 minutes; treated with protease K ([PK] Millipore) for 5 minutes; and stained with 12F10 antibody (Dako) using the Dako autostainer platform. The sections were pretreated in PT Link using a high pH/low pH target retrieval solution (Dako). The staining was conducted on the DakoLink 48 utilizing the EnVision Flex+ detection kit (Dako). KG9 Before the KG9 was applied, the sections were pretreated with PK. The KG9 staining was performed with the Ventana Benchmark Ultra utilizing the UltraView/Optiview detection kit (Roche Diagnostics, Basel, Switzerland). DNA Extraction For the fresh frozen brain tissue, 2 methods were applied for DNA extraction: 1) NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany), and 2) the phenol-chloroform DNA precipitation method, which was also used for FFPE tissues. The latter method is based on samples being deparaffinized with xylene, their subsequent homogenization with a lysis buffer (pH 8.5, 0.5% 20 mM Tris buffer, 0.5% NP-40, sodium deoxycholate) and PK; followed by DNA precipitation with phenol, water, chloroform, and glycogen; and DNA washing with 70% ethanol and RNAse-free water. DNA from the blood samples was extracted with the QIAamp DNA Blood Midi Kit following the manufacturer’s recommendations. DNA from 4 mL plasma was extracted using the QiaSymphony circulating DNA Kit according to the manufacturer’s specifications (Qiagen, Hilden, Germany). PCR and Sequencing Several short overlapping PCR amplicons covering the coding region of PRNP were designed. Allele-specific primers were designed to identify the allele carrying the P102L-mutation. For P102L detection in PRNP, the son’s DNA was extracted from a blood sample and used as a positive control. All primers were designed using NM_183079 (chr20: 4686094–4701590; UCSC Genome Browser, GRCh37/hg19 assembly) as the reference sequence. Primers were designed with the addition of T3 and T7 tails to the forward and reverse primers, respectively, which were later used for the sequencing of the amplified products. The primers used for amplification of the sex-determining region Y (SRY) have previously been described (23). All primers used in the current study are shown in Table 2. TABLE 2. Primer Sequences for PRNP and SRY Coding Regions PRNP . Forward primers with a polynucleotide tail Reverse primers with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′ATG GGA CTC TGA CGT TCT CC3′ 5′GCT TAC TCG GCT TGT TCC AC3′ 5′CAC CCA CAG TCA GTG GAA CA3′ 5′CGC GCT CCA TCA TCT TAA CG3′ 5′GCT GGG GTC AAG GAG GTG3′ 5′TCC CTC AAG CTG GAA AAA GA3′ 5′TCA CAA TCA AGC AGC ACA CG3′ 5′AAT GTA TGA TGG GCC TGC TC3′ 5′GGA CAG CCT CAT GGT GGT3′ 5′CGG TGC ATG TTT TCA CGA TA3′ 5′CTG GGG TCA AGG AGG TGG3′ 5′AAC GGT GCA TGT TTT CAC GA3′ Forward primers (no tail) Reverse primers (no tail) 5′TGA TAC CAT TGC TAT GCA CTC ATT C3′ 5′GAC ACC ACC ACT AAA AGG GCT GCA G3′ Forward primer (no tail) Reverse primers (no tail) 5′AGG TGG CAC CCA CAG TCA GT3′ 5′CAC TAA AAG GGC TGC AGG TG3′ 5′CGA TAG TAA CGG TCC TCA TA3′ Forward primer for mutated allele analysis Reverse primer for mutated allele analysis 5′CCA CAG TCA GTG GAA CAA GAC3′ 5′TCA TGG CAC TTC CCA GCT T3′ SRY Forward primer with a polynucleotide tail Reverse primer with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′GAA TAT TCC CGC T CT CCG G3′ 5′ACA ACC TGT TGT CCA GTT GC3′ PRNP . Forward primers with a polynucleotide tail Reverse primers with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′ATG GGA CTC TGA CGT TCT CC3′ 5′GCT TAC TCG GCT TGT TCC AC3′ 5′CAC CCA CAG TCA GTG GAA CA3′ 5′CGC GCT CCA TCA TCT TAA CG3′ 5′GCT GGG GTC AAG GAG GTG3′ 5′TCC CTC AAG CTG GAA AAA GA3′ 5′TCA CAA TCA AGC AGC ACA CG3′ 5′AAT GTA TGA TGG GCC TGC TC3′ 5′GGA CAG CCT CAT GGT GGT3′ 5′CGG TGC ATG TTT TCA CGA TA3′ 5′CTG GGG TCA AGG AGG TGG3′ 5′AAC GGT GCA TGT TTT CAC GA3′ Forward primers (no tail) Reverse primers (no tail) 5′TGA TAC CAT TGC TAT GCA CTC ATT C3′ 5′GAC ACC ACC ACT AAA AGG GCT GCA G3′ Forward primer (no tail) Reverse primers (no tail) 5′AGG TGG CAC CCA CAG TCA GT3′ 5′CAC TAA AAG GGC TGC AGG