Allele count in gnomAD control population: 9
Cases in literature: 3 (Italy)
Penetrance calculation: n/a
Variant first reported by Bernardi et al1 in two Italian patients.
Patient 11: 67-year-old male, employed as a barman. Aged 58, he increased his alcohol consumption and in the following two years developed depressive symptoms. Aged 60 years, difficulties in word recruitment and writing appeared. Impairment of short-term memory and attention and transient topographic disorientation occurred 1 year later. At 63 years, he was consulted because of prominent behavioural disturbances and executive dysfunctions. The patient appeared emotionally flat, blunted, apathetic and absent-minded; he started to live in his van, sleeping and hoarding rubbish. He showed an imitative behaviour, environmental dependency, hyperorality and binge-eating. Insight was absent and judgement impaired. He was completely dependent on his family for daily activities of living. He developed sleep disturbances and a short-tempered mood. At time of reporting, he had a severe frontal dementia, with behaviour that is rigid, aimless and compulsive, with loss of autonomy and incontinence.
Patient 21: 78-year-old male previously employed as a truck driver. He had no family history suggestive of dementia, although his mother was referred to as an irritable and wilful person. She was impulsive and bossy like the patient and in later years of life showed signs of hand tremor. The patient’s past medical history included coronary heart disease, hypertension, diabetes mellitus and dyslipidaemia. The clinical onset was age 75 years with apathy, reduction in speech, emotional flatness and anhedonia. He developed mental rigidity and perseverative. He had difficulties in modulating his behaviour in response to sudden changes. Cognitive impairment followed after a few months; the patient developed disorientation and attention impairment, reduced working memory, difficulties on abstract reasoning, planning, and problem solving. At the same time his speech became progressively reduced in fluency, with difficulties in naming, word retrieval, syntaxes and oral comprehension of structured sentences, as well as echolalia. At 77 years, he was referred to neurological consultation because of failure in activities of daily living and ideomotor apraxia. He was socially disinhibited, showing restlessness and wandering, hyperphagia and utilisation behaviour.
Both patients shared a frontal dementia dominated by a dysexecutive syndrome and severe behavioural disturbances. Neither patient developed the typical clinical signs associated with prion diseases such as myoclonus or cerebellar ataxia. Neuroimaging findings showed marked frontal and temporal atrophy with a peculiar involvement of mesial frontal regions and left temporal lobe. The topography of cortical atrophy was concordant with the behavioural and language disorders. Hyperintense alterations of cortical and subcortical structures in FLAIR and Diffusion-weighted MR imaging were not seen. The patients (1 and 2) did not show the typical EEG of prion disease with periodic sharp wave complexes.
The Pro39Leu mutation was also found in another Italian patient with frontotemporal dementia, when a screen of seven hundred and sixty-one patients diagnosed with frontotemporal dementia and 719 controls was undertaken, by another group2. This patient (patient 3) was a 67-year-old male, who worked as a bricklayer until the age of 58. Family history was positive for dementia: his mother died aged 70 years with a three-year history of dementia. His past medical history included hypertension, type II diabetes mellitus, dyslipidaemia, chronic bronchitis and chronic renal failure. He had a history of alcoholism and was a smoker. From the age of 66 years, he developed apathy, short-term memory deficit and postural instability, with three falls in one month. The symptoms rapidly progressed, requiring admission to hospital within a month of onset. Six-months from symptom onset the family reported emotional bluntness, apathy, anhedonia and episodes of aggressiveness, but he remained clinically stable. He was dependent on his family for all activities of daily living.
Of note, other mutations in the PRNP gene have been reported to induce a clinical phenotype imitating frontotemporal dementia: Pro102Leu3, Glu196Lys4, His187Arg5 and Thr183Ala6, Gln217Arg7; as well as the nonsense mutation Gln227Stop8 (See Truncating Mutations).
Patient 11 showed frontal signs and non-fluent aphasia. Cognitive dysfunction with stereotyped ritualistic behaviours, inappropriateness, mirror sign (inability to recognise ones’ own reflection), dressing and constructive apraxia. He became echolalic and uncommunicative, speech was almost unintelligible.
Patient 21 examination revealed a frontal syndrome, non-fluent aphasia and mild-to-moderate akinetic rigid Parkinsonism with camptocormia. In the following years, the patient worsened with extrapyramidal signs, severe gait disturbance and freezing, dysarthria and dysphagia. The patient died aged 78 years due to pneumonia.
