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Inherited prion disease with six octapeptide repeat insertional mutation—molecular analysis of phenotypic heterogeneity

Simon Mead, Mark Poulter, Jon Beck, Thomas E. F. Webb, Tracy A. Campbell, Jacqueline M. Linehan, Melanie Desbruslais, Susan Joiner, Jonathan D. F. Wadsworth, Andrew King, Peter Lantos, John Collinge
DOI: http://dx.doi.org/10.1093/brain/awl226 2297-2317 First published online: 21 August 2006

Summary

By far the largest known kindred with an inherited prion disease caused by a prion protein (PrP) octapeptide repeat insertion mutation originates from southeast England. This extended family shows very marked phenotypic heterogeneity and provides a unique opportunity to characterize this diversity and examine possible modifying factors amongst a large number of individuals in whom prion disease has been initiated by the same defined genetic mutation. As the inherited prion diseases comprise a significant proportion of familial early-onset dementia, an appreciation of their wide range of clinical presentation is important for differential diagnosis. Genealogical and clinical record review, together with the characterization of the mutation-linked single nucleotide polymorphism and microsatellite haplotype, suggested a single founder for both this large kindred and a smaller family in the mid-18th century. Here we report the phenotype of 86 affected individuals; at least another 84 individuals are known to be at risk of inheriting the disease. Clinical onset, typically with cognitive impairment, can be strikingly early in this kindred when compared with other inherited or sporadic prion diseases. We have investigated the effect of PrP genotype, candidate genes and prion strain type on clinical, neuroradiological and neuropathological phenotype. The transmission characterisitics of prions from affected individuals resembled those of classical sporadic Creutzfeldt–Jakob disease. One surprising finding was a strong inverse correlation between age of onset and disease duration. The PrP gene polymorphic codon 129 was found to confer 41% of the variance in age of onset but interestingly this polymorphism had no effect on disease duration suggesting different molecular mechanisms are involved in determining disease onset and rate of clinical progression.

  • 144 bp
  • CJD
  • early-onset dementia
  • insertion
  • OPRI
  • prion

Introduction

Prion diseases are biologically unique with inherited, sporadic and transmissible aetiologies (Collinge, 2001). Inherited forms comprise 15% of recognized prion disease, and a significant proportion of familial early-onset dementia (Finckh et al., 2000). Inherited prion disease may be caused by point mutation in the C-terminal domain of the prion protein (PrP), premature STOP codon mutation or insertion of additional octapeptide repeats in the N-terminal domain (Collinge and Palmer, 1997).

The normal prion protein gene (PRNP) octapeptide repeat region lies between codons 51 and 91, and comprises a nonapeptide followed by a tandem repeat of four copies of an octapeptide. The normal structure of the five repeats has been designated R1-R2-R2-R3-R4 (Goldfarb et al., 1991). R2, R3 and R4 are each distinguished from R1 by non-coding nucleotide differences. The region contains a high affinity binding site for copper (Jackson et al., 2001), but the functional significance of this is unknown. Only the central three octapeptide units are inserted by mutation. Mutations of up to nine additional repeats have been characterized; 1- 2- or 3-OPRI (octapeptide repeat insertion) mutations are single case reports; none have been described in large prion disease pedigrees, and as a result the evidence that they are pathogenic remains questionable (Goldfarb et al., 1993; Laplanche et al., 1995; Van Harten et al., 2000). 4-OPRI or larger have been described in families with autosomal dominant inherited prion disease (Oda et al., 1995; Van Gool et al., 1995; Cochran et al., 1996; Laplanche et al., 1999).

Typically, OPRI-associated prion disease manifests as a slowly progressive multifocal dementia often with cerebellar ataxia. Considerable variation between individuals within the same family is consistently reported, in terms of both clinical features and age of onset. Some families however, with identical mutant proteins, appear to have consistently atypical clinical features. Laplanche has reported a large pedigree with predominantly psychiatric features and an 8-OPRI (Laplanche et al., 1999). Moore has reported a large family with a Huntington's disease phenocopy and an 8-OPRI (Moore et al., 2001). It is difficult to draw firm conclusions however, as different clinicians may place emphasis on different clinical features and case series are small.