TG3′ 5′CGA TAG TAA CGG TCC TCA TA3′ Forward primer for mutated allele analysis Reverse primer for mutated allele analysis 5′CCA CAG TCA GTG GAA CAA GAC3′ 5′TCA TGG CAC TTC CCA GCT T3′ SRY Forward primer with a polynucleotide tail Reverse primer with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′GAA TAT TCC CGC T CT CCG G3′ 5′ACA ACC TGT TGT CCA GTT GC3′ Open in new tab TABLE 2. Primer Sequences for PRNP and SRY Coding Regions PRNP . Forward primers with a polynucleotide tail Reverse primers with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′ATG GGA CTC TGA CGT TCT CC3′ 5′GCT TAC TCG GCT TGT TCC AC3′ 5′CAC CCA CAG TCA GTG GAA CA3′ 5′CGC GCT CCA TCA TCT TAA CG3′ 5′GCT GGG GTC AAG GAG GTG3′ 5′TCC CTC AAG CTG GAA AAA GA3′ 5′TCA CAA TCA AGC AGC ACA CG3′ 5′AAT GTA TGA TGG GCC TGC TC3′ 5′GGA CAG CCT CAT GGT GGT3′ 5′CGG TGC ATG TTT TCA CGA TA3′ 5′CTG GGG TCA AGG AGG TGG3′ 5′AAC GGT GCA TGT TTT CAC GA3′ Forward primers (no tail) Reverse primers (no tail) 5′TGA TAC CAT TGC TAT GCA CTC ATT C3′ 5′GAC ACC ACC ACT AAA AGG GCT GCA G3′ Forward primer (no tail) Reverse primers (no tail) 5′AGG TGG CAC CCA CAG TCA GT3′ 5′CAC TAA AAG GGC TGC AGG TG3′ 5′CGA TAG TAA CGG TCC TCA TA3′ Forward primer for mutated allele analysis Reverse primer for mutated allele analysis 5′CCA CAG TCA GTG GAA CAA GAC3′ 5′TCA TGG CAC TTC CCA GCT T3′ SRY Forward primer with a polynucleotide tail Reverse primer with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′GAA TAT TCC CGC T CT CCG G3′ 5′ACA ACC TGT TGT CCA GTT GC3′ PRNP . Forward primers with a polynucleotide tail Reverse primers with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′ATG GGA CTC TGA CGT TCT CC3′ 5′GCT TAC TCG GCT TGT TCC AC3′ 5′CAC CCA CAG TCA GTG GAA CA3′ 5′CGC GCT CCA TCA TCT TAA CG3′ 5′GCT GGG GTC AAG GAG GTG3′ 5′TCC CTC AAG CTG GAA AAA GA3′ 5′TCA CAA TCA AGC AGC ACA CG3′ 5′AAT GTA TGA TGG GCC TGC TC3′ 5′GGA CAG CCT CAT GGT GGT3′ 5′CGG TGC ATG TTT TCA CGA TA3′ 5′CTG GGG TCA AGG AGG TGG3′ 5′AAC GGT GCA TGT TTT CAC GA3′ Forward primers (no tail) Reverse primers (no tail) 5′TGA TAC CAT TGC TAT GCA CTC ATT C3′ 5′GAC ACC ACC ACT AAA AGG GCT GCA G3′ Forward primer (no tail) Reverse primers (no tail) 5′AGG TGG CAC CCA CAG TCA GT3′ 5′CAC TAA AAG GGC TGC AGG TG3′ 5′CGA TAG TAA CGG TCC TCA TA3′ Forward primer for mutated allele analysis Reverse primer for mutated allele analysis 5′CCA CAG TCA GTG GAA CAA GAC3′ 5′TCA TGG CAC TTC CCA GCT T3′ SRY Forward primer with a polynucleotide tail Reverse primer with a polynucleotide tail 5′AAT TAA CCC TCA CTA AAG GG3′ 5′TAA TAC GAC TCA CTA TAG GG3′ 5′GAA TAT TCC CGC T CT CCG G3′ 5′ACA ACC TGT TGT CCA GTT GC3′ Open in new tab For the PCR reactions, DNA samples were diluted to 10 ng/µL and 100 ng/µL. To validate the SRY detection assay, male and female DNA extracted from the blood samples from patients without neurological pathologies and male and female DNA extracted from fresh frozen CJD brain tissues were included as positive and negative controls, respectively. PCR reactions for the PRNP and SRY coding regions amplification were performed using the following parameters: 94°C for 5 minutes, then the following cycle at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes repeated 39 times followed by 72°C for 5 minutes, then stored at 4°C. PCR products were visualized using QIAxcel Advanced equipment together with QIAxcel ScreenGel software, and the PCR products were isolated using the QIAquick PCR purification kit (Qiagen). For sequencing using the T3 and T7 primers, 25 ng of the purified PCR products was used for BigDye incorporation using the BigDye Terminator 3.1 Cycle Sequencing Kit and was sequenced on an Applied Biosystems ABI3500 Dx Genetic Analyzer in accordance with the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). Restriction endonuclease NsP1 analysis was performed to verify polymorphism at codon 129 (methionine/valine) and validate the quality of the DNA. PCR reaction was performed, and PCR products were visualized on 1% agarose gel electrophoresis run with ethidium bromide, as described