Wednesday, September 3, 2008

Phenotypic heterogeneity and genetic modification of P102L inherited prion disease in an international series

Brain Advance Access published September 1, 2008

Phenotypic heterogeneity and genetic modification of P102L inherited prion disease in an international series

T. E. F.Webb,1,2 M. Poulter,1 J. Beck,1 J.Uphill,1 G. Adamson,1 T. Campbell,1 J. Linehan,1 C. Powell,1 S. Brandner,1,2 S. Pal,1,2 D. Siddique,1,2 J. D.Wadsworth,1 S. Joiner,1 K. Alner,2 C. Petersen,2 S. Hampson,2 C. Rhymes,2 C. Treacy,2 E. Storey,3 M. D.Geschwind,4 A. H. Nemeth,5 S.Wroe,1,2 J. Collinge1,2 and S. Mead1,2 1MRC Prion Unit and Department of Neurodegenerative Disease,UCL Institute of Neurology, 2National Prion Clinic and National Hospital for Neurology & Neurosurgery, Queen Square, London,WC1N 3BG, 3Department of Medicine (Neuroscience), Monash University, Melbourne, Australia, 4Department of Neurology,University of California, San Francisco (UCSF), San Francisco, CA,USA and 5Department of Clinical Genetics, Churchill Hospital and Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,Oxford,OX3 9DU, UK Correspondence to: Prof. John Collinge, Department of Neurodegenerative Disease and MRC Prion Unit, UCL Institute of Neurology, National Hospital for Neurology and Neurosurgery, Queen Square, LondonWC1N 3BG, UK E-mail:

The largest kindred with inherited prion disease P102L, historically Gerstmann-Stra«ussler-Scheinker syndrome, originates from central England, with e¤migre¤ s now resident in various parts of the English-speaking world. We have collected data from 84 patients in the large UK kindred and numerous small unrelated pedigrees to investigate phenotypic heterogeneity and modifying factors.This collection represents by far the largest series of P102L patients so far reported.Microsatellite and genealogical analyses of eight separate European kindreds support multiple distinct mutational events at a cytosine-phosphate diester-guanidine dinucleotide mutation hot spot. All of the smaller P102L kindreds were linked to polymorphic human prion protein gene codon 129M andwere not connected by genealogy ormicrosatellite haplotype background to the large kindred or each other. While many present with classical Gerstmann-Stra«ussler-Scheinker syndrome, a slowly progressive cerebellar ataxia with later onset cognitive impairment, there is remarkable heterogeneity. A subset of patients present with prominent cognitive and psychiatric features and some have met diagnostic criteria for sporadic Creutzfeldt-Jakob disease. We show that polymorphic human prion protein gene codon 129 modifies age at onset: the earliest eight clinical onsets were all MM homozygotes and overall age at onset was 7 years earlier for MM compared with MV heterozygotes (P=0.02).Unexpectedly, apolipoprotein E4 carriers have a delayed age of onset by10 years (P=0.02).We found a preponderance of female patients comparedwithmales (54 females versus 30 males,P=0.01), which probably relates to ascertainment bias.However, thesemodifiers had no impact on a semi-quantitative pathological phenotype in 10 autopsied patients.These data allow an appreciation of the range of clinical phenotype, modern imaging andmolecular investigation and should inform genetic counselling of at-risk individuals, with the identification of two genetic modifiers.