Patient 32 was examined at 6 months from clinical onset and found to be disorientated in space and partially in time. Spontaneous speech was reduced, but he could answer questions when stimulated. The speech was monotonous, with reduced vocabulary. He had difficulty sustaining attention and had an apathetic attitude. Cranial nerves were intact, in particular his pursuit and saccades were normal. He did not have any motor or sensory deficit. Deep tendon reflexes were reduced symmetrically. Tone was mildly increased, especially in the lower limbs. He was unstable on standing with mild multi-directional oscillations. His gait was slow and cautious. There was no evidence of dysmetria. Jaw jerk, glabellar and palmo-mental reflexes were present. Myoclonus was absent. At one year from onset he developed a preference for sweet food. This case also presented with frontal dementia dominated by a dysexecutive syndrome, as reported for patients 1 and 2, however, severe behavioural disturbance was absent.
Patient 11, CT head and MR brain showed diffuse cortical atrophy prominent in the mesial frontal, temporal, and posterior parietal regions, mainly in the left side. EEG and EMG studies were normal. Patient 21, MR brain showed a pattern of cortical atrophy that was similar to patient 1, with marked involvement of mesial frontal, temporal and posterior parietal regions in the left side, besides some lacunar ischaemic subcortical lesions. Brain single-photon emission computed tomography confirmed these findings, revealing a hypoperfusion of fronto-temporal-parietal cortical areas with relative sparing of the occipital lobe, brainstem and cerebellum. EEG did not show any typical periodic complexes of prion diseases. The clinical course for both patients was not rapidly progressive given the duration of disease was over 18 months. These clinical findings do not satisfy the diagnostic criteria for inherited prion disease.
Patient 32, underwent neuropsychological testing which showed an MMSE of 17/30, multiple EEGs did not reveal periodism, EMG revealed a mild chronic sensorimotor axonal polyneuropathy and MR brain showed bilateral frontal lobe atrophy with confluent lesions in the white matter and no alteration in DWI MR imaging. Of note, neuroimaging showed that atrophy was mainly frontal without the temporal or left parietal atrophy noted in patients 1 and 2. This is consistent with the absence of language problems in this patient. Lumbar puncture showed a weak positivity for protein 14.3.3. CSF amyloid-β level was 1031 pg/mL (reference range: ≥ 550 pg/mL) and total tau was 540 pg/mL (refence range: ≤ 375 pg/mL). Paired autoantibodies in plasma and CSF were all negative. Brain MR showed neither cortical ribboning nor basal ganglia hyperintensities on DWI. Multiple lesions in the white matter, interpreted as vascular lesions, were observed, together with bilateral frontal lobe atrophy. FDG-PET showed hypometabolism in the prefrontal cortex and insula bilaterally and in the right caudate. EEG showed no periodic complexes. A repeat lumbar puncture showed the absence of protein 14.3.3, Amyloid-β levels were 714 pg/mL and total tau was 152 pg/mL, phosphorylated tau level was 28 pg/mL (reference range: ≤ 52 pg/mL). No oligoclonal bands were present in the CSF. In addition, autoantibodies were absent. The results of these investigations together with examination findings, again do not support the diagnosis of prion disease.
Patients 11 and 21: c. 116C>T (CCG to CTG). A heterozygous transition in the second base of codon 39. The absence of this substitution was assessed in 200 cognitively healthy controls. Genotype at codon 129: Patient 1 was Met/Met homozygous and patient 2 was Met/Val heterozygous (it is not known which is the in cis allele in patient 2). Mutations in dementia causative genes PSEN1, PSEN2, MAPT, GRN and C9orf72 were excluded.
Patient 32: Pro39Leu mutation with Met/Met homozygosity at codon 129. Genetic testing was negative for MAPT, GRN and C9orf72 gene mutations.
Neuropathological studies: None undertaken.
Structure-based protein function annotation:
Proline 39 lies in the N-terminal domain between the fist polybasic charged cluster (CC1) and the octapeptide repeat region, and is perfectly conserved amongst mammalian sequences9,10. Biophysical evidence converges on the finding that that the partially structured N-terminus and the globular C-terminal domain directly interact in a functionally important manner11,12. This cis interaction is mediated by both Cu2+ 12-15 and Zn2+ 16 binding to the octapeptide repeat domain, as well as electrostatic interactions between the polybasic N-terminus clusters CC1 and CC2 and a negatively charged pocket on the globular C-terminal domain contributed predominantly by helices α2 and α312,14,16-19 (see Architecture of PrP).
