Ximelis et al. Alz Res Therapy (2021) 13:176 https://doi.org/10.1186/s13195-021-00912-6 RESEARCH Open Access Homozygous R136S mutation in PRNP gene causes inherited early onset prion disease Teresa Ximelis1†, Alba Marín‑Moreno2†, Juan Carlos Espinosa2, Hasier Eraña3, Jorge M. Charco3, Isabel Hernández4, Carmen Riveira5, Daniel Alcolea6, Eva González‑Roca7, Iban Aldecoa1,8, Laura Molina‑Porcel1,9, Piero Parchi10,11, Marcello Rossi11, Joaquín Castilla3,12, Raquel Ruiz‑García7,9, Ellen Gelpi1,13, Juan María Torres2* and Raquel Sánchez‑Valle1,9* Abstract Background: More than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date. Methods: We describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro ­PrPSc propagation studies were performed using recombinant- adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease. Results: The index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated ­PrPSc fragment, consistent with a Gerstmann-Sträussler- Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of P ­ rPSc studies failed to show disease transmissibility. Conclusion: In conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be “probably damaging”, heterozygous carriers are protected, at least from an early onset pro‑ viding evidence for a potentially recessive pattern of inheritance in human prion diseases. Keywords: Gene PRNP, GSS, Homozygous, Neuropathology, Prion *Correspondence: jmtorres@inia.es; rsanchez@clinic.cat Background † Teresa Ximelis and Alba Marín-Moreno contributed equally to this work. Genetic prion diseases represent ~ 15% of human prion 2 Centro de Investigación en Sanidad Animal (CISA-INIA-CSIC), 28130 diseases [1] and are caused by genetic variants in the Valdeolmos, Madrid, Spain 9 Alzheimer’s Disease and Other Cognitive Disorders Unit, Neurology PRNP gene, located on the chromosome 20. More than Service, Hospital Clínic de Barcelona, IDIBAPS, University of Barcelona, 40 rare variants have been described to cause genetic Villarroel, 170 08036 Barcelona, Spain prion disease. Importantly, these mutations are linked Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ximelis et al. Alz Res Therapy (2021) 13:176 Page 2 of 13 to the polymorphism at codon 129, coding either for obtained from medical records or was provided by the methionine or valine, which frequently determines the family members themselves. phenotypic presentation. Genetic prion diseases usu- ally present family history of disease. However, in some Genetic studies genetic cases, the familial tree [2] could not identify other PRNP exon 2 was amplified by PCR of genomic DNA cases in the family or suggests incomplete penetrance, extracted from whole peripheral blood as previously which has been attributed to underdiagnosis or low described [15] at Hospital Clínic de Barcelona. We used clinical expression during the lifespan [3, 4]. Mutations two pathogenic prediction softwares: SIFT [16] and Poly- induce misfolding of the cellular prion protein (­PrPC), morphism Phenotyping v2 (Polyphen-2 (score 1.0)) [17] and the accumulation of the disease associated isoform to predict the potential pathogenic effect of the observed ­PrPSc [5, 6]. According to the clinic-neuropathological variant. Copy number variation analysis was performed features, inherited prion diseases can be classified as using Affymetrix SNP6.0 arrays. genetic Creutzfeldt-Jakob disease, Gerstmann-Sträussler- Scheinker (GSS) syndrome, Fatal Familial Insomnia, Neuropathology and immunoblotting and Prion diseases associated to octapeptide insertions Neuropathologic examination was performed according although there are also some variants presenting an to standardized protocols at Neurological Tissue Bank intermediate clinical phenotype or not fitting into these of Hospital Clínic-IDIBAPS in Barcelona, Spain. The categories [7]. The clinical diagnosis of genetic prion dis- right brain hemisphere was dissected in coronal sections orders might be challenging, especially in patients with and frozen while the left brain hemisphere was fixed by non-prototypical phenotypes. Analysis of the human immersion in 4% formalin for 3 weeks and after fixation PRNP gene is recommended whenever such as disease and cutting were treated with 98% formic acid for 1 h. form is suspected [8, 9]. Upon several water washes, brain material covering at Up to date, all described pathogenic variants cause the least 30 different brain areas was postfixed in 10% forma- disease in heterozygous carriers, being the disease inher- lin for at least 48 h and embedded in paraffin. For his- ited with an autosomal dominant pattern. Very few indi- tologic evaluation, 5-μm-thick sections were stained with viduals have been reported to be homozygous for PRNP hematoxylin and eosin and periodic acid-Schiff (PAS). variants [10–13]. Most of them are homozygous carriers Immunohistochemistry (IHC) was performed using the of well-known pathogenic mutations which cause also Autostainer System method (Dako) with various antibod- the disease in heterozygous carriers from areas of high ies including anti-PrP (12F10; Bertin-bioreagent, France), prevalence of the mutation. In 2006, Pacheco et al. [14] anti-βA4 (6F/3D, Dako, Glostrup, Denmark), anti-tau reported at the Prion 2006 meeting a Portuguese patient (AT8; Thermo Scientific, USA), anti-α-synuclein (5G4; carrying the homozygous R136S variant with family his- Analytik Jena, Germany), anti-ubiquitin (P4D1; Cell sig- tory of disease in two siblings, but not additional infor- nalling, USA), anti-α-internexin (2E3, Invitrogen, USA), mation is available in the literature. and anti-TDP-43 (2E2-D3, Abnova, Taiwan). Disease In this report, we describe the clinical, neuropathologi- evaluation was performed according to international cal, and biochemical phenotype of genetic prion disease consensus criteria [18]. associated with a biallelic R136S substitution in the PRNP Samples of frontal, temporal, parietal and occipital gene in a Spanish family. We also present the results of cortices, hypothalamus, thalamus, and cerebellum were in vivo and in vitro transmissibility and propagation stud- processed for Western blotting. Human brain tissue was ies of this rare variant. homogenized in 10% (w/v) in lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxy- cholate, 100 mM Tris) at pH 6.9. To detect brain P ­ rPres Materials and methods brain homogenate were subjected to digestion with 2 U/ Clinical data ml of proteinase K (PK) (Roche Diagnostics) at 37 °C for The index case and two asymptomatic siblings were 1 h. After blocking PK activity with phenylmethylsulfo- studied at the Alzheimer’s disease and other cognitive nyl fluoride (PMSF, final concentration 3.6 mM), sam- disorders unit at Hospital Clínic de Barcelona, Barce- ples were boiled in sample buffer (final concentration: 3% lona, Spain. Clinical information also included data from SDS, 4% β-mercaptoethanol, 10% glycerol, 2 mM EDTA, other centers specialized in cognitive disorders where the 62.5 mM Tris) for 6 min at 100 °C. Digested samples were patient was evaluated and followed after diagnosis. The loaded into 12% Tris-Glycine gel (Criterion, Bio-Rad), youngest sister and also an affected patient were exam- after electrophoretic transference of proteins onto poly- ined at Hospital of Leon, Leon, Spain. The clinical infor- vinydene fluoride membranes (Bio-Rad) and after block- mation associated with other members of the family was ing the membrane 1 h at 37 °C. After electrophoretic Ximelis et al. Alz Res Therapy (2021) 13:176 Page 3 of 13 transference of proteins onto polyvinylidene fluoride expressing the Val129-PrPC variant (TgVal129) [22]. membranes (Bio-Rad) membranes were incubated with These transgenic mouse lines express 4-fold and 8-fold 3F4 (1:30.000; Millipore). 