The existence of a further clinical variant, a premorbid personality disorder, characterized by aggression, hypersexuality and antisocial behaviour has been proposed (Collinge et al., 1992). The existence of this personality disorder remains an unresolved but important issue. An abnormality detectable early in life might suggest a neurodevelopmental problem, perhaps related to a hemizygous loss of normal function of the PrP and a phenotype related to loss of normal synaptic transmission, abnormal neurodevelopment or copper metabolism. There have been other reports of abnormal personalities in OPRI families: some patients in the Laplanche pedigree (Laplanche et al., 1999) had very prolonged psychiatric prodromes to neurological illness but the author did not conclude these were similar to the personality disorder described in (Collinge et al., 1992). Cochran et al. (1996), reported a family in which two members had premorbid personalities very similar to that described by Collinge et al. (1992). These authors suggest that personality disorder is an early sign of disease rather than caused by a life-long loss of protein function. Goldfarb has also described a similarly abnormal personality (Goldfarb et al., 1991). Small numbers and the problems with retrospective opinions of different clinicans complicate these reports.

Three reports concern the large UK 6-OPRI family investigated in this paper (Owen et al., 1989; Collinge et al., 1992; Poulter et al., 1992). Genealogical investigation in the early 1990s identified a common ancestor of four families living in southeast England with a dementing illness. The cloned OPRI was sequenced in four individuals; all carrying a 1222323g2234 sequence of repeats, for comparison, other 6-OPRI reports are shown in Table 1. A strong genetic susceptibility factor for all prion diseases, the polymorphic codon 129 genotype, was available from 13 affected individuals from the large family. Using this necessarily small sample size, an association between codon 129 genotype and age of onset was found.

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Table 1

Reports of 6-OPRI inherited prion disease

Lead authorYearCountry of originInsertion typeNumber of patients reportedLinked codon 129 allele
Poulter/Collinge1992UK2323g2247M
Nicholl1995UK323g23g2>2M
Oda1995Japan3g223g226M
Capellari1997Spain2222223M
Gelpi2005Austria2222221M

Here we apply advances in prion disease molecular biology to an updated and expanded pedigree in order to further our understanding of the molecular basis of phenotypic variability. We were able to quantify the variation in phenotype attributable to the codon 129 polymorphism with implications for genetic counselling. We present the molecular strain typing (Hill et al., 2003) in the family. The disease has also been transmitted from affected family members into wild-type and transgenic mouse models. Candidates for genetic modification of phenotype [PRNP codon 129, the PRNP single nucleotide polymorphism (SNP) haplotype and apolipoprotein E gene (APOE) genotype] have been examined.

Methods

Genealogy and clinical ascertainment

This study was approved by the Institute of Neurology/National Hospital for Neurology and Neurosurgery Local Research Ethics Committee. Genealogical work was carried out with the assistance of the Family Record Centre, London. Clinical records were obtained with the consent of the local medical record management. Family members were considered as affected if there was evidence of a dementing disease with onset prior to age 60 in the absence of an alternative cause.

Molecular genetics

The 6-OPRI mutation in the large pedigree was characterized previously (Poulter et al., 1992). A molecular diagnosis was made in known members of the large pedigree by size discrimination of a PCR amplicon on agarose gels. In affected individuals not obviously related to the large family, the mutation was characterized in one of three ways. First and most simply, by discrimination of mutant and wild-type allele on a single automated fluorescent sequencing electropherogram trace (Applied Biosystems-377). Second, by subcloning the mutant allele as described previously (Campbell et al., 1996). Third, by sequencing of an allele specific PCR amplicon in individuals heterozygous for methionine-valine at codon 129. This methodology uses primers flanking the octapeptide repeat region with the reverse primer positioned such that the most 3′ nucleotide of the primer anneals to the position of the polymorphic nucleotide of codon 129 allowing for specific amplification of the 6-OPRI allele.

SNP genotypes were determined by restriction digestion as described previously (Mead et al., 2001). For individual family members, SNP haplotypes were determined empirically, a methodology that has been validated by comparison with pedigree based ascertainment in control samples (Mead et al., 2001).