Binding of divalent ions Cu2+ and Zn2+ drives an intra-molecular contact between the N- and C-terminal domains; a number of mutations linked to familial prion disease reside in regions of contact. NMR experiments demonstrate that mutations D–178N and E–200K+ systematically weaken the cis N-C interaction, likely through reduction of negative surface on the docking surface of the C-terminus, possibly contributing to the disease phenotype12,16. The weakened cis interaction observed in mutants P102L and V210I cannot be explained by charge disruption; it is possible that loss of proline alters conformational dynamics required to mediate effective docking of the N-terminus onto the C-terminal pocket16. Interestingly, the Q219K polymorphism, associated with resistance to sporadic CJD in the Japanese population20, was seen induce a moderate strengthening of the N-C interaction16. Therefore, it may be possible that increased stability of the PrPC quaternary N-C interaction creates a barrier to misfolding and is, in turn, protective against prion disease.
Proline is the only amino acid whose sidechain is connected to the protein backbone twice, forming a five-membered nitrogen-containing ring; therefore, proline is an imino acid, as in its isolated form, it contains an NH2+ rather than an NH3+ group, this leaves the backbone at this point with no amide hydrogen so that no hydrogen bonding is possible21. Additionally, the Pro five-membered ring imposes rigid constraints on N-Cα rotation, and thus imparts conformational rigidity22. It has previously been proposed that an extended poly(L-proline) II (PPII) helix structure can form in the octapeptide repeat region of PrP23 of a type involved in regulatory, multiple weak interactions when present in other proteins24-25. PPII helix is the predominant secondary structure in proteins with a high degree of conformational flexibility26 and this structure is noted to impart a rheomorphic (flowing) character27.
The PPII structure is an extended left-handed helix with three-fold rotational symmetry; the average dihedral angles of residues ϕ and ψ being -77° and 145°, respectively22. The initial suggestion of a PPII helix in the N-terminus23 had been largely overlooked, as NMR studies on full-length recombinant PrP found no evidence of for such a structure28, additionally, this peptide is rich in tryptophan, which can contribute substantially to CD spectra in the far UV29-30. However, lack of main-chain hydrogen bonding in PPII helices makes NMR determination of this type of structure difficult, especially if it is in equilibrium with random coil or other types of secondary structure, and retrospective analysis of crystallographic and NMR PrPC structures does in fact find PPII helices forming most of the N-terminus31. CD spectroscopy of synthetic peptides comprising residues 48-9323 and 57-9130, 58-9132, and 60-9133 containing the four octapeptide repeats have given spectra with features diagnostic of a PPII conformation; a strong minimum at ~195 nm and weaker maximum at ~220 nm34, which is lost upon addition of copper ions to this metal binding motif30,32-33. The presence of an N-terminus PPII helix, interspersed with β-turns, has recently been confirmed by both Raman optical activity35 and the finding that the specific sequence Ser-Pro44-Gly-Gly can form a PPII structure in aqueous buffers, act as a substrate for prolyl 4-hydroxylation in CHO cells and in brain cells of mice infected with prions, strongly suggest that the polypeptide forms an extended PPII structure in vivo36. Hydroxylation to 4-hydroxyproline by polyl 4-hydroxylase occurs within the consensus sequence X-Pro-Gly37 and requires, principally, a PPII conformation38, as well as a partial β-turn, to extend the -X-Pro-Gly- motif into the catalytic site for hydroxylation39.
Therefore, the PPII structure and factors influencing its dynamic flexibility, may be critical for a role of PrP in normal cellular functioning and signalling36. Although, the role of this region in the prion disease process has been somewhat overlooked, primarily, because transgenic mice that are devoid of much of the N-terminal region still support prion replication40, indicating that the N-terminal region is not required a priori for the PrPC-PrPSc conversion process. The presence of a large amount of PPII structure in the N-terminal region, is consistent with the flexibility and conformational adaptability required for its ability to bind metal ions, modulate properties of full-length protein via interactions with the structured C-terminal region, as well as facilitating molecular interactions with myriad ligands within the intra- or extra-cellular environment41.
With respect to Pro39Leu, it is our conclusion42 (and others’43) that this variant constitutes a polymorphic variant, that is common in controls and rare in cases, which has coincidentally co-segregated in the three patients with frontotemporal dementia detailed above. However, if this residue participates in a PPII structure, its substitution may subtly modify dynamical properties of the N-terminus that permit ligand binding and correct orientation of the N-C interaction, although in this case, not to an extent that facilitates its misfunction. Such a mechanism, however, may underlie the pathogenic loss-of-proline mutants: Pro84Ser, Pro102Leu, Pro105Leu/Ser/Thr (LINK).