3F4 recognizes the 109-112 the level of ­ PrPC expression in human brain respec- epitope of the human-PrPC sequence. Immunocomplexes tively. Six to nine individually identified mice 6–10 weeks were detected by 1 h membrane incubation with horse- old were anesthetized and inoculated with 2 mg of 10% radish peroxidase conjugated antimouse IgG (GE Health- brain homogenate in the right parietal lobe by using a care Amersham Biosciences) and development with 25-gauge disposable hypodermic needle. After inocula- enhanced chemiluminescence in ECL Select (GE Health- tion, mice were observed daily and their neurologic sta- care Amersham Biosciences). For P ­ rPSc deglycosylation, tus was assessed weekly. At the established experimental N-Linked glycans were removed by using a peptide-N- endpoint (700 days postinoculation), animals were eutha- glycosidase (PNGaseF+) F kit (New England Biolabs) nized. After necropsy, part of the brain was fixed by using according to the manufacturer’s instructions. immersion in neutral-buffered 10% formalin (4% 2-for- maldehyde) and used it for histopathology analysis while In vitro propagation of PrP by PMCA the rest of the tissue was frozen at − 20 °C and used for ­ rPSc propagation studies were performed at the In vitro P proteinase K–resistant ­PrPSc ­(PrPres) detection by WB. Prion Research Lab at CICbioGune, in Bizkaia, Spain. For each inoculation experiment, mean survival times Two different types of experiments were performed using with standard deviation was calculated as well as the recombinant-adapted protein misfolding cyclic ampli- attack rate defined as the proportion of mice that scored fication (recPMCA) technique as previously described positive for ­PrPres divided by the number of total inocu- [19]. In the experiment focused on the in vitro propaga- lated mice. tion of human rec-PrPSc using R136 vs. S136 human rec- PrP (23-231) as substrates, samples were complemented Western blotting in mouse bioassay with chicken brain homogenate and seeded with different Western blotting analysis was done as previously dilutions of recPMCA-adapted CJD MM1 misfolded rec- described [23]. Briefly, 175 + 20 mg of frozen mouse PrPSc (seed obtained through serial passages in PMCA of brain tissue were homogenized in 5% glucose distilled an sCJD MM1 isolate on a substrate containing recombi- water in grinding tubes (Bio-Rad) and adjusted to 10% nant human M129 PrP), from ­10−1 to ­10−8 and subjected (wt/vol) by using a TeSeE Precess 48TM homogenizer to a unique 24-h round of standard recPMCA. Specifi- (Bio-Rad). To detect brain ­PrPres 100 μL of 10% (wt/vol) cally, the propagation capacity of this seed was evaluated brain homogenate were subjected to digestion with 40 in three different substrates human R136 rec-PrP, human μg/mL of PK in buffer 5% sarkosyl, 5% Triton X100, 1 M S136 rec-PrP, and a mix (1:1) of both proteins, all of them Urea, and 16 mM Tris–HCl (pH 9.6) at 60 °C for 15 min. coupled to M129 polymorphism. The second experiment, Digested samples were loaded samples into 12% Bis-Tris focused on the evaluation of the spontaneous propensity Gel (Criterion XT; Bio-Rad). After electrophoretic trans- to misfold of the R136S variant, was performed twice ference of proteins onto polyvinylidene fluoride mem- using four replicates of four and compared with other branes (Millipore) and blocking overnight with 2% bovine human mutated rec-PrP (GSS-associated variants P105L serum albumin blocking buffer, membranes were incu- and A117V) and wt rec-PrP. Samples were subjected to bated with 12B2 [24] and Sha31 [25] mAb at a concen- 15 serial rounds of recPMCA in the absence of seed and tration of 1 μg/mL. 12B2 recognizes the 89-WGQGG-93 with dilution 1:10 in each round [20]. Amplified samples epitope of the human-PrPC sequence while Sha31 recog- were digested with 50–80 μg/ml of PK and analyzed by nizes the 145-WEDRYYRE-152 epitope of the human- Western blot using monoclonal antibody D18 (1:5000). PrPC sequence. Immunocomplexes were detected by 1 h membrane incubation with horseradish peroxidase Transmission studies in human PrP transgenic mice conjugated antimouse IgG (GE Healthcare Amersham Inoculation of transgenic mice was performed at Centro Biosciences) and development with enhanced chemi- de Investigación en Sanidad Animal (CISA-INIA-CSIC), luminescence in ECL Select (GE Healthcare Amersham Madrid, Spain. Inocula were prepared from fresh frozen Biosciences). Images were captured using the ChemiDoc cerebellar tissue from the proband and from one inde- WRS+ System and processed them by using Image Lab pendent case from the Portuguese family [14] carrying 5.2.1 software (both Bio-Rad). the homozygous R136S variant, as 10% (wt/vol) homoge- nates in 5% glucose distilled water. Two different mouse Histological analysis in mouse bioassay models for human-PrP were inoculated: HuPrP-Tg340- Mouse brain samples were immediately fixed in neutral- Met129 (TgMet129) mouse line expressing the Met129- buffered 10% formalin (4% 2-formaldehyde) during nec- PrPC variant [21] and HuPrP-Tg362-Val129 mouse line ropsy, coronally trimmed, and processed routinely. After Ximelis et al. Alz Res Therapy (2021) 13:176 Page 4 of 13 embedded in paraffin-wax, tissues were cut to 4-μm with short steps, generalized bradykinesia, hypertonia, thickness, de-waxed, and rehydrated by standard proce- and brisk reflexes. No sensory problems were observed. dures. Tissue slides were subjected to two different his- At the age of 46, she presented akinetic mutism, dif- tological methods: hematoxylin and eosin staining for fuse myoclonus, and severe generalized hypertonia. The lesion profile evaluation by standard methods [26] and patient died at the age of 48, but the postmortem neuro- IHC. Lesion profile evaluation was done by semi-quan- pathological examination was not performed. The other titative assessment of spongiform degeneration scoring four siblings were neurologically preserved (present ages vacuolization in the following brain areas: cerebral cor- from 63 to 74 years) (Fig. 