by Dyrbye et al (24). Sequencing libraries were prepared, and shallow sequencing on the Ion Proton platform (Thermo Fisher Scientific) was performed to quantify the amount of high-quality mapping reads (MAPQ ≥ 80) aligning to chr.Y, as described by Johansen et al (25). Real-Time Quaking-Induced Conversion (RT-QuIC) The CJD patient’s CSF sample was delivered to the National Creutzfeldt-Jakob Disease Research and Surveillance Unit in Edinburgh, Scotland for a RT-QuIC analysis, which was previously described by McGuire et al (26). Western Blot Fresh frozen brain tissues were manually homogenized in 2 mL Eppendorf tubes using pestles. The lysis buffer volume and tissue weight ratio was 9:1. One-fortieth volume of 2.0 mg/mL PK (Millipore) was added to supernatants and incubated at 37°C for an hour. PK activity was stopped by adding 1/100 volume of Pefabloc SC (Roche). To ensure the detection of variably protease-sensitive types of CJD, 50 µL of supernatants were centrifuged for 1 hour, and the pellets were resolved in a 15 µL lysis buffer. All supernatant aliquots containing the LDS sample buffer and reducing agent were resolved on a 10% Bis-Tris gel (Thermo Fisher Scientific) for 40 minutes at 200 V. Then, the proteins from the gel were transferred to the PVDF membrane (Bio-Rad, Hercules, CA) and immunoblotted. The monoclonal antibody 3F4 (1:10 000, Millipore) was used as the primary antibody, and Amersham ECL antimouse HRP-conjugated antibody (1:50 000, GE Healthcare, Little Chalfont, United Kingdom) was used as the secondary one. The blots were developed using the Amersham ECL Prime substrate (GE Healthcare) and visualized with VersaDoc, 3000 (Bio-Rad). All procedures were performed in a biosafety level-3 laboratory. For Western blot analysis, fresh frozen CJD type 1, type 2A (Parchi’s classification [27, 28]), and variably protease-sensitive prionopathy (vPSPR) samples were used as controls. The vPSPR control tissue was provided by MRC Edinburgh Brain & Tissue Bank, Scotland. RESULTS Clinical History and Findings The CJD patient’s medical records did not state invasive cranial or eye surgeries or treatments, blood transfusions, or growth hormone and gonadotropin treatments. Therefore, the diagnosis of iCJD gained via known PrPSc transmission routes could not be supported. Although the CJD patient did not have a special diet and was consuming a variety of meat and dairy products, the Western blot results do not support the diagnosis of variant CJD, which is caused by consumption of bovine meat infected with PrPSc that normally is type 2B. Clinical findings in the CJD and GSS patients were not characteristically distinctive. The main differences were observed in the patients’ age at disease onset and duration and in the CSF findings. The CJD patient’s disease duration was 6 months, while all the GSS patients in the family had a disease duration longer than 4 years. The CJD patient’s CSF showed decreased β-amyloid 1–42, elevated Tau, 14-3-3 protein, and NSE, and positive PrPSc amplification; whereas the son’s CSF showed elevated Tau and neurofilament light polypeptide. The son’s CSF sample was not used for RT-QuIC analysis. Neuropathology Macroscopically, the brain from the GSS proband (III-5) appeared to be normal. Microscopically, spongiform changes were seen throughout the gray matter (Figs. 2a, c, 3e). The changes were the most noticeable in the neocortex, where the cortical layering was often lost (Fig. 2a, e). In the striatum, thalamus, and cerebellum, spongiform changes were moderate and differed from area to area. Neuronal loss was severe, and astrogliosis was prominent in the cortex, basal ganglia, and cerebellum. Microgliosis was variable, minimal in the cerebral cortex, and more pronounced in the basal ganglia and cerebellum. In the cerebellum, distinct uni and multicentric plaques were found throughout the molecular layer and in the granular layer (Fig. 3a), and plaques were also seen in the H&E stain in the cortex and striatum (Figs. 2c, 3e). 