Microsatellite polymorphism allele sizes were determined by size discrimination of fluorescent labelled PCR amplicon (Applied Biosystems-377). Allele sizes were standardized according to the most frequent allele found in the Centre d'Etude du Polymorphisme Humain (CEPH) database. In the results tables, microsatellites are displayed 5′–3′ left–right, with PRNP situated between D20S97 and D20S895.

The location of SNP and microsatellite markers relative to PRNP are shown in Table 2.

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Table 2

Neuropathology

The neuropathology from many of these cases has been reported previously (Collinge et al., 1992; King et al., 2003). Blocks from the formalin fixed brain from the following areas were sampled where available: frontal lobe, temporal lobe (including hippocampus), medial occipital lobe, parietal lobe, caudate nucleus, lentiform nucleus, thalamus, cerebellum (vermis and hemisphere), midbrain, pons and medulla oblongata. These blocks were decontaminated in 98% formic acid for 1 h prior to processing and embedding in paraffin wax.

In cases where only limited blocks were available those closest matching the above blocks were used. All blocks were cut at 7 μm thickness and stained with Haemotoxylin and Eosin and with immunohistochemistry for PrP antibody, 3F4 (1/1500 dilution overnight, Signet Laboratories, MA), and/or 12F10 (1/250 dilution overnight, gift of Professor Hunsmann, German Primate Centre, Goettingen), using the ABC immunohistochemical technique. Pretreatment protocols for PrP staining involved autoclaving in distilled water at 121°C for 5 min followed by 98% formic acid for 5 min then 4 M guanidine thiocyanate for 2 h at 4°C.

PrP immunoblotting

10% (w/v) brain homogenates were prepared in Dulbecco's phosphate-buffered saline (PBS) lacking Ca2+ or Mg2+, or lysis buffer (10 mM Tris, 10 mM EDTA, pH 7.4 containing 100 mM NaCl, 0.5% w/v NP-40 and 0.5% w/v sodium deoxycholate) by serial passage through needles of decreasing diameter (Collinge et al., 1996; Wadsworth et al., 1999; Hill et al., 2003). Aliquots were analysed with or without proteinase K digestion (50 μg/ml final protease concentration, 1 h, 37°C) by immunoblotting using anti-PrP monoclonal antibody 3F4 (Kascsak et al., 1987) as described previously (Wadsworth et al., 2001; Hill et al., 2006).

Transmission studies

Care of mice was according to institutional guidelines. Transgenic mice homozygous for a human PrP 129V transgene array and murine PrP null alleles (Prnpo/o) designated Tg (HuPrP129V+/+ Prnpo/o)-152 mice, (129VV Tg152 mice) (Collinge et al., 1995; Hill et al., 1997; Wadsworth et al., 2004) and inbred FVB/NHsd mice were inoculated intracerebrally with human brain homogenates using methods described previously (Asante et al., 2002). Mice were examined daily and were killed if exhibiting signs of distress or once a diagnosis of clinical prion disease was established (Carlson et al., 1986). Brains from all inoculated mice were analysed by immunoblotting and/or neuropathological examination. Table 4 reports attack rate as the total number of both clinically affected and sub-clinically infected mice as a proportion of the number of inoculated mice.

Statistical analysis

Statistics were performed using the SPSS package 12.0.1 for Windows (SPSS). Means were compared using the independent samples t-test. Z-scores for each codon 129 genotype were calculated using Excel (Microsoft). Linear regression and survival statistics were performed using SPSS. Histograms with normal distributions or cumulative normal curves were plotted in SPSS and modified in Adobe Illustrator.

Results

Genealogy

Historical genealogy and clinical research was pursued at the Family Records Centre, London and various hospitals and other sources of medical records to establish common ancestry between the previously published pedigree and four further families. Two families were linked to descendents of III.2 and one family linked to descendents of V.10; the details are presented below as part of pedigree 1 (Fig. 1). Common ancestry between the fourth family (pedigree 2) and pedigree 1 could not be established by genealogy. The first generation individuals of both these pedigrees were born less than five miles from one another.