1A, II.1–II.4). tex, striatum, hippocampus, thalamus, hypothalamus, The proband’s parents were born in a small village in midbrain, cerebellar cortex, and pons/medulla oblongata. the North-west of Spain and were probably consanguin- For the IHC analysis, tissue slides were subjected to anti- eous but the degree of kinship is unknown. The mother gen retrieval and quenching of hydrogen peroxide, lat- (Fig. 1A, subject I.2) died at the age of 89 years and was ter incubated with the Sha31 mAb [25] and subsequent neurologically healthy according to their family mem- steps of the IHC procedure were performed by a com- bers. The father (Fig. 1A, subject I.1) died at the age of 84 mercial immunoperoxidase technique (Vector-Elite ABC with dementia with a clinical onset at the age of 75 that kit, Vector Laboratories) as per manufacturer’s instruc- had been classified as vascular cognitive impairment by tions finally counterstaining the sections with Harri’s the treating physician. hematoxylin. Genetic studies Results The genetic study of the Spanish patient and her young- Clinical characterization and family history est sister showed the presence of a substitution of gua- The index patient was a 51-year-old female who was nine for thymine (AGG to AGT) at codon 136 of the referred to the Neurology department due to progressive PRNP in the two alleles, causing the missense mutation memory decline, language problems, and subtle changes R136S in homozygosis. This was linked to methionine in behavior (apathy and childish behavior) beginning at homozygosis at codon 129. No deletions or duplications the age of 49 years (Table 1 and Fig. 1A, subject II.5). At were detected in the 100-kb region which includes the first examination, her Minimental State Examination PRNP gene. Score was 28/30. The cognitive evaluation revealed mod- Of the four asymptomatic siblings, two underwent erate generalized cognitive impairment including mild genetic testing. One asymptomatic 74-year-old sibling verbal memory impairment and visuospatial, language, (Fig. 1A, subject II.1) carried the R136S mutation in hete- and executive disturbances. The neurological examina- rozygosis, which was also linked to homozygous methio- tion was normal except for mild dysarthria. Nerve con- nine at codon 129. Another sibling showed a normal duction studies were not performed. MRI examination PRNP sequence and was also methionine homozygous at was unremarkable (Fig. 2). Cerebrospinal fluid showed codon 129. elevated total tau levels (1262 pg/ml), Abeta42 (701 pg/ According to pathogenic prediction software, the ml) within the normal range, slightly elevated phospho- R136S variant is considered to be damaging (SIFT) [16] tau (68.5 pg/ml), and reduced p-tau/t-tau ratio (0.054). or probably damaging (Polymorphism Phenotyping v2 The 14-3-3 Western blot test was negative. The patient (Polyphen-2 (score 1.0)) [17]. deteriorated progressively over the next years with wors- ening of both cognitive and motor functions develop- Neuropathological findings and immunoblotting ing severe ataxia. At the last neurological examination, The brain of the index patient was removed after death the patient presented severe inattention, only partial at the Neurological Tissue Bank of the IDIBAPS Biobank response to verbal stimuli, mutism, and generalized in Barcelona. Unfixed brain weight was 1030 g. Gross hypertonia and hyperreflexia and clonus in lower extrem- examination showed a moderate diffuse brain atrophy ities. She died at the age of 53 after disease duration of 4 accentuated in the frontal lobes and mild nigral pallor. years. Histology revealed a severe involvement of grey mat- The youngest sibling of the index patient (Table 1 and ter that showed prominent neuronal loss, astrogliosis, Fig. 1A subject II-6) also suffered from cognitive decline, and microglial proliferation, especially in frontal cor- motor impairment, and myoclonus with onset in her tex (Fig. 3A). Despite observing a superficial laminar late 30s (38 years). Neurological examination, MRI scan, spongiosis, only mild bona fide spongiform change was and CSF 14-3-3 were normal at first examination. At detected in grey matter which was characterized by a few the age of 45, the neurological examination disclosed a small-sized vacuoles in cortical regions and basal ganglia. MMSE score of 24/30, dysarthria, apraxia, gait disorder The most striking feature was the presence of abundant Table 1 Demographic and clinical features of homozygous PRNP mutations in the literature Present study Spanish family Portuguese family [14] Beck et al [11] Komatsu et al [12] Simon et al [10] Hassan et al [27] Ximelis et al. Alz Res Therapy Gender 2 female 2 male/1 female Female Female 3 male/2 female Male Country Spain Portugal Ireland Japan Libya China Family history Yes Yes (siblings) No (grandmother with No Yes Unknown dementia in later life) PRNP mutation R136S R136S Q212P V203I E200K E200D (2021) 13:176 Polymorhism codon 129 MM MM MM MM Unknown MM Phenotype Gerstmann-Sträussler- Gerstmann-Sträussler- Unknown genetic Creutzfeldt Jakob’s genetic Creutzfeldt Jakob’s Unknown Scheinker Scheinker disease disease Cosanguinity Yes Unknown Probable Yes Unknown Probable Age at onset (years) 43.5 ± 5.5 52 (female) 36 73 50.4 ± 6.2 61 Age of death (years) 50.5 ± 2.5 49.6 ± 6.4 Unknown 75 50.8 ± 8.2 61 Disease duration 7.25 ± 2.75 years 4 years 4 years 2 years 15.8 ± 15.9 years 10 weeks Clinical symptoms Progressive memory Psychiatric symptoms, Dysarthria, ataxia, nys‑ Gait disturbance, cogni‑ Dementia (5/5), personal‑ Confusion, dysarthria, ataxia, decline (2/2), language myoclonous, Parkinsonian tagmus, and executive tive dysfunction ity change (2/5), parkin‑ myoclonus, and hallucina‑ (1/2), executive distur‑ symptoms, and progres‑ dysfunction sonism (4/5); myoclonus tions bances (2/2) sive dementia (2/5), gait ataxia (2/5), and dysarthria (3/5) 14-3-3 protein Negative Negative Unknown Positive Unknown Negative MRI brain Normal Normal Moderate cerebral Hyperintensity in basal Unknown Abnormal symmetric atrophy ganglia and right frontal, restricted bilateral diffusion parietal and occipital in the striatum and cortex lobes EEG Unknown Lentified Mildly abnormal Diffuse slowing of waves 3/5 abnormal Mild slowing only, with no periodic sharp ware complexes Neuropathology Multicentric amyloid PrP Multicentric amyloid PrP N/A N/A 1/2 did not display ­PrPres N/A plaques, prominent neu‑ plaques, mild neuritic ronal loss, astrogliosis, and component, and micro‑ microglial proliferation glial activation PrP Western blotting ~ 8 kDa band 8 kDa and 5 kDa band N/A N/A N/A N/A In vivo transmissibility Negative N/A N/A N/A N/A N/A studies In vitro propagation Negative N/A N/A N/A N/A N/A studies Page 5 of 13 Ximelis et al. Alz Res Therapy (2021) 13:176 Page 6 of 13 Fig. 1 Genetic pedigrees. A Visual representation of the R136S Spanish family history. B Visual representation of the homozygous R136S Portuguese family (inferred from Pacheco et al. [14]). Created with BioRe​nder.