12F10 and KG9 immunostaining demonstrated strong reactions for PrPSc in both plaques and coarse granular deposits (Figs. 2e, 3c, g). The pontine nuclei, the inferior olivary nuclei, and the spinal tracts were all unaffected. Throughout the cerebrum and cerebellum, the white matter seemed rarefied although not obviously damaged. In the brain stem, pons, and medulla, the myelin looked quite normal. Few, but very distinct, tau-positive tangles were seen in the hippocampal gyrus. Dots and rod-like ubiquitin-positive structures were found throughout the cerebrum and cerebellum. FIGURE 2. Open in new tabDownload slide Histopathological findings in the GSS and CJD patients’ neocortices. (A). Neocortex in the GSS patient with severe spongiosis and atrophy. H&E; scale bar: 200 µm. (B) Neocortex in the CJD patient with sparse spongiosis and atrophy. H&E; scale bar: 200 µm. (C) Neocortex with multicentric prion plaques in the GSS patient; H&E; scale bar: 20 µm. (D) Perineuronal and synaptic prions deposition in layers 4–5 in the CJD patient; KG9; scale bar: 20 µm. (E) Multicentric prion plaques in the GSS patient; KG9; scale bar: 200 µm. (F) Linear deposition of prions in cortical layers 4–5 in the CJD patient; KG9; Scale bar: 200 µm. FIGURE 2. Open in new tabDownload slide Histopathological findings in the GSS and CJD patients’ neocortices. (A). Neocortex in the GSS patient with severe spongiosis and atrophy. H&E; scale bar: 200 µm. (B) Neocortex in the CJD patient with sparse spongiosis and atrophy. H&E; scale bar: 200 µm. (C) Neocortex with multicentric prion plaques in the GSS patient; H&E; scale bar: 20 µm. (D) Perineuronal and synaptic prions deposition in layers 4–5 in the CJD patient; KG9; scale bar: 20 µm. (E) Multicentric prion plaques in the GSS patient; KG9; scale bar: 200 µm. (F) Linear deposition of prions in cortical layers 4–5 in the CJD patient; KG9; Scale bar: 200 µm. FIGURE 3. Open in new tabDownload slide Histopathological findings in the GSS and CJD patients’ cerebellums and putamen. (A) Multicentric prion plaques (arrows) in the cerebellum of the GSS patient. H&E; scale bar: 20 µm. (B) Spongiosis and neuron loss but no plaques in the cerebellum of the CJD patient. H&E; scale bar: 20 µm. (C) Multiple prion plaques in the cerebellum of the GSS patient. 12F10; scale bar: 200 µm. (D) Diffuse synaptic prions’ staining in both the molecular and granular layers of the cerebellum in the CJD patient. 12F10; scale bar: 200 µm. (E) Prion plaques in the basal ganglia of the GSS patient. H&E; scale bar: 20 µm. (F) Spongiosis in the striatum of the CJD patient. H&E; scale bar: 20 µm. (G) Both plaques and diffuse distribution of prions in the gray matter of the striatum in the GSS patient. KG9; scale bar: 200 µm. (H) Diffuse synaptic prions’ staining in the gray matter of the striatum in the CJD patient. KG9; scale bar: 200 µm. FIGURE 3. Open in new tabDownload slide Histopathological findings in the GSS and CJD patients’ cerebellums and putamen. (A) Multicentric prion plaques (arrows) in the cerebellum of the GSS patient. H&E; scale bar: 20 µm. (B) Spongiosis and neuron loss but no plaques in the cerebellum of the CJD patient. H&E; scale bar: 20 µm. (C) Multiple prion plaques in the cerebellum of the GSS patient. 12F10; scale bar: 200 µm. (D) Diffuse synaptic prions’ staining in both the molecular and granular layers of the cerebellum in the CJD patient. 12F10; scale bar: 200 µm. (E) Prion plaques in the basal ganglia of the GSS patient. H&E; scale bar: 20 µm. (F) Spongiosis in the striatum of the CJD patient. H&E; scale bar: 20 µm. (G) Both plaques and diffuse distribution of prions in the gray matter of the striatum in the GSS patient. KG9; scale bar: 200 µm. (H) Diffuse synaptic prions’ staining in the gray matter of the striatum in the CJD patient. KG9; scale bar: 200 µm. Macroscopically, the brain from the CJD patient (III-6) showed a light degree of atrophy, especially in the cerebellum. Microscopically, spongiform changes, neuronal loss, astrogliosis, and microgliosis were seen throughout the gray matter. Prion depositions