The patients presented here constitute by far the largest study of the classical GSS-associated PRNP mutation, P102L. Modern investigation, molecular genetic techniques and advances in pathological examination have allowed us to revisit a historical British pedigree and other smaller kindreds. We find evidence of a mutation hot spot at codon 102, a subgroup presenting with rapid cognitive decline and the significant modification of phenotype by two single nucleotide polymorphisms. The microsatellite haplotyping presented here demonstrates the existence of multiple separate and worldwide P102L kindreds of UK origin, confirmed in most patients by established genealogical links that had not been identified previously. However, the identification of a further six UK P102L kindreds along with two others of European origin suggests that multiple mutational events have occurred in the recent past. This might be supported by the identification of at least one of these patients (6.VIII.1) in an individual without suggestive family history or of known P102L patients living nearby, suggesting that this might represent a novel mutation. Given the identification of IPD patients in otherwise typical sCJD presentations, as well as in patients without a family history of neurodegeneration, novel mutational events may occur more commonly than previously thought. The commonest point mutations associated with IPD occur at cytosine-phosphate diesterguanidine dinucleotide sites hypothesized to be mutation ‘hot spots’ in human DNA (Vollmert et al., 2006). These hot spots are related to the spontaneous demethylation of cytosine to thymidine. Cytosine-phosphate diester-guanidine dinucleotide sites are associated with E200K, D178N and P102L point mutations, which between them account for the bulk of IPD patients worldwide (Dagvadorj et al., 2002; Mead, 2006). An alternative possibility to multiple separate mutational events being responsible for these apparently unconnected P102L IPD kindreds is that these small pedigrees do share a common ancestor with the large pedigree but that this was long enough in the past for linkage to the disease haplotype to have broken down. We estimate a probability of recombination of the 3MB microsatellite haplotype as about 10% per generation (assuming a genetic:physical ratio of 3.7). The unexpected finding that significantly greater numbers of females were identified with P102L IPD and the consequently higher number of individuals having an affected mother rather than father as the parent from whom the mutation was inherited is puzzling. This discrepancy has been reported once previously in this family, albeit with smaller numbers of individuals and without available genetic techniques, allowing the linkage of kindreds not known to share ancestry (Baker et al., 1985). It has also been observed in the large 6-OPRI IPD kindred from the South-East of England (Mead et al., 2006). In the earlier study in P102L, the finding was explained by postulating that affected females had significantly more children than their unaffected siblings and that greater than half these were daughters. Such an explanation is intriguing, though hard to explain. Alternatively, because of possible illegitimacy, we suspected that genealogical research might more readily identify affected mothers than affected fathers. In order to consider the possibility of ascertainment bias, the gender of members of the kindred identified from parish records or census entries, but who have not been identified as having suffered from IPD P102L, were collected. An excess of males in these untraced individuals was indeed identified. When these were added to the presumed affected male and female individuals, an excess of females to males remained (82–68), although this was not significantly different from an expected proportion of 0.5. It seems likely that ascertainment bias is responsible for this gender difference, although the intriguing possibility of a biological explanation remains. While the majority of P102L IPD patients here present with progressive ataxia accompanied by mild or absent cognitive symptoms and signs, a subset present with a predominantly cognitive and or psychiatric onset, with mild or absent cerebellar signs initially. Psychiatric involvement has been severe enough to necessitate antipsychotic medication in four patients (VII.1, VII.8, VIII.4 and 3.VIII.1) and inpatient treatment in two (VII.1 and VII.8). The existence of these distinct phenotypes (cognitive and psychiatric versus cerebellar onset) is supported by the finding of early prominent frontal executive impairment on neuropsychology in these patients not seen in other patients so tested. Neuropsychological assessment is now routine at the National Prion Clinic but in the past this was more usually performed only when clinical evidence of cognitive impairment existed. In addition to these neuropsychological differences, there is a suggestion that the cognitive and Fig. 9 Photograph of original 1871 asylum entry relating to admission and care of III.3 from Fig. 2. His condition is described as hereditary and reference ismade to his relatives’ care in the same institution.He is reported as being ‘scarcely able to move one leg in ront of another’ and his countenance is described as ‘imbecile’. He appears to have had dementia as well as weakness: ‘Can only answer in monosyllables & then incoherently; is much paralysed’. He was treated with daily doses of brandy until he died. ‘Paralysis’ was listed as the cause on the death certificate. Page 12 of 15 Brain (2008) T. E. F.Webb et al. psychiatric presentations have an earlier age at onset and death. Neuropsychology also highlights, however, that most if not all patients have cognitive deficits when psychology is performed, even if the findings are subtle, although selection bias may limit this conclusion. No pathological correlates of these two syndrome types have been identified, although numbers compared are small. Kuru and growth hormone-associated prion disease are notable for their onset with cerebellar symptoms (Collinge, 2001), which prompts speculation that cerebellar onset in P102L might result from the onset of prion replication outside the central nervous system. The range of tissue available did not permit the thorough testing of this hypothesis. The high degree of clinical heterogeneity and the lack of characteristic findings on commonly available clinical neurological investigations make correct diagnosis of P102L IPD challenging. The finding of positive CSF 14-3-3 combined with the albeit unusual occurrence of periodic sharp wave complexes on EEG and an sCJD-like phenotype in some individuals raises the possibility of missed diagnosis. It also supports our clinical practice of advising PRNP analysis routinely in all those presenting with otherwise undiagnosed pre-senile dementing or ataxic illnesses. Peripheral sensory symptoms and muscle weakness appear almost universal during the course of P102L IPD. The demonstration of muscle denervation and axonal sensorimotor neuropathy suggest peripheral