​com Fig. 2 Magnetic resonance imaging scan of the index patient amyloid plaques consisting of multiple densely packed PrPSc typing by Western blotting displayed the asso- cores (multicentric plaques) that were variably PAS posi- ciation of a low molecular weight (mw) fragment of ~ 8 tive and strongly immunoreactive for ­PrPSc (12F10 anti- kDa with less defined upper bands in the ~ 21–30 kDa body) after pretreating the tissue sections for removal range (Fig. 4A, B). After de-glycosylation with PNGase of physiological P­ rPC. These multicentric plaques were F, the ~ 8 kDa remained unchanged whereas the upper detected in all cortical areas — with less involvement of bands merged in a single band of ~ 21 kDa, migrating primary visual cortex, in basal ganglia, thalamic nuclei, slightly above the unglycosylated fragment of P ­ rPSc type and brainstem and in the molecular layer of cerebel- 1 detected in CJD (Fig. 4A). The relative intensity of the lar cortex. Moreover, multicentric PrP amyloid plaques upper and lower fragments varied between different induced a moderate neuritic component that was immu- brain regions. At the visual inspection, the frontal cortex noreactive for phosphorylated tau (AT8). contained larger amounts of P ­ rPSc. Ximelis et al. Alz Res Therapy (2021) 13:176 Page 7 of 13 Fig. 3 Neuropathological characterization. A1 Frontal cortex shows prominent neuronal loss and gliosis with loss of cortical structure and mild superficial spongiosis (H&E stain). A2 Anti-PrP immunohistochemistry (12F10 antibody) shows a very high density of multicentric PrP plaques covering the whole cortical thickness. A3 These large multicentric plaques are well identified in the PAS stain and the clusters of large amyloid plaques are observed by immunohistochemistry with the 12F10 anti-PrP antibody (A4). A5 Immunohistochemistry for hyperphosphorylated tau shows marked dystrophic neurites surrounding the plaques in the hippocampus (AT8 immunohistochemistry). A6 Abundant multicentric plaques are detected in the molecular layer of the cerebellum (anti-PrP immunostaining, 12F10 antibody) In vitro propagation of ­PrPSc propagation ability of the R136 (wt) vs. the mutant R136S In order to evaluate the effect of the R136S mutation rec-PrP or the mix 1:1 of both proteins (Fig. 5). in the in vitro propagation of human rec-PrPSc, human To evaluate the spontaneous propensity of the R136S 129M 136R (wt) rec-PrP, human 129M 136S rec-PrP, and recPrP to misfold compared to the wt recPrP and other a mix of both rec-PrPs in ratio 1:1 were used as substrates GSS-related mutated recPrP (P105L and A117V), a serial for recPMCA and seeded with different dilutions of recP- rec-PMCA (15 rounds) was performed in the absence MCA-adapted CJD MM1 misfolded rec-PrP (10-1–10- of ­PrPSc seed and performing 1:10 dilutions of the pre- 8), and subjected to a unique 24-h round of standard vious passage after each round (Fig. 6). All cases were recPMCA. No significant differences were found in the associated with methionine at codon 129. The wt and the Ximelis et al. Alz Res Therapy (2021) 13:176 Page 8 of 13 Fig. 4 Western blot ­PrPresR136S profile in fresh-frozen tissue of the index patient. In contrast to typical sCJDMM1 (lanes 12 and 15), PK-treated tissue homogenates from different brain regions (lanes 3–9: frontal cortex (3), temporal cortex (4), parietal cortex (5), occipital cortex (6), hippocampus (7), thalamus (8), cerebellum (9)) of the index patient show the association of a low molecular weight fragment of ~ 8 kDa with less defined upper bands in the ~ 21–30 kDa range. After de-glycosylation with PNGase F (lanes 10 and 11), the ~ 8 kDa is unchanged. In contrast, the upper bands merge in a single band of ~ 21 kDa migrating slightly above the unglycosylated fragment of PrPSc type 1 detected in sCJDMM1. Lanes 1 and 2 show a brain homogenate from a control with (1) or without (2, ­PrPC profile) PK digestion. The comparison between the ­PrPSc profiles of the index patient and a GSS patient carrying the P102L mutation, in which only the hallmark ~ 8 kDa peptide is seen, is shown in lanes 13 and 14 Fig. 5 In vitro human misfolded PrP propagation by recPMCA using R136 vs. S136 human as substrates. Human 129M 136R (wt) rec-PrP, human 129M 136S rec-PrP, and a mix of both rec-PrPs in ratio 1:1 were complemented with chicken brain homogenate, seeded with different dilutions of recPMCA-adapted CJD MM1 misfolded rec-PrP (10-1–10-8) and subjected to a unique 24 h round of standard recPMCA. Amplified samples were digested with 50 μg/ml of Proteinase-K and analyzed by Western blot using monoclonal antibody D18 (1:5000). No significant differences were found in the propagation ability of the R136 (wt) vs. the mutant R136S rec-PrP or the mix 1:1 of both proteins R136S PrP linked to the methionine polymorphic variant sacrificed at the end of their lifespan without showing any at codon 129 did not show spontaneous misfolding, in clinical sign suspicious of prion disease and remaining contrast to the other GSS-related recPrP. uninfected (Fig. 7). No ­PrPres was found in mice brains either by WB or IHC (data not shown). Vacuolization Bioassay in human‑PrP transgenic mice was no different from the one associated with the natural Brain material from the two patients (the Spanish index aging process of non-inoculated control mice of the same case and the Portuguese index case) were intracrani- transgenic lines by histology (data not shown). ally inoculated in TgMet129 and TgVal129 transgenic mice (26) to assess the transmissibility of this rare inher- Discussion ited form of prion disease (Table 2). These mouse lines We report here that the biallelic R136S PRNP mutation expressed in homozygosis the Met129-PrPC variant causes inherited human prion disease with early onset or the Val129-PrPC variant respectively. All mice were of symptoms (30s–40s). The same mutation presented Ximelis et al. Alz Res Therapy (2021) 13:176 Page 9 of 13 Fig. 6 Spontaneous generation of human misfolded recombinant proteins. Graphical representation of the emergence of spontaneous protein misfolding evaluated through Western blot analysis in SDS-PAGE for each round of recPMCA (outlined as R01–R15). Different 129M human recombinant proteins were grouped according to the substitution. Every experiment contained four tubes (intra-experimental duplicates) and was performed in duplicate as shown. The percentage of positive tubes (tubes showing a protease resistant signal after digestion with 80 mg/ml of PK) after each round of recPMCA was noted with different intensities of grey, as shown in the legend below the figure. Neither wild-type (wt) 129M not R136S 129M were able to misfold spontaneously. WT, wild type human rec-PrPSc Table 2 Transmission of Spanish and Portuguese R136S