were found in the immunostains, and microgliosis often followed the same pattern; the changes were the most severe in the limbic structures, striatum, cerebellum, and brainstem nuclei (Fig. 3b, f). The changes were only focally present in the neocortex (Fig. 2b). Immunostaining demonstrated strong reactions for both 12F10 and KG9 in a synaptic and perineuronal pattern (Figs. 2d, 3d, h). In the neocortex, the immunostaining was patchy, but when present, the deposition of PrPSc often had a linear pattern in layers 4 and 5 (Fig. 2f). Supplementary staining for ubiquitin, p62, α-synuclein, β-amyloid, and Tau revealed few Tau-positive tangles located only in the transentorhinal cortex. β-Amyloid plaques were seen throughout the neocortex and also in the striatum but not in mesencephalon or cerebellum. CERAD scoring of neuritic plaques was not performed. In conclusion, the level of Alzheimer-related neuropathological changes, according to the ABC classification system, was low (ABC score: A3, B1, Cx) (29). Furthermore, neither Lewy body nor vascular neuropathological changes were found. Molecular Genetics The PCR that was performed with the specific primers needed for SRY detection in the DNA from the CJD patient’s brain tissues from 15 different regions did not yield positive results. The shallow sequencing of the CJD patient’s plasma indicated 137 high-quality chr.Y reads in 25 302 122 raw reads. However, normally, pregnant women carrying male fetuses present with an average of 938 high-quality chr.Y reads (30). The sequencing of the PRNP coding region revealed that the CJD patient (III-6) was homozygous for valine (V) at codon position 129 (c.385G, p.M129V) and had a nonpathogenic silent mutation at codon position 117, which codes for the amino acid alanine (c.351A>G, p.A117A) and is linked to allele coding for V at codon position 129 (Fig. 4a) (24). V homozygosity at codon 129 was also confirmed by restriction endonuclease NsP1 analysis. FIGURE 4. Open in new tabDownload slide Polymorphic sites of the PRNP coding region of the CJD patient and her son. (A) The CJD patient (from left to right): Normal codon 102 sequence coding for proline; a silent A117A mutation; and codon 129 homozygosity for valine. (B) The son (from left to right): Mutation at codon 102, causing the production of leucine instead of proline; a silent A117A mutation; and codon 129 heterozygosity for methionine and valine. The mutations and polymorphism sites are indicated with the red arrow in the nucleotides' sequence and the corresponding double-peaked wave. FIGURE 4. Open in new tabDownload slide Polymorphic sites of the PRNP coding region of the CJD patient and her son. (A) The CJD patient (from left to right): Normal codon 102 sequence coding for proline; a silent A117A mutation; and codon 129 homozygosity for valine. (B) The son (from left to right): Mutation at codon 102, causing the production of leucine instead of proline; a silent A117A mutation; and codon 129 heterozygosity for methionine and valine. The mutations and polymorphism sites are indicated with the red arrow in the nucleotides' sequence and the corresponding double-peaked wave. The son (IV-1) is heterozygous for methionine and valine (MV) at codon 129 (c.385A>G, p.M129V). He has a pathogenic missense mutation replacing the amino acid proline with leucine at codon 102 (c.305C>T, p.P102L). The missense mutation is located on the allele, which encodes M at codon position 129 (P102L-129M) because the allele coding for V was mutation-free. He also carries a silent mutation at codon 117 (c.351A>G, p.A117A; Fig. 4b). The P102L mutation was also identified in the GSS proband (III-5), but there was no information about this patient’s genotype at codons 117 and 129. Therefore, we sought to extract the GSS proband’s DNA from FFPE and formalin fixed brain tissues and then amplify and sequence it. However, extraction of a satisfactory quality DNA was not possible because of a long tissue fixation period (since 1999). Western Blot Western blot analysis of the