Allele count in gnomAD: 0
Cases in literature: 1 (UK)
Penetrance calculation: pathogenicity not established
Pro84Ser mutation described by Jones et al in 20141.
A 61-year-old Caucasian gentleman presented in the UK with a one-year history of memory disturbance, fatigue, apathy and agitation (age of onset 60 years). Past medical history is notable for haemochromatosis. He does not take regular medication. Family history a includes maternal history of Parkinson’s disease (the details of which are unknown) and there is no strong history to indicate genetic prion disease. He was initially diagnosed with early-onset Alzheimer disease. The patient deteriorated rapidly from time of presentation with subsequent onset of aggression and paranoia. Further investigative results revised the diagnosis to immune-mediated encephalitis, for which 60 mg OD prednisolone was commenced. He was also treated with IVIg at a dose of 2 g/Kg. Plasma exchange was attempted but not tolerated. The patient failed to respond to treatment despite two further doses of IVIg at months two and three of admission. Mycophenolate mofetil was started four months into his admission, however, at five months he deteriorated further with minimal spontaneous activity, flat affect and poor comprehension. He was immobile and developed focal seizures, although not faciobrachial dystonic seizures, that were anti-epileptic drug responsive. The patient also developed limb and trunk dystonia, thought secondary to antipsychotic therapy for behavioural management. Rituximab 1g was administered at five months into his admission; no clinical improvement was seen, and VGKC-complex antibody levels remained persistently elevated. The patient continued to relentlessly deteriorate and died 14 months following initial presentation (26 months from onset). A diagnosis of GSS was made post-mortem.
GSS classically presents with a slowly progressive cerebellar ataxia, associated with distal pain and loss of sensation, absent ankle reflexes, mild abnormalities of frontal lobe functioning and late cognitive impairment; mean disease duration is four years (range 0.5-12 years)2-3. The typical ataxic presentation is associated with mutant Pro102Leu2,4, which is the most common GSS-causing mutation worldwide5. However, at least seventeen missense mutations in total have been associated with GSS (Pro84Ser, Pro102Leu, Pro105Leu/Ser, Ala117Val, Gly131Val, Ser132Ile, Ala133Val, Val176Gly, His187Arg, Phe198Ser, Asp202Asn, Glu211Asp, Gln212Pro, Gln217Arg, Tyr218Asn and Met232Thr), as well as the nonsense mutation Gln227X (see Truncating Mutations).
Examination reveals only scant fasciculations in the lower limbs. Repeat examination following deterioration finds mild limb ataxia1.
Initial routine blood tests, MR brain imaging (including DWI and FLAIR sequences) and nerve conduction studies were normal. EMG confirmed fasciculations without active denervation changes. Neuropsychological assessment revealed deficits in memory, language and visual perception. Following deterioration, voltage-gated potassium channel (VGKC)-complex antibodies were found to be strongly positive (4,159 pM, normal range < 100 pM), but leucine rich, glioma inactivated protein 1 (LGI1) and contactin associated protein-like 2 (CASPR2) antibodies were negative. VGKC-complex antibody titre remained persistently high throughout his admission at > 2000 pM. Paraneoplastic neuronal antibody panel was negative and whole-body CT scanning revealed no underlying malignancy (these both remained negative on repeat testing). EEG showed non-specific non-encephalopathic change. Of note, repeat MR brain was also normal with no medial temporal lobe changes. CSF analysis was not performed1.
Pro84Ser mutation (c.250C>T resulting in CCT to TCT change)1; in addition, the patient harboured the synonymous, non-pathogenic Ala117Ala polymorphism6 (allele count in gnomAD, 7525). He was heterozygous (Met/Val) at codon 1291, although it is not known which is the in cis allele.
General post-mortem analysis revealed no systemic malignancy. The proximal cause of death was necrotising bronchopneumonia. Macroscopic examination of the brain showed no evidence of cerebral or cerebellar atrophy, and no focal abnormalities. Histologic analysis showed numerous multicentric amyloid plaques in the cerebral cortex, basal ganglia, thalamus, and cerebellum. Spongiform changes were not seen in the cerebral cortex and subcortical grey matter, and only patchy spongiform change was noted in the cerebellar molecular layer. Immunohistochemistry for prion protein revealed intense labelling of the amyloid plaques. Occasional small prion protein deposits (nut no plaques) were present in the brainstem and in the substantia gelatinosa of the spinal cord, but not in the spinal nerve roots or dorsal root ganglia. No systemic prion protein deposition was observed. Staining for -amyloid and -synuclein proteins was negative. Tau staining labelled only a few fine neuritic processes around the PrP amyloid plaques, without the presence of neurofibrillary tangles1.
Western blot analysis of homogenised frontal and temporal cortices showed two low-molecular-mass proteinase-K resistant PrPSc bands in the 7 kDa and 9 kDa range1,7.
Structure-based protein function annotation:
Proline 84 contributes the first residue of the fourth sequential PHGGGWGQ octapeptide motif in the repeat region (aa 60-91), that is known to coordinate copper and zinc with high affinity8-14. Residues HGGGW in each repeat form the Cu(II)-binding unit. The four histidine residues (His61/His69/His77/His85) contained within the octapeptide repeats are able to coordinate a single Cu2+ ion in square planar geometry, with sub-nanomolar affinity15. At higher Cu2+ concentrations, each of the individual tandem repeats coordinates a single Cu2+ ion (to a total of four coordinated Cu2+ ions) with weaker micromolar affinity15-16 (see Architecture of PrP).