cases in presented with an autosomal recessive pattern of inherit- TgMet129 and TgVal129 [8x] mice ance, keeping the proven or obligated heterozygous fam- Inocula Mice survival (attack rate) ­STa (n/ ily members protected from developing the disease, at n0)b least, at early ages. In vitro propagation and in vivo trans- TgMet129 TgVal129 [8x] missibility studies suggest that the ­PrPSc variant resulting from the R136S mutation failed to propagate both in vivo Portuguese R136S case > 650 days (0/5) > 650 days (0/5) and in vitro in the conditions analyzed, suggesting that Spanish R136S case > 650 days (0/5) > 650 days (0/5) this variant is biologically less active than other muta- Sporadic CJD type I (Met129) 219 ± 17 (6/6)c 295 ± 21 (6/6)d tions causing genetic prion diseases. Sporadic CJD type II (Val129) 618 ± 81 (6/6)c 228 ± 7 (5/5)d Previous described pathogenic mutations cause a Mean survival time human prion disease in the heterozygous state and b Attack rate was calculated as the relation between brain ­PrPres positive animals show an autosomal dominant inheritance pattern. (= n) and total number of inoculated animals (= n0) There are scarce examples in the literature of patients c Previously published in Cassard et al. [28], Fernández-Borges et al. [29], and Marín-Moreno et al. [30] affected by prion diseases carrying homozygous genetic d Previously published in Fernández-Borges et al. [29] variants. Homozygous E200K carriers are associated with consanguinity in areas of high prevalence of this variant. Homozygous carriers of the E200K mutation in heterozygous state in other family members did not present an earlier age of onset than heterozygous carri- induce the same phenotype. Clinically, the patients pre- ers but no other relevant differences in clinical features sented with early-onset dementia developing motor dis- compared with heterozygous carriers [10]. One case of turbances at follow-up. Neuropathological examination prion disease linked to homozygous Q212P has been revealed the presence of abundant multicentric PrP amy- described, but also another case has been reported in a loid plaques with mild neuritic component, along with heterozygous carrier [11]. Komatsu et al. [12] reported severe neuronal loss, gliosis, and microglial proliferation the case of a homozygous V203I patient, in addition to with only mild spongiform change, which is consistent three previously reported heterozygous patients for this with a GSS neuropathological phenotype. In contrast to variant. However, the lack of family history of disease previous description of homozygous mutations in PRNP in all the V203I and the presence of this variant in nor- [10–13, 27, 31] (Table 1), the disease in R136S families mal controls causes that its pathogenicity is still under Ximelis et al. Alz Res Therapy (2021) 13:176 Page 10 of 13 Fig. 7 Visual representation of the survival curves in the transgenic mouse lines A ­TgMet129 and B ­TgVal129 discussion [3, 11, 31]. Recently, Hassan et al. [27] also The expression of the disease only in homozygous reported a case with CJD and a homozygous E200D subjects could be explained by a dose effect as it has mutation, similar to Q212P [11]. been suggested for an earlier age of onset in homozy- Minikel et al. [3] reported in 2016 the presence of gous E200K carriers [10]. A larger number of R136S PrP the R136S variant in heterozygosis in two alleles from molecules (or the absence of wild-type protein) in the 60.706 population control exomes but not in patients, homozygous patient’s brain may increase the chance of suggesting that this represents a very rare variant in spontaneous conversion. Even if homozygous patients the normal population even if the R136S variant is are at higher risk for the spontaneous conversion to ­PrPSc predicted to be “probably damaging” in several patho- than heterozygous patients, on the other hand, the subse- genicity prediction software. In our center, we had not quent infective process based on ­PrPSc-PrPC interaction identified this genetic variant in more than 300 Span- might be less effective as there is only mutant ­PrPSc lead- ish subjects (including both patients and controls) ing to longer disease duration. analyzed so far. In 2006, Pacheco et al. [14] described Even if we cannot rule-out a late onset of the disease, one Portuguese patient (Fig. 1B, subject II.1) with simi- the presence of known or obligated heterozygous car- lar clinical and neuropathological features of those of riers in neurological preserved family members that our patients (early-onset dementia, motor symptoms were already in their 70s, 80s, or 90s make this possibil- in her early 50s and a GSS neuropathological pheno- ity much less probable during the usual lifespan. In this type) linked to the presence of the homozygous R136S sense, we believe that both family trees (Fig. 1) provide variant. Two of her siblings presented history of simi- evidence for a recessive pattern of inheritance in human lar disease in their early 50s, but not the parents who prion diseases. should be obligated carriers. The clinical phenotype in the patients resembled that In both families (the present family and the Portuguese of insertion mutations (OPRI), which are characterized family) described heterozygous carriers were apparently by early-onset progressive dementia with late motor protected from disease. As the genetic study in the Por- dysfunction [6], whereas the neuropathological features tuguese index case’s mother (Fig. 1B, subject I.2) and were typical of GSS. The observed clinical phenotype is another family member neurologically preserved in their atypical for GSS, which more commonly present with 80s and 90s revealed the presence of heterozygous R136S motor signs followed by dementia, although it has also linked to 129MV, the authors suggested that the presence been linked to other point mutations (Y218N) [6] and of valine in the trans-allele could prevent the expression OPRI [31, 32]. Irrespectively of the clinical syndrome of the disease. In contrast, in the Spanish family, one het- and the relative regional distribution of lesions, GSS erozygous carrier, homozygous for methionine at codon is defined by the presence of multicentric PrP amyloid 129 is neurologically preserved in their 70s, suggesting plaques in the cerebral and cerebellar cortices [7, 18]. that the expression of the disease does not depend on the In contrast to other human prion diseases, the presence codon 129 of the trans-allele but in the homozygous state of spongiform change can be variable, from absent to or “dosage” of the mutant allele. severe, which could explain the lack of signal intensity Ximelis et al. Alz Res Therapy (2021) 13:176 Page 11 of 13 changes in diffusion-weighted imaging in MRI scans. In slower misfolding rate of the wild-type allele that may addition, Western blot studies typically show, as in the finally