samples from the CJD patient’s frontal cortex and cerebellum revealed the presence of PrPSc type 2A according to Parchi’s classification system (Fig. 5). FIGURE 5. Open in new tabDownload slide Western blotting revealed that the CJD patient had PrPSc type 2A. (Lane 1) marker; (lane 2) 15 µL of the patient’s frontal cortex (FC) homogenate; (lane 3) type 1A CJD control; (lane 4) type 2A CJD control; (lane 5) 0.5 µL of the patient’s cerebellum (CB) homogenate; (lane 6) concentrated patient’s frontal cortex (FC) homogenate; (lane 7) concentrated variably protease-sensitive prionopathy (vPSPr) control; (lane 8) concentrated patient’s cerebellum (CB) homogenate. FIGURE 5. Open in new tabDownload slide Western blotting revealed that the CJD patient had PrPSc type 2A. (Lane 1) marker; (lane 2) 15 µL of the patient’s frontal cortex (FC) homogenate; (lane 3) type 1A CJD control; (lane 4) type 2A CJD control; (lane 5) 0.5 µL of the patient’s cerebellum (CB) homogenate; (lane 6) concentrated patient’s frontal cortex (FC) homogenate; (lane 7) concentrated variably protease-sensitive prionopathy (vPSPr) control; (lane 8) concentrated patient’s cerebellum (CB) homogenate. DISCUSSION This is the first report of presumed sporadic CJD occurring in a person who married into a GSS family. The estimated prevalence of GSS is in the range of 2–5 per 100 million people worldwide, and the annual mortality rate for sCJD in Denmark is 1.46 per 1 million people (31). The population of Denmark consists of 5 740 185 individuals, and there are 2 registered GSS cases that belong to the same family. The Danish GSS family is only the thirty-fourth known GSS family in the world (32). One could assume that the risk for a Danish man with GSS to have a wife or a mother who would develop CJD in her seventies is as high as for any other man. On the basis of the mortality rate for sCJD, and assuming that the incidence of sCJD is the same among married and unmarried people, we could state that 1 man out of 684 932 men has a risk of marrying a woman who would develop CJD. However, in this case, the man a priori had GSS, which means that it would take 1 man out of 684 932 men with GSS for such a pairing to occur. Considering the worldwide rarity of GSS cases, the likelihood for co-occurrence of GSS and sCJD in one family is hence very low and warrants an investigation for the possible transmission of prions routes. Four cases of a random co-occurrence of prion diseases in the same family were reported before (12–15). In 3 of those reports, evaluation of the likelihood of prion diseases co-occurrence by chance implies a considerably low probability. For example, the occurrence of sCJD in a husband and wife has been reported in the United States in 1998. In the 15-year surveillance period, the evaluation of the statistical likelihood for a couple in the United States to develop sporadic CJD within 5 years from each other was presented as a 2.1% risk, given the size of the population and its assumed structure at the time (13). In a case study from Switzerland, the estimated probability for a sibling of an iCJD patient to develop any kind of CJD was 1.4 × 10−3, and the probability that one patient with sporadic fatal insomnia occurs by chance in a fatal familial insomnia family in Italy ranged from 0.78–1.56 × 10−4 (14, 15). In the current study, a thorough neuropathological investigation of the CJD patient’s and GSS proband’s brain was performed and revealed a classic sCJD type V2 and GSS, respectively (33). In the CJD patient, the main histopathological findings were a characteristic diffuse synaptic and perineuronal PrPSc distribution throughout the brain, spongiosis, and neuronal loss, whereas in the GSS proband, the major finding was the presence of uni and multicentric PrPSc plaques, especially in the cerebellum. To ensure that the CJD patient was not exposed to known routes of PrPSc transmission and did not carry pathogenic mutations, her clinical history was studied, the PrPSc type was defined, and the coding region of her PRNP