It has been shown that the octapeptide repeat region takes up copper in three distinct coordination modes, referred to as components 1, 2 and 3; controlled by the precise molar ratio of Cu2+ to protein17. Component 3 coordination, which is observed at low copper occupancy, involves three to four octapeptide repeats binding through the histidine imidazoles. Component 1 arises at full copper occupancy and involves Cu2+ coordination to the HGGGW residues within each octapeptide repeat through the imidazole nitrogen (1) of histidine, deprotonated amide nitrogens from the two following glycines, and the amide carbonyl oxygen from the second glycine. Tryptophan participates through the formation of a hydrogen bond between the indole NH to an axially coordinated water13,15. The intervening GQP segments are thought to act as flexible links between the HGGGW domains18. Copper also binds residues His96 and His111, outside of the octapeptide region14,19-21.
Importantly, binding of divalent metal ions to PrPC promotes a quaternary cis interaction between the N-terminus and globular C-terminal domain, interruption of which, may play a role in prion pathogenesis. This cis interaction is mediated by both Cu2+ 22-25 and Zn2+ 26 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 α-helices 2 and 322,24,26-29. It has been observed that the N-terminal domain, and in particular the first nine residues (aa 32-31, encompassing charged cluster CC1) acts as a latent, toxic effector, whose activity is under autoinhibitory control by the cis N-C interaction that sequesters the metal bound N-terminus – thus, PrPC is involved in a neuroprotective self-regulatory process23-24,30. Indeed, mutations causing familial CJD (Glu200Lys – the most common cause worldwide) and either familial CJD or FFI (Asp178Asn, dependent upon whether the PRNP cis codon 129 polymorphism is Met or Val31) have demonstrated weakened N-C cis interactions, likely through reduction of negative charge on the C-terminal docking surface22,24,26.
How copper co-localises the two subdomains of PrPC has recently been investigated by the Millhauser group32; their work reveals a mechanism whereby an N-terminal histidine is exchanged for one of two highly conserved C-terminal histidine residues, His140 or His177, and in this manner, the copper ion is co-bound between the N and C-termini, maintaining its square planar (octahedral) geometry, and creating an intermolecular tether that stabilises N-C quaternary structure32. Disruption of electrostatic interactions by mutants Glu200Lys and Asp178Asn, that are in close proximity to His176, are likely to also impair C-terminal copper binding, which would further destabilise the N-C interaction.
Pro84 is adjacent to the metal ion-binding His85; the effect of its substitution is two-fold. First, the proline ring forms part of the protein backbone, and restricts available backbone conformations; this limited conformational freedom may be necessary to maintain correct coordination geometry18,33, although the proline itself is not directly involved in metal ion coordination. Second, the octapeptide repeat region is thought to form an extended poly(L-proline) II helix structure34 and substitution of Pro84 may interfere with the conformational adaptability imparted by such a structure, that may be required to correctly orientate the N- and C-termini (see Pro39Leu analysis). Therefore, the loss of coordination geometry mediated by substitution of Pro84 to serine, as well as possible disruption of the PPII structure in this region, would be expected to have a deleterious effect on metal-ion binding and consequently weaken the neuroprotective N-C interaction, thereby, contributing to GSS pathogenesis in this mutant. The small size of the substituted serine is unlikely to cause steric hindrance or significant structural perturbation at this site35. Interestingly, the aromatic-to-aromatic His85Tyr variant is not pathogenic3, consistent with the notion that copper stabilises the N-C interaction by binding with three histidine residues from the octapeptide repeat region and one histidine from the C-terminus32, thus, this variant leaves His61, His69, His77 available for N-terminus metal coordination. This is also in agreement with data that a deletion mutation of one octapeptide repeat is not toxic in humans and is seen as a common polymorphism6,36, whereas deletion of two or more octapeptide repeats results in prion disease37-39.
Interestingly, PRNP is thought to act as a modifier gene in Wilson disease, as part of a ‘multi-hit’ hypothesis that impacts disease phenotype40-41. PrPC Met129Val genotype frequency was not found to be different between patient’s with Wilson disease and the general population, indicating that this polymorphism has no influence on penetrance. However, the PrP Met/Met129 genotype was established as an important factor in delaying the onset of neurological and hepatic symptoms42; the age of onset of Wilson disease and that of the neurological presentation was noted to be delayed five years and seven years, respectively, in patients homozygous for Met at codon 129 compared with subjects with at least one Val at codon 129 in the PRNP gene42. Furthermore, Met/Met129 homozygosity was found to be associated with more severe neurological symptoms in Wilson disease43. Additional data that PRNP may act as a modifier gene in Wilson disease, is provided by a recent case44 of a Wilson disease patient with rapidly progressive neurological decline and a genotype of ATP7B compound heterozygosity (c.2165dupT; c.4039G>A) and PRNP non-pathogenic variant Gly54Ser (c.160G>A) with Val/Val homozygosity at position 129 (c.385A>G). The patient’s sister was asymptomatic (despite exhibiting a classical Wilson disease biochemical phenotype) was also compound heterozygous for the same two ATP7B mutations but lacked the Gly54Ser PrPC variant and was Met/Val heterozygous at codon 12944. A further case report45 of Wilson disease, with co-morbid clinical and biochemical evidence of prion disease, also seeks to co-localise the effects of PrPC on Wilson’s disease phenotype, however notably, genetic analysis was not undertaken in this patient45. These data raise the interesting possibility of a synergistic interaction between ATP7B and PRNP variants, mediated through effects on copper metabolism, to modulate onset and severity of neuropathology in Wilson disease.
In silico Pathogenicity predictions:
- Probability of pathogenicity: 0.474
- Standard error: 0.075
- Prediction: Unknown
- Score: 0.848
- Prediction: Pathogenic
A stringent REVEL score threshold of 0.75 is applied, above which the variant is classified as pathogenic.
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