contribute to disease development at elder ages present case, PK-resistant non-glycosylated P ­ rPSc frag- cannot be ruled out. ments with a low molecular weight that varies between The in vitro propagation and spontaneous misfolding 6 and 8 kDa depending on the PRNP pathogenic vari- proneness studies, performed by recPMCA, indicate that ant. Occasionally, however, the immunoblot profile also the R136S mutation does not confer an enhanced mis- shows larger truncated P ­ rPres fragments with a molecu- folding propensity to the human recombinant PrP. No lar weight of 19–21 kDa, as observed in other sporadic or differences were detected on misfolding proneness of this genetic prion diseases [33–36]. protein compared to the wild type, neither induced or Animal modeling of human inherited prion diseases spontaneously. In fact, prion ability to cause direct cellu- is not easy. Practically all attempts to generate trans- lar damage (toxicity) has often been dissociated from the genic mouse models for human inherited prion diseases capacity of successfully transmit from an infected host using the human-PrPC sequence had been unsuccessful to an uninfected recipient (transmissibility) [42]. This, apart from the recent reported exception of an A117V added to the difficulty of modelling inherited prion dis- GSS model [37]. The rest of successful transgenic mouse ease that is further discussed below, may explain the lack models that develop spontaneously neurologic disor- of in vivo and in vitro propagation of the prion disease ders when harboring mutations that have been reported associated to R136S mutation. The synthesis of peptides in human inherited prion diseases have been made in containing the mutation and testing their effect on cul- mouse or bank vole PrP sequences or in chimeric mouse/ tured cells, apart from allowing determining differences human PrP proteins [38]. It seems that mouse and bank in the misfolding proneness of mutated proteins as the vole PrP proteins are more prone to spontaneous mis- PMCA assays carried out here, could allow evaluating folding while human PrP is apparently more resistant. potential changes in their neurotoxic properties [43]. Alternatively, interactions between mouse PrP and other than PrP mouse factors may be important for the spon- taneous generation of prions. In general terms for GSS Limitations modelling, models overexpressing mouse, mouse/human This report has some limitations. First, the diagnosis of chimeric, and cow PrP harboring the respective equiva- genetic prion disease is a hurdle task and some mem- lents to human P102L mutation spontaneously developed bers of both extended families might have been under- a prion disease along with neuropathological changes diagnosed, lowering the possibilities of describing this [38]. However, brain ­PrPres detection and transmissibil- mutation in other patients. Second, due to the lack of ity to wild-type mice was only achieved in the particular neuropathological evaluation in any of the asymptomatic case of equivalent P113L mutation in cow-PrPC sequence heterozygous carriers, we could not rule-out subclinical producing a prion agent with features resembling those prion disease at late ages, although this possibility seems of classical bovine spongiform encephalopathy [39]. less probable. When the A117V mutation was overexpressed in the mouse sequence (A116V) at 4–6x levels, transgenic mice showed signs of prion disease including neuropa- Conclusions thology markers but no ­PrPres were detected [40]. How- The biallelic R136S substitution is a rare PRNP mutation ever, A117V GSS cases were successfully transmitted that induces inherited human prion disease presenting into A117V human PrP transgenic mice Tg30 and Tg31 with early-onset dementia and motor impairment and (2x and 3x overexpression respectively) [37, 41] from neuropathological and molecular features consistent with which Tg30 developed a spontaneous disease that was GSS. Even if the R136S variant is predicted to be “prob- later transmissible to both Tg30 and Tg31 mice as well ably damaging”, heterozygous carriers are protected, as to wild-type V129 human PrP transgenic mice [37]. It at least, from an early-onset, providing evidence for a would be of interest to further investigate the effects of potentially recessive pattern of inheritance in human this homozygous mutation in an animal model, as well as prion diseases. the production of heterozygous animals to further inves- tigate to which extent the presence of the wild-type allele Abbreviations dumpers the spontaneous misfolding of the mutated one. PrPC: Cellular prion protein; GSS: Gerstmann-Sträussler-Scheinker; TgMet129: The recessive inheritance pattern displayed by this dis- HuPrP-Tg340-Met129; IHC: Immunohistochemistry; NP: Neurologically pre‑ ease in humans points to a protective effect exerted by served; OP: Obligated carrier; PAS: Periodic acid-Schiff; PNGaseF+: Peptide-N- glycosidase; PMSF: Phenylmethylsulfonyl fluoride; recPMCA: Protein misfolding the wild-type variant and the in vitro studies performed cyclic amplification; PK: Proteinase K; PrPres: Proteinase K–resistant ­PrPSc; in this work also supports this hypothesis. However, a TgVal129: Val129-PrPC variant. Ximelis et al. Alz Res Therapy (2021) 13:176 Page 12 of 13 Acknowledgements Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic We are indebted to the Neurological Tissue Bank of Hospital Clínic-IDIBAPS de Barcelona, IDIBAPS, University of Barcelona, Villarroel, 170 08036 Barcelona, Biobank for sample and data procurement. We are grateful with Dr Paula Spain. 10 Department of Experimental, Diagnostic and Specialty Medicine Pacheco, Departamento de Genética Humana, Instituto Nacional de Saúde (DIMES), University of Bologna, 40138 Bologna, Italy. 11 IRCCS, Istituto delle Doutor Ricardo Jorge, Portugal, and Dr Leonor Orge, Laboratório de Patologia, Scienze Neurologiche di Bologna, 40139 Bologna, Italy. 12 IKERBasque Basque Instituto Nacional de Investigação Agrária e Veterinária, Portugal, for providing Foundation for Science, 48009 Bilbao, Spain. 13 Division of Neuropathology the frozen samples from an affected subject from Portugal for the transmis‑ and Neurochemistry, Department of Neurology, Medical University of Vienna, sibility studies. 1090 Vienna, Austria. Authors’ contributions Received: 30 April 2021 Accepted: 3 October 2021 RSV and JMT designed the study. AMM, HE, JMC, EG, PP, and MR performed experiments. JCE, PP, JC, RRG, EG, JMT, and RSV interpreted the results. JCE, HE, JMC, IH, CR, DA, EGR, IA, LMP, PP, MR, RRG, and EG contributed to the sample collection and characterization. EG, JMT, JC, LMP, and RSV provided extensive feedback to the manuscript to improve it significantly. TX and AMM wrote the References manuscript draft. The authors read and approved the final manuscript. 