In silico Pathogenicity predictions
- Probability of pathogenicity 0.539
- Standard error: 0.058
- Prediction: Unknown
- Score: 0.883
- Bernardi L, Cupidi C, Frangipane F et al. Novel N-terminal domain mutation in prion protein detected in 2 patients diagnosed with frontotemporal lobal degeneration syndrome. Neurobiology of Aging 2014; 35: 2657.e7-2657.e11. (PMID: 25022973)
- Oldoni E, Fumagalli GG, Serpente M et al. PRNP P39L Variant is a Rare Cause of Frontotemporal Dementia in Italian Population. Journal of Alzheimers Disease 2016; 50(2): 353-357. (PMID: 26757195)
- Giovagnoli AR, Di Fede G, Aresi A et al. Atypical frontotemporal dementia as a new clinical phenotype of Gerstmann-Straussler-Scheinker disease with the PrP-P102L mutation. Description of a previously unreported Italian Family. Neurological Sciences 2008; 29(6): 405-410. (PMID: 19030774)
- Clerici F, Elia A, Girotti F et al. Atypical presentation of Creutzfeldt-Jakob disease: the first Italian case associated with E196K mutation in the PRNP gene. Journal of the Neurological Sciences 2008; 275(1-2): 145-147. (PMID: 18706660)
- Hall DA, Leehey MA, Filley CM et al. PRNP H187R mutation associated with neuropsychiatric disorders in childhood and dementia. Neurology 2005; 64(7): 1304-1306. (PMID: 15824374)
- Nitrini R, Teixeira da Silva LS, Rosemberg S et al. Prion disease resembling frontotemporal dementia and parkinsonism linked to chromosome 17. Arquivos de Neuropsiquitria 2001; 59(2-A): 161-164. (PMID: 11400017)
- Woulfe J, Kertesz A, Frohn I et al. Gerstmann-Staräussler-Scheinker disease with the Q217R mutation mimicking frontotemporal dementia. Acta Neuropathologica 2005; 110: 317-319.
- Jansen C, Parchi P, Capellari S et al. Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP. Acta Neuropathologica 2010; 119(2): 189-197. (PMID: 19911184)
- Wopfner F, Weidenhöfer G, Schneider R et al. Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. Journal of Molecular Biology 1999; 289(5): 1163-1178. (PMID: 10373359)
- van Rheede T, Smolenaars MMW, Madsen O, de Jong WW. Molecular Evolution of the Mammalian Prion Protein. Molecular Biology and Evolution 2003; 20(1): 111-121.
- Evans EGB and Millhauser GL. Copper- and Zinc-Promoted Interdomain Structure in the Prion Protein: A mechanism for Autoinhibition of the Neurotoxic N-terminus. Progress in Molecular Biology and Translational Science 2017; 150: 35-56. (PMID: 28838668)
- McDonald AJ, Leon DR, Markham KA et al. Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally relevant Mutations: Analysis by Cross-Linking, MS/MS and NMR. Structure 2019; 27(6): 907-922.e5. (PMID: 30956132)
- Wu B, McDonald AJ, Markham K et al. The N-terminus of the prion protein is a toxic effector regulated by the C-terminus. eLife 2017; 6: e23473. (PMID: 28527237)
- Evans EGB, Pushie MJ, Markham KA et al. Interaction between Prion Protein’s Copper-Bound Octarepeat Domain and a Charged C-Terminal Pocket Suggests a Mechanism for N-Terminal Regulation. Structure 2016; 24(7): 1057-1067. (PMID: 27265848)
- Thakur AK, Srivastava AK, Srinivas V et al. Copper alters aggregation beahviour of prion protein and induces novel interactions between its N- and C-terminal regions. Journal of Biological Chemistry 2011; 286(44): 38533-38545. (PMID: 21900252)
- Spevacek AR, Evans EGB, Miller JL et al. Zinc Drives a Tertiary Fold in the Prion Protein with Familial Disease mutation Sites at the Interface. Structure 2013; 21(2): 236-246. (PMID: 23290724)
- Martínez J, Sánchez R, Castellanos M et al. PrP charge structure encodes interdomain interactions. Scientific Reports 2015; 5: 13623. (PMID: 26323476)
- Markham KA, Roseman GP, Linsley RB et al. Molecular Features of the Zn2+ Binding Site in the Prion Protein Probed by 113Cd NMR. Biophysical Journal 2019; 116(4): 610-620. (PMID: 30678993)
- Roseman GP, Wu B, Wadolkowski MA et al. Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region. FASEB Journal 2020; 34(6): 8734-8748. (PMID: 32385908)
- Shibuya S, Higuchi J, Shin RW et al. Codon 219 Lys allele of PRNP is not found in sporadic Creutzfeldt-Jakob disease. Annals of Neurology 1998; 43(6): 826-828. (PMID: 9629853)
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- Bernardi L and Bruni AC. Mutations in Prion Protein Gene: Pathogenic Mechanisms in C-Terminal vs. N-Terminal Domain, a Review. International Journal of Molecular Sciences 2019; 20(14): 3606. (PMID: 31340582)
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