was sequenced. Neither the clinical history nor the biochemical findings of the CJD patient’s tissues provided evidence of iCJD or variant CJD. No established pathogenic mutations were found in the CJD patient’s PRNP coding region. However, she had a silent A117A mutation, which is present in only 5%–10% of the population in Western countries and is considered nonpathogenic because the evidence for its impact on prion diseases pathogenesis is lacking (34). Yet alanine-to-valine replacement at the same codon position (p.A117V) is known to cause GSS (35, 36). Whether or not the CJD patient’s possession of a silent A117A mutation in PRNP could have favored the CJD susceptibility in the given GSS family case remains to be elucidated, but if its presence was a cofactor, this could explain the rarity of CJD cases in GSS families. Given the unusual circumstances, we hypothesized that the cells of the fetus, which carried the P102L mutation in PRNP and PrPSc, might be transmitted to the mother’s brain via acquired microchimerism, thus triggering the conversion of maternal PrPC into PrPSc. The microchimerism would have marked the start of the CJD incubation period. Although more than a 50-year incubation period seems lengthy for sCJD, there were cases of iCJD and Kuru incubation lasting up to 40 and 56 years, respectively, underlining the possible variability of the incubation period of prion diseases (37, 38). During pregnancy, both the fetus’ and mother’s blood-brain barriers exhibit increased permeability, allowing the exchange of DNA and cells (16). Microchimerism in female brains has been reported in 63% of all women who carried a male fetus, and it was detectable when measured 2 decades after the pregnancy (16). We know that the CJD patient who married into the Danish GSS family was pregnant with the male fetus with GSS 52 years before her death. Therefore, her DNA samples from 15 different brain regions and blood plasma was extracted to search for the male sex-determining chr.Y. However, no male SRY was detected in the maternal brain. Besides, the number of high-quality chr.Y reads in the maternal plasma was too small to conclusively prove the presence of genuine male fetus’ cells (30, 39). Different factors may have influenced the negative results: 1) the fetus cells might have vanished from the mother’s body because of the 52-year period since the putative microchimerism; 2) the sensitivity of the chr.Y detection methods might not have been high enough for the presumed low level of the target Y-sequence; and 3) although the samples were taken from 15 brain regions, it could be possible that the fetal cells were missed (16, 20). Even though no fetal cells were detected, according to the hypothesis, it is likely that PrPSc from the fetal cells acted as seeds and started the process of maternal PrPC misfolding and, once triggered, this process could go on for many years, even after the microchimeric fetal cells were lost again. It is known that CJD susceptibility and phenotypes can differ greatly because of a person’s polymorphism at codon 129 in PRNP (40–42). In this case, the CJD patient was homozygous for V at codon 129 and had PrPSc type 2, which is the second most common sporadic CJD type, suggesting that V homozygosity may have favored PrPC misfolding and CJD pathogenesis (35, 43). The PrPSc biochemical and histological picture of the V homozygote CJD patient is very different from that of GSS although, hypothetically, the initial PrPSc seed was from the P102L-129M GSS fetus. This could be explained by the host PrPSc strain mutation and/or a strain selection phenomenon, which also might have contributed to the lengthy CJD incubation period. Strain mutation means that a distinct PrPSc strain can be propagated on the passage to a new host, even in the same species, if the PrPC primary sequence differs from that of the host. In humans, the propagation of certain PrPSc strains is thought to be influenced by variations in the host’s PRNP and likely in other genes that shape the microenvironment