1. Head MW, Ironside JW, Ghetti B, et al. Prion diseases. In: Love S, Budka H, Ironside JW, Perry A, editors. Greenfield’s neuropathology. 9th ed. Boca Funding Raton: CRC Press; 2015. p. 1016–86. This work was funded by the Instituto de Salud Carlos III (grant no. 2. Budka H, Aguzzi A, Brown P, et al. Neuropathological diagnostic criteria PI20/00448 to RSV), Spanish Ministerio de Ciencia e Innovación (grant no. for Creutzfeldt-Jakob disease (CJD) and other human spongiform PDI2019-105837RB-I00 to J.M.T. and J.C.E., and RTI2018-098515-B-I00 to J.C.), encephalopathies (prion diseases). Brain Pathol. 1995;5(4):459–66. https://​ Fundació La Marató de TV3 (grant no. 201821-31 to J.C.E.), Instituto Nacional doi.​org/​10.​1111/j.​1750-​3639.​1995.​tb006​25.x PMID: 8974629. de Investigación y Tecnología Agraria y Agroalimentaria (fellowship INIA-FPI- 3. Minikel EV, Vallabh SM, Lek M, et al. Quantifying prion disease pen‑ SGIT-2015-02 to A.M.M.) and Dr Baselga funds for CJD research. The funders etrance using large population control cohorts. Sci Transl Med. had no role in study design, data collection and analysis, decision to publish, 2016;8(322):322ra9. https://​doi.​org/​10.​1126/​scitr​anslm​ed.​aad51​69 PMID: or preparation of the manuscript. 26791950; PMCID: PMC4774245. 4. Kovács GG, Puopolo M, Ladogana A, et al. Genetic prion disease: the Availability of data and materials EUROCJD experience. Hum Genet. 2005;118(2):166–74. https://​doi.​org/​ The datasets used and/or analyzed during the current study are available from 10.​1007/​s00439-​005-​0020-1 Epub 2005 Nov 15. PMID: 16187142. the corresponding author on reasonable request. 5. Kovács GG. Genetic background of human prion diseases. Ideggyog‑ yaszati Szemle; 2007;60(11-12):438–46 Hungarian. PMID: 18198790. 6. Alzualde A, Indakoetxea B, Ferrer I, et al. A novel PRNP Y218N mutation in Declarations Gerstmann-Sträussler-Scheinker disease with neurofibrillary degenera‑ tion. J Neuropathol Exp Neurol. 2010;69(8):789–800. https://​doi.​org/​10.​ Ethics approval and consent to participate 1097/​NEN.​0b013​e3181​e85737 PMID: 20613639. The study was approved by the ethical committee of the Hospital Clínic de 7. Ritchie DL. Chapter fourteen - neuropathology of human prion diseases. Barcelona. In: Legname G, Vanni S, editors. Progress in molecular biology and trans‑ Animal experiments were conducted in accordance with the Code for lational science, vol. 150: Academic; 2017. p. 319–39. https://​doi.​org/​10.​ Methods and Welfare Considerations in Behavioral Research with Animals 1016/​bs.​pmbts.​2017.​06.​011. Epub 2017 Aug 3. PMID: 28838666. (Directive 2010/63/EU) and every effort was made to minimize animal suffer‑ 8. Brown P, Cathala F, Castaigne P, et al. Creutzfeldt-Jakob disease: clinical ing. Transmission experiments were developed at Centro de Investigación en analysis of a consecutive series of 230 neuropathologically verified cases. Sanidad Animal (CISA-INIA-CSIC, Madrid, Spain) and they were evaluated by Ann Neurol. 1986;20(5):597–602. https://​doi.​org/​10.​1002/​ana.​41020​0507 the Committee on the Ethics of Animal Experiments of the Instituto Nacional PMID: 3539001. de Investigación y Tecnología Agraria y Alimentaria and approved by the Gen‑ 9. Windl O, Dempster M, Estibeiro JP, et al. Genetic basis of Creutzfeldt- eral Directorate of the Madrid Community Government (permit nos. PROEX Jakob disease in the United Kingdom: a systematic analysis of predis‑ 291/19 and PROEX 094/18). posing mutations and allelic variation in the PRNP gene. Hum Genet. 1996;98(3):259–64. https://​doi.​org/​10.​1007/​s0043​90050​204 PMID: Consent for publication 8707291. The participants or their next-of-kin provided written informed consent for the 10. Simon ES, Kahana E, Chapman J, et al. Creutzfeldt-Jakob disease profile use of samples for diagnostic and research purposes. in patients homozygous for the PRNP E200K mutation. Ann Neurol. 2000;47(2):257–60 PMID: 10665501. Competing interests 11. Beck JA, Poulter M, Campbell TA, et al. PRNP allelic series from 19 years RSV reports personal fees from Wave pharmaceuticals and Ionis Pharma‑ of prion protein gene sequencing at the MRC prion unit. Hum Mutat. ceuticals for attending Advisory board meetings, personal fees from Roche 2010;31(7):E1551–63. https://​doi.​org/​10.​1002/​humu.​21281 PMID: diagnostics, Janssen, and Neuraxpharm for educational activities, and research 20583301. grants to her institution from Biogen and Sage Therapeutics outside the sub‑ 12. Komatsu J, Sakai K, Hamaguchi T, et al. Creutzfeldt-Jakob disease associ‑ mitted work. The other authors declare that they have no competing interests. ated with a V203I homozygous mutation in the prion protein gene. Prion. 2014;8:336–8. https://​doi.​org/​10.​4161/​19336​896.​2014.​971569 PMID: Author details 1 25495585; PMCID: PMC4601383. Neurological Tissue Bank of the Biobanc-Hospital Clinic-Institut 13. Nitsan Z, Cohen OS, Chapman J, et al. Familial Creutzfeldt-Jakob disease d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, homoasanteasantzygous to the E200K mutation: clinical characteristics Spain. 2 Centro de Investigación en Sanidad Animal (CISA-INIA-CSIC), 28130 and disease course. J Neurol. 2020;267(8):2455–8. https://​doi.​org/​10.​ Valdeolmos, Madrid, Spain. 3 Center for Cooperative Research in Biosciences 1007/​s00415-​020-​09826-z Epub 2020 May 4. PMID: 32367297. (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia 14. Pacheco P, Orge L, Head H, et al. Novel prion disease mutation R136S has Technology Park, 48160 Derio, Spain. 4 Fundació ACE, Barcelona Alzheimer an incomplete penetrance dependent upon codon 129 trans allele. Prion Treatment and Research Center, 08028 Barcelona, Spain. 5 Neurology Service, 2006. Abstract book. Neuroprion https://www.neuroprion.org/ (available Hospital de León, 24071 León, Spain. 6 Memory Unit, Hospital de la Santa Creu upon request). In: Strategies, advances and trends towards protection of i Sant Pau, 08041 Barcelona, Spain. 7 Immunology department, Biomedi‑ society. cal Diagnostic Center, Hospital Clínic de Barcelona, 08036 Barcelona, Spain. 8 15. Sanchez-Valle R, Nos C, Yagüe J, et al. Clinical and genetic features of Pathology Department, Biomedical Diagnostic Center, Hospital Clínic de human prion diseases in Catalonia. Eur J Neurol. 2004;11:649–55. https://​ Barcelona, University of Barcelona, 08036 Barcelona, Spain. 9 Alzheimer’s doi.​org/​10.​1111/j.​1468-​1331.​2004.​00967.x PMID: 15469448. Ximelis et al. Alz Res Therapy (2021) 13:176 Page 13 of 13 16. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synony‑ 31. Goldfarb LG, Brown P, Vrbovská A, et al. An insert mutation in the chromo‑ mous variants on protein function using the SIFT algorithm. Nat Protoc. some 20 amyloid precursor gene in a Gerstmann-Sträussler-Scheinker 2009;4(7):1073–81. https://​doi.​org/​10.​1038/​nprot.​2009.​86 Epub 2009 Jun family. J Neurol Sci. 1992;111(2):189–94. https://​doi.​org/​10.