Fig. 1

The UK 6-OPRI prion disease pedigree The smaller pedigree 2 shares the same microsatellite and SNP haplotype background as the larger pedigree. Only affected individuals (filled symbol) or possibly affected (question mark) are shown, vertical bar through symbol denotes unknown affected status but transmitted the mutation. If not already reported in full (Collinge et al., 1992), case reports for each deceased individual are given in the Appendix.

Update of the 6-octapeptide repeat family

Clinical and investigation details are provided in the Appendix for deceased affected individuals, some are updated from the previous paper (Poulter et al., 1992), in which case the original pedigree number is provided in parentheses. Details of living patients have been omitted to preserve anonymity. Details of many of the autopsied cases have been published separately (King et al., 2003).

Clinical features are summarized in a phenotype table (Table 3). The core clinical feature remains a progressive cortical dementia often presenting with aggressive or apathetic behavioural change suggestive of frontal lobe dysfunction. Detailed neuropsychological assessments in the early stages of disease were infrequently obtained. Despite this, a deficit in episodic memory loss, semantic or expressive language dysfunction, apraxia and dyscalculia were frequently observed early neuropsychological features. Cerebellar ataxia and pyramidal tract signs were the most common additional neurological signs. Some individuals were clinically diagnosed with Huntington's disease during life (VI.5, VI.23 for example). The most commonly noted extrapyramidal signs were reduced arm swing, truncal or limb rigidity, dystonic posturing, and less commonly, chorea. Other differential diagnoses offered included Alzheimer's disease, Parkinson's disease, Pick's disease, frontotemporal dementia, Unverricht–Lundberg disease (VI.3), progressive myoclonic epilepsy, and in earlier cases, disseminated sclerosis or general paralysis of the insane. On at least one occasion the progression of disease was so rapid that the diagnosis made was classical or sporadic Creutzfeldt–Jakob disease (CJD) (V.11). Aside from myoclonus, which was frequently documented, epileptic seizures were uncommon.

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Table 3

Phenotype table

Pedigree numberSexAge at onset(years)Age at death (years)Duration (years)Codon 129 wild-type alleleWild-type haplotypeApoE genotypePremorbid personality disorderPersonality/PsychiatricDementiaMyoclonusPyramidal signCerebellarExtrapyramidal featuresSeizuresChorea
II.1F47++
II.2F61+++
II.3M32364++++
III.2F62++
III.3M36393+++
III.5F38446++++++
III.6M37++
III.7M39++
IV.1F42++
IV.3F58++++
IV.5M395718++
IV.6F39401++++++
IV.7F263913+++++++++++++
IV.8F54++++++
V.1F46++
V.2F43463+++
V.4F41(++)
V.5F43++
V.6M56++
V.7M35++
V.8F324311+++
V.9F53574+++++++++
V.10F40(++)++
V.11M36382++++++++
V.12M48524V+++++++
V.13F38468+++++++++++++++
VI.1F53++
VI.2F46++++
VI.3F26304++++++
VI.4M283810+++++
VI.5F38++++
VI.6F45505VRare+++++++
VI.7F28379M++++++++
VI.8F44++
VI.9M40++
VI.10M46++++++++++
VI.11F46++
VI.13M43++
VI.14F38413+++
VI.15M39489++++++
VI.16M435411VA2,4+++++++
VI.17M34439M+++++++
VI.18F36459++++++++
VI.19F47525++++
VI.20M46559VA3,4++++++++
VI.21M40455+++++++++++
VI.22M49512MD3,4+++++
VI.23F335219MB3,3+++++++++++
VI.24F32419+++++++
VI.25F42++++
VII.1F34439++++++++++
VII.2F405313V3,3+++++++++
VII.3F476518VA3,3+++++++
VII.4F37458++++
VII.5M324412MB3,3++++++++++++
VII.6M324210MB3,4++++++++++
VII.7F31409MB3,3++++++++++
VII.8F233916M+++++++++++++
VII.9F203515MD2,3++++++++++
VII.10F37ME++++
VII.11F30ME++++++++
VII.12F24M++++++++-
VII.13F325321MB3,4+++++
VII.14M314514MB3,4++++++
VII.15F364610MB++++-++++
VII.16M30MB+++++++++
VII.17M42420V+++++
VII.18F31VL++++++++++
VII.20M28ME+++++++++
VII.21F233815MB3,3++++++++++++++
VII.22F34M+++++
VII.23M30ME+++++++++
VII.24F36MR+++++
VII.25M35449MB3,3+++++++
VII.26M243612MD3,3+++++++++++
VII.27M31398MB3,3++++++++++
VII.28M304414MB3,4+++++++
VIII.1M27336MK++++++++++
VIII.2M35MD+++++++++++
VIII.3F30M+++
2III.1F384911V++
2III.2F425715+++++++
2III.3F32397++++++
2IV.1F37447ME3,4++++
2IV.2M39MB+++++
2IV.3F37V++
  • Phenotype table of the large UK 6-OPRI family.−denotes an absent feature, + denotes a mildly present feature, ++ denotes a moderate-severe feature. (++) denotes a probable feature but clinical records were not complete.