of the brain too (44, 45). For example, M at codon 129 is associated with the production of type 1 PrPSc and V with the production of type 2 PrPSc (46). Cases of PrPSc type 1 and type 2 co-occurring in the same patients prove that several PrPSc conformations defining the PrPSc types or strains can be found in a brain affected by prion disease (28, 35, 46). Moreover, it has been demonstrated that several rodents inoculated with the same human P102L GSS brain homogenate developed distinct pathologies and 2 different PrPSc strains corresponding to 8 and 21 kDa fragments (47, 48). Strain selection phenomenon refers to the propagation of one PrPSc strain over another that is selected by preferential “templating” against the host’s PrPC sequence and genetic background (44, 47). Studies on the transmissibility and conformation of synthetic PrPSc strains propose that when the host is introduced to a mixture of different PrPSc strains, those that are the most compatible with the host’s PrPC propagate the fastest and dominate the clinical symptoms (49). Detailed investigation of the CJD and GSS patients’ genomes and proteomes would perhaps reveal more possible explanations for CJD and GSS co-occurrence in one family. However, this would require fresh brain tissues from the GSS proband and the son. Studies of transgenic animal models with genotypes and PrPSc types corresponding to the Danish GSS and CJD family model could be the next step in resolving the possibility of PrPSc transmission via microchimerism. Moreover, knowing that the CJD patient’s husband was a PrPSc carrier too, the possibility of other new PrPSc transmission routes, such as body fluids exchange during sexual relations, should be considered. In conclusion, the current study is the first report of presumed sCJD and GSS presenting in the same family. We analyzed the clinical, neuropathological, and genetic findings of the affected family members; however, the co-occurrence of such rare diseases in the same family could not be explained by these findings. Thus, we have proposed and investigated a novel possible PrPSc transmission route in humans. We suggested that via acquired microchimerism, PrPSc could be freely transmitted from the fetus to the mother and possibly vice versa. Despite the data we have obtained, the time of sampling since the PrPSc seeding and the overall sensitivity of the genetic measurements necessarily limit the support for our novel transmission hypothesis in this extremely rare Danish case. However, the hypothesis should not be disregarded without further investigation. Exploring new possible prion infection routes and the biological mechanisms favoring them in animal models with controlled genetic and proteomic characteristics would help in elucidating the co-occurrence of CJD and GSS in one family. The family case presented in the current study, and the 4 other previously reported family cases of rare prion diseases co-occurrence foster the necessity to explore the complexity of possible routes for prions transmission, as well as the biological and environmental factors contributing to the susceptibility of prion diseases. An in-depth comprehension of these aspects of prion diseases is essential for their prevention and for the proper genetic counseling of affected families. ACKNOWLEDGMENTS The authors thank the patients and their families for the opportunity to use their samples for this research. Laboratory technician Elena Projva is acknowledged for her excellent technical assistance. No external funds were received. 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TI - Sporadic Creutzfeldt-Jakob Disease in a Woman Married Into a Gerstmann-Sträussler-Scheinker Family: An Investigation of Prions Transmission via Microchimerism
JF - Journal of Neuropathology & Experimental Neurology
DO - 10.1093/jnen/nly043
DA - 2018-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/sporadic-creutzfeldt-jakob-disease-in-a-woman-married-into-a-gerstmann-0M94IY509v
SP - 673
EP - 684
VL - 77
IS - 8
DP - DeepDyve
ER -