​1016/​0022-​ 25. PMID: 19561590. 510x(92)​90067-u PMID: 1431985. 17. Adzhubei IA, Schmidt S, Peshkin L, et al. Nat Methods. 2010;7(4):248–9. 32. Kovács GG, Trabattoni G, Hainfellner JA, et al. Mutations of the prion pro‑ https://​doi.​org/​10.​1038/​nmeth​0410-​248 PMID: 20354512; PMCID: tein gene phenotypic spectrum. J Neurol. 2002;249(11):1567–82. https://​ PMC2855889. doi.​org/​10.​1007/​s00415-​002-​0896-9 PMID: 12420099. 18. Parchi P, de Boni L, Saverioni D, et al. Consensus classification of human 33. Gambetti P, Kong Q, Zou W, Parchi P, Chen SG. Sporadic and familial CJD: prion disease histotypes allows reliable identification of molecular sub‑ classification and characterisation. Br Med Bull. 2003;66:213–39. https://​ types: an inter-rater study among surveillance centres in Europe and USA. doi.​org/​10.​1093/​bmb/​66.1.​213 PMID: 14522861. Acta Neuropathol. 2012;124(4):517–29. https://​doi.​org/​10.​1007/​s00401-​ 34. Rossi M, Baiardi S, Parchi P. Understanding prion strains: evidence from 012-​1002-8 Epub 2012 Jun 30. PMID: 22744790; PMCID: PMC3725314. studies of the disease forms affecting humans. Viruses. 2019;11(4):309. 19. Elezgarai SR, Fernández-Borges N, Eraña H, et al. Generation of a new https://​doi.​org/​10.​3390/​v1104​0309 PMID: 30934971; PMCID: infectious recombinant prion: a model to understand Gerstmann- PMC6520670. Sträussler-Scheinker syndrome. Sci Rep. 2017;7(1):9584. https://​doi.​org/​ 35. Pirisinu L, Di Bari MA, D’Agostino C, et al. Gerstmann-Sträussler-Scheinker 10.​1038/​s41598-​017-​09489-3 PMID: 28851967; PMCID: PMC5575253. disease subtypes efficiently transmit in bank voles as genuine prion 20. Lund C, Olsen CM, Tveit H, Tranulis MA. Characterization of the prion diseases. Sci Rep. 2016;6:20443. https://​doi.​org/​10.​1038/​srep2​0443 Pub‑ protein 3F4 epitope and its use as a molecular tag. J Neurosci Methods. lished 2016 Feb 4. PMID: 26841849; PMCID: PMC4740801. 2007;165(2):183–90. https://​doi.​org/​10.​1016/j.​jneum​eth.​2007.​06.​005 36. Parchi P, Chen SG, Brown P, et al. Different patterns of truncated prion pro‑ Epub 2007 Jun 13. PMID: 17644183. tein fragments correlate with distinct phenotypes in P102L Gerstmann- 21. Padilla D, Béringue V, Espinosa JC, et al. Sheep and goat BSE propagate Sträussler-Scheinker disease. Proc Natl Acad Sci U S A. 1998;95(14):8322– more efficiently than cattle BSE in human PrP transgenic mice. PLoS 7. https://​doi.​org/​10.​1073/​pnas.​95.​14.​8322 PMID: 9653185; PMCID: Pathog. 2011;7(3):e1001319. https://​doi.​org/​10.​1371/​journ​al.​ppat.​10013​ PMC20974. 19 Epub 2011 Mar 17. PMID: 21445238; PMCID: PMC3060172. 37. Asante EA, Linehan JM, Tomlinson A, et al. Spontaneous generation of 22. Notari S, Xiao X, Espinosa JC, et al. Transmission characteristics of variably prions and transmissible PrP amyloid in a humanised transgenic mouse protease-sensitive prionopathy. Emerg Infect Dis. 2014;20(12):2006–14. model of A117V GSS. PLoS Biol. 2020;18(6):e3000725. https://​doi.​org/​10.​ https://​doi.​org/​10.​3201/​eid20​12.​140548 PMID: 25418590; PMCID: 1371/​journ​al.​pbio.​30007​25 PMID: 32516343; PMCID: PMC7282622. PMC4257788. 38. Marín-Moreno A, Espinosa JC, Torres JM. Transgenic mouse models for 23. Aguilar-Calvo P, Espinosa JC, Andréoletti O, et al. Goat K222-PrPC the study of prion diseases. Prog Mol Biol Transl Sci. 2020;175:147–77. polymorphic variant does not provide resistance to atypical scrapie in https://​doi.​org/​10.​1016/​bs.​pmbts.​2020.​08.​007 Epub 2020 Sep 11. PMID: transgenic mice. Vet Res. 2016;47(1):96. https://​doi.​org/​10.​1186/​s13567-​ 32958231. 016-​0380-7 PMID: 27659200; PMCID: PMC5034450. 39. Torres JM, Castilla J, Pintado B, et al. Spontaneous generation of infectious 24. Yull HM, Ritchie DL, Langeveld JP, et al. Detection of type 1 prion protein prion disease in transgenic mice. Emerg Infect Dis. 2013;19(12):1938–47. in variant Creutzfeldt-Jakob disease. Am J Pathol. 2006;168(1):151–7. https://​doi.​org/​10.​3201/​eid19​12.​130106 PMID: 24274622; PMCID: https://​doi.​org/​10.​2353/​ajpath.​2006.​050766 PMID: 16400018; PMCID: PMC3840888. PMC1592651. 40. Yang W, Cook J, Rassbach B, et al. A new transgenic mouse model of 25. Féraudet C, Morel N, Simon S, et al. Screening of 145 anti-PrP monoclonal Gerstmann-Straussler-Scheinker syndrome caused by the A117V muta‑ antibodies for their capacity to inhibit PrPSc replication in infected cells. tion of PRNP. J Neurosci. 2009;29(32):10072–80. https://​doi.​org/​10.​1523/​ J Biol Chem. 2005;280(12):11247–58. https://​doi.​org/​10.​1074/​jbc.​M4070​ JNEUR​OSCI.​2542-​09.​2009 PMID: 19675240; PMCID: PMC2749997. 06200 Epub 2004 Dec 23. PMID: 15618225. 41. Asante EA, Linehan JM, Smidak M, et al. Inherited prion disease A117V 26. Fraser H, Dickinson AG. Studies of the lymphoreticular system in the is not simply a proteinopathy but produces prions transmissible to pathogenesis of scrapie: the role of spleen and thymus. J Comp Pathol. transgenic mice expressing homologous prion protein. PLoS Pathog. 1978;88(4):563–73. https://​doi.​org/​10.​1016/​0021-​9975(78)​90010-5 PMID: 2013;9(9):e1003643. https://​doi.​org/​10.​1371/​journ​al.​ppat.​10036​43 Epub 101558. 2013 Sep 26. PMID: 24086135; PMCID: PMC3784465. 27. Hassan A, Campbell T, Darwent L, et al. Case report of homozygous 42. Senesi M, Lewis V, Kim JH, et al. In vivo prion models and the discon‑ E200D mutation of PRNP in apparently sporadic Creutzfeldt-Jakob nection between transmissibility and neurotoxicity. Ageing Res Rev. disease. BMC Neurol. 2021;21(1):248. https://​doi.​org/​10.​1186/​s12883-​021-​ 2017;36:156–64. https://​doi.​org/​10.​1016/j.​arr.​2017.​03.​007 Epub 2017 Apr 02274-w Published 2021 Jun 28. 24. PMID: 28450269. 28. Cassard H, Torres JM, Lacroux C, et al. Evidence for zoonotic potential of 43. Forloni G, Angeretti N, Malesani P, et al. Influence of mutations associated ovine scrapie prions. Nat Commun. 2014;5:5821. https://​doi.​org/​10.​1038/​ with familial prion-related encephalopathies on biological activity of ncomm​s6821 PMID: 25510416. prion protein peptides. Ann Neurol. 1999;45(4):489–94. https://​doi.​org/​ 29. Fernández-Borges N, Espinosa JC, Marín-Moreno A, et al. Protective effect 10.​1002/​1531-​8249(199904)​45:4%​3c489::​aid-​ana10%​3e3.0.​co;2-o PMID: of Val129-PrP against bovine spongiform encephalopathy but not variant 10211473. Creutzfeldt-Jakob disease. Emerg Infect Dis. 2017;23(9):1522–30. https://​ doi.​org/​10.​3201/​eid23​09.​161948 PMID: 28820136; PMCID: PMC5572891. 30. Marín-Moreno A, Huor A, Espinosa JC, et al. Radical change in zoonotic Publisher’s Note abilities of atypical BSE prion strains as evidenced by crossing of sheep Springer Nature remains neutral with regard to jurisdictional claims in pub‑ species barrier in transgenic mice. Emerg Infect Dis. 2020;26(6):1130–9. lished maps and institutional affiliations. https://​doi.​org/​10.​3201/​eid26​06.​181790 PMID: 32441630; PMCID: PMC7258450.