There was marked variation in clinical phenotype between individuals. This is best exemplified by case history. Compare the aggressive disease in V.11 and his two sons, VI.21 and VI.22 [mean duration of disease 3 years (range 2–5)] with the slowly progressive disease in three sisters 2III.1–3 [mean duration of disease 11 years (range 7–15)]. This clustering of phenotype in first-degree relatives prompted us to investigate the heritability of disease onset and genetic modifying factors. The age of onset and death data fit a normal distribution using standard statistical tests (Figs 2 and 3). The mean age of onset was 34.9 years (range 20–53, SD = 6.93, n = 63), mean age of death was 45.1 years (range 30–65, SD = 7.3, n = 73).

Fig. 2

(A) Cumulative age of onset and age at death curves; (B) cumulative age of onset curves for the two genotypes at codon 129. Data are shown for each individual patient onset and death, together with a normative curve (SPSS).

Fig. 3

(A) Age at onset and (B) age at death histograms with curves modelling the data assuming a normal distribution. Genotypes at codon 129 are not available for the majority of the deceased patients. 129MV patients comprise the majority of those presenting aged over 40 years or dying aged over 50 years.

Of historical interest is possibly the first autopsy case of prion disease, although this was not histologically studied at the time (histological features of CJD were not defined until much later) (Fig. 4). Presentation was in 1895 with severe post-natal depression and abnormal behaviour. She progressively demented and died, aged 44. Autopsy showed a markedly atrophic brain and cerebral convolutions. Histology of brain was undertaken, but the ‘fresh method was not good as the brain was friable.’ Cause of death was stated to be an ‘organic disease of the brain.’

Fig. 4

Of historical interest is an autopsy report of an individual in the pedigree (III.5) who was admitted in 1897 and died in 1901. She was noted to have a poor memory and a heritable disease. Underlined in the original report were the ‘convolutions which were markedly atrophied.’ The hemispheres each weighed 13 oz, the cerebellum 4 oz and the pons and medulla ¾ oz. Unfortunately the histology was not useful as the ‘fresh method not good as brain friable’ although the sections showed ‘degeneration of the large pyramids.’ Conclusion: ’Organic disease of brain.’ Figure courtesy of West Sussex Record Office.

The existence of an early premorbid personality disorder in this kindred was not strongly confirmed but needs formal evaluation and further study is planned. The present assessment is based on an absence of documented premorbid symptoms in clinical reports and could not compare to a detailed psychiatric and personality assessment in the presymptomatic phase. It is also possible that recent changes to allow open access of patients to their medical records may have led to underreporting of some of these features. Of interest, one family member presented to the National Prion Clinic displaying behavioural changes thought suggestive of the previously described personality disorder but was found to be negative for the mutation on testing.

The Department of Health have recommended that individuals with prion disease have precautions taken with surgery, dental work and other invasive procedures in order to avoid iatrogenic transmission. It is notable that a significant number of patients in the 6-OPRI pedigree (VI.4, VI.19, VII.10, VII.15, VII.18 and VIII.2) had procedures undertaken without necessary precautions, including dental and general surgery. In addition, although a family history of inherited prion disease is routinely sought by screening questions by the Blood Transfusion Service, several patients and at-risk family members have been regular blood donors until appropriately counselled. All affected and at-risk individuals are counselled on infection control issues and asked to notify other at-risk family members. These incidents have required referral to the CJD Incidents Panel (Health Protection Agency).