neurological pathology is responsible. Experimental mice over-expressing wild-type PrP have shown demyelinating peripheral nerve pathology (Westaway et al., 1994). However, evidence of peripheral neuropathy in P102L IPD was inconsistent and where present was axonal, a form that is not uncommon in the population from unrelated causes. No prion protein (scrapie isoform) positivity could be demonstrated on two muscle biopsies examined here and although an indirect pathological process could still be responsible, peripheral findings in P102L IPD may be incidental, while sensory symptoms could be centrally rather than peripherally driven. Presented for the first time here is the finding that P102L IPD patients with methionine homozygosity at codon 129 present significantly earlier than codon 129 heterozygotes. Earlier age at onset is well recognized in codon 129 homozygotes in other IPD mutations (Collinge et al., 1992; Poulter et al., 1992; Mead et al., 2006), although the association with IPD associated with point mutations rather than OPRIs is less clear (Dlouhy et al., 1992). The less strong effect in P102L with respect to 6-OPRI may explain why this observation has not been made before where smaller numbers of patients have been examined (Hainfellner et al., 1995; Barbanti et al., 1996). There are several possible mechanisms of action of codon 129 genotype on clinical phenotype. It has long been known that the interaction between prion protein, scrapie isoform (PrPSc) and prion protein, normal cellular isoform (PrPc) occurs most efficiently when the proteins have an identical primary structure (Palmer et al., 1991); therefore, prion replication may occur more rapidly and clinical onset earlier in 129MM individuals. As a further complexity, the primary structure of PrP determines the permissible conformations of PrPSc. The extent to which a pathogenic 102L-129M PrP conformer is permitted by wild-type PrPc with 129V or 129M may also be important [see Collinge, 2007 for a recent review (Collinge and Clarke, 2007)]. 102L-129M PrP may be able to adopt several different pathogenic conformations, which may be permissible to a greater or lesser extent by the wild-type protein (Parchi et al., 1998; Hill et al., 2006). The involvement of wild-type protein is known to be a variable phenomenon in P102L and a possible determinant of phenotype (Wadsworth et al., 2006). The identification of the P102L mutation on a methionine allele in all of the patients presented here with adequate haplotype data probably relates simply to the frequency of this allele in the background population. P102L patients existing on a codon 129 valine allele have been reported, apparently occurring in the context of a distinct phenotype with prominent psychiatric features and seizures, different from classical P102L codon 129 methionine patients (Young et al., 1997; Bianca et al., 2003). Such a phenotypic difference could relate to different prion strain propagation originating from the valine allele. Genotype–phenotype correlations presented here, corrected for codon 129, demonstrate an effect of APOE genotype on age at onset, with individuals carrying the E4 allele having a significantly later age at onset than those without. While this evidence appears in contrast to the strong association of E4 with risk of Alzheimer’s disease (Corder et al., 1993) and to published work on the possible impact of APOE polymorphisms in sCJD showing an overrepresentation of the E4 allele in sCJD patients (Amouyel et al., 1994; Van Everbroeck et al., 2001), such findings have not been replicated in all published studies (Nakagawa et al., 1995; Zerr et al., 1996) nor in our own observations (387 sCJD patients versus unaffected controls, unpublished data). Recent reports present a similar result in frontotemporalobar degeneration, with later disease onset in those with the APOE E4 genotype in the context of progranulin mutations (Gass et al., 2006; Beck et al., 2008). APOE may thus have contrasting effects in the context of different neurodegenerative disease types. Most of the P102L IPD patients shared common features on pathological analysis. Spongiform change, astrocytosis and PrP deposition, both as multicentric plaques and synaptic deposits, were seen in the majority. However, the degree of severity of spongiform change and plaque deposition were very variable. This did not seem to correlate with age at onset or duration of illness. Neither did predominantly cerebellar or cognitive clinical presentations seem to correlate with pathological findings. PRNP codon 129MM homozygotes and MV heterozygotes were equally represented in the pathology series but no significant differences were seen between these two groups. However, the small Inherited prion disease P102L Brain (2008) Page 13 of 15 numbers in each subgroup and the age of some of the samples examined (with tissue up to 30 years old) may prevent correlations from being made. A single highly atypical clinical patient showed no significant histological differences from the rest of the series, while an atypical pathological patient has limited clinical data, although what is known about this individual is not obviously different from the other patients examined. These results suggest no clear clinicopathological correlations, but sample size was necessarily small. Immunoblots of proteinase K-digested brain homogenate from P102L patients demonstrate a spectrum of involvement of protease-resistant wild-type PrP in P102L IPD (Wadsworth et al., 2006). Four of the seven P102L IPD patients examined in this study were negative for prion protein (scrapie isoform), which most probably relates to sampling issues and the differing density of PrP deposition in tissue samples both within and between patients. Our large study therefore does not expand the existing data and we were unable to test whether protease-resistant PrP diversity might contribute to clinical heterogeneity. This remains a plausible concept in IPD that requires a large series of fresh frozen brain tissue and molecular analysis of PrP type following partial protease digestion. The need for public health control measures, together with the evident diagnostic challenges that IPD heterogeneity causes, make a strong argument for including PRNP gene analysis in he list of investigations for suspected prion disease of any type and indeed of all undiagnosed familial dementia or cerebellar syndromes. Successful diagnosis allows clinicians to provide more accurate prognostic information to patients, to allow participation in the clinical trials and reduce the risk of iatrogenic transmission of disease. As a consequence of these geographically highly mobile ancestors, and the large number of untraced individuals in the nineteenth century who were clearly at risk of inheriting the P102L mutation, it remains likely that further patients and at-risk individuals exist who have yet to be identified. It is hoped that the data presented here will help to raise awareness of P102L IPD and its associated presentations.


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