Imaging, EEG and other investigations

Neither routine CT nor MRI of the brain showed any specific diagnostic features, only generalized cerebral and cerebellar atrophy (see Fig. 5). A time series of volumetric MRI has been obtained in a limited number of affected family members, showing a gradually progressive atrophy of cerebrum and cerebellum (details to be published separately).

Fig. 5

MRI appearances of 6-OPRI prion disease These show cerebral and cerebellar atrophy (A) Coronal T1 at the level of the hippocampus, (B) Axial T2 at the level of the basal ganglia and (C) Sagittal T1 (all from patient VII.20).

Similarly EEG typically showed either non-specific abnormalities such as low amplitude, an excess of theta or was normal. In two cases, spike and wave abnormalities led to a diagnosis of myoclonic epilepsy.

CSF examination for basic constituents, performed in three patients, was unremarkable. Other proteins which may be elevated in prion disease, such as 14-3-3 and S100b were not looked for in these samples. Peripheral nerve conduction, performed in one patient, was normal.

Pathology

Thirteen cases were available for examination (in one case only a small number of blocks were available). The 12 brains examined neuropathologically varied in weight between 804 and 1375 g. The histopathology revealed variable spongiosis and astrocytosis, not only between different cases but also between different areas of the same brain. All cases except VII.1 showed extensive deposition of PrP in the molecular layer of the cerebellum in the form of patches mainly orientated perpendicular to the pial surface (Fig. 6A). The exception VII.1 showed deposition in the form of occasionally congophilic ill-defined plaques, perineuronal and synaptic granular staining in the granular and molecular layer (Fig. 6B). In seven cases, there were immunopositive dense plaque-like deposits within the parahippocampal gyrus and/or subiculum. In addition to this in one case, there was strong immunopositivity within the occipital lobe. Only two of the cases examined neuropathologically had a codon 129MV genotype. The histology of these cases showed the typical PrP patches in the molecular layer of the cerebellum.

Fig. 6

Neuropathology from two autopsy cases of 6-OPRI prion disease. PrP immunohistochemistry using anti-PrP monoclonal antibody 3F4. (A) Cerebellum with deposition of PrP in the molecular layer in the form of patches, as exhibited in the majority of cases studied neuropathologically Scale bar = 100mm (B) Cerebellum with deposition of PrP in the form of plaques (arrows), synaptic and perineuronal staining as seen in one case. Inset shows high power of small poorly-formed plaque in the molecular layer. Scale bar = 75mm (inset 20mm).

The SNP haplotype background of 6-OPRI inherited prion diseases

All affected family members with DNA available for molecular genetic analysis possess at least one of the less frequent SNP alleles immediately upstream of PRNP exon 1: 1368C and 10870A (positions refer to clone U29185, GenBank). The 6-OPRI mutation in the large family therefore occurs on the common European methionine haplotype designated B (Mead et al., 2001). This haplotype is overrepresented amongst patients with sporadic prion disease (Mead et al., 2001). The prion-like doppel (PRND) gene polymorphism on the mutation chromosome, although not associated with prion disease susceptibility (Mead et al., 2000), was studied and the more common 174T allele was found in all patients studied (n = 26).

The microsatellite haplotype background of OPRI mutations

Thirteen microsatellites linked to PRNP (5′–3′ D20S181, 193, 473, 867, 889, 116, 482, 97, PRNP, 895, 849, 873, 95, 194) were genotyped, their locations relative to PRNP codon 129 are shown in Table 2 along with alleles of affected individuals. The 6-OPRI mutation occurs on a 156-152-178-279-268-107-159-271-214-228-191-84-202 haplotype (in order of marker above). This haplotype is also shared by the small 6-OPRI pedigree 2. Assuming a genetic:physical ratio of 3.7 in the region of PRNP and considering the rarity of the mutation-linked alleles at the 5′ and 3′ extremes of the haplotype and a physical distance of 3 Mb across the haplotype, then the chance of disruption of the mutation-linked haplotype by recombination is ∼10% per generation, giving a ∼50% chance of disruption of haplotype in six generations. There are wide confidence intervals around estimation of the most recent common ancestor, but our genetic data supports a common ancestor within the documented history of the large pedigree. Although a common ancestor between the smaller family and the larger family could not be identified genealogically, the sharing of OPRI mutation type, an identical linked SNP and microsatellite haplotype, and geographical proximity of the families is strong evidence for a single ancestral mutation.

The three additional families with an identical 6-OPRI, which were incorporated into pedigree 1 by genealogy were unsurprisingly also found to share the disease microsatellite haplotype. None of the unaffected family members or the predicted wild-type haplotypes of the affected individuals possess the mutation-associated haplotype. It is also notable that no other insertion mutation characterized at the Medical Research Council (MRC) Prion Unit shares this haplotype background, the important implication being that the 6-OPRI mutation is stable over many generations. The insertion mutation stability may be contrasted with insertion mutations with smaller repeat lengths such as the triplet repeat disorders, which may show anticipation associated with an increase in repeat length over successive generations.

Two recombinations were seen in branches of the larger pedigree. They are shown in Table 2. A 5′ recombination was found in two descendants of V.9 between 116 and 482 (160–614 kb 5′ to codon 129) probably affecting four individuals in total. A 3′ recombination was found in three descendants of IV.7 between 849 and 873 (527–930 kb 3′ to codon 129) probably affecting 14 individuals in total. There are data from mouse and human studies in support of a PRNP-linked susceptibility factor in addition to that conferred by amino-acid coding changes at PRNP (Manolakou et al., 2001; Mead et al., 2001; Lloyd et al., 2002; McCormack et al., 2002). We therefore compared the age of onset and death in the recombinant lines with the remainder of the pedigree hypothesising that a linked susceptibility factor might modify inherited prion disease phenotype, and that if a recombination changed the linked susceptibility allele then a modified phenotype might be shared in all affected descendents of the recombinant. In order to take into account the strong modifying effect of codon 129, the age of onset and death were modelled as normal distributions for each 129 genotype. Patient data from recombinant branches of the pedigree were analyzed by Z-score (the distance of each individual's age of onset or death from the pedigree mean, expressed in units of the standard deviation, positive values reflecting a later onset or death for the recombinant individuals). For the 5′ recombinants the Z-score for onset was 0.23 (n = 4), Z-score for death was 1.08 (n = 3). For the 3′ recombinant line the Z-score for onset was 0.26 (n = 8), Z-score for death was 0.55 (n = 4). None of the observations for recombinants were statistically significantly different from the rest of the pedigree.

Duration of disease correlates with age of onset

Patients presenting at an early age tended to have a longer disease duration when compared with older age of onset patients [Fig. 7, for deceased individuals Pearson correlation, age of onset (years) versus duration (years) r = −0.377, P = 0.007]. This is illustrated by Kaplan–Meier survival curves for four quartiles of age of onset (Fig. 8), incorporating individuals surviving to date. Surprisingly, there was not a similar correlation between duration of disease and age of death.

Fig. 7

Pearson correlation of age of onset versus duration of disease. These data are significantly correlated, r = −0.377 (P = 0.007), n = 50.

Fig. 8

Kaplan–Meier survival curves. Younger presenting patients with 6-OPRI prion disease survive longer, although there are individuals in the older presenting group that do survive over a decade. The youngest and oldest presenting quartiles are shown (total n = 63).

PrPsc types in 6-OPRI disease

The PrPSc type present in the brain of case VII.2 (PRNP codon 129MV) or VI.23 (PRNP codon 129MM), respectively (Fig. 9), was identical to PrPSc type 2 or PrPSc type 3 seen in sporadic CJD using the London classification of PrPSc types (Hill et al., 2003). Neither VII.2 nor VI.23 had an unusual disease phenotype (see Appendix). PrPSc was undetectable in other available frozen samples of human 6-OPRI frontal cortex (VII.25, VI.17, VII.21). In this regard it is notable that immunohistochemisty of the 6-OPRI cases shows PrP is more readily detected in the cerebellum than frontal cortex.

Fig. 9

Molecular PrPSc typing in 6-OPRI prion disease (A) Immunoblot with anti-PrP monoclonal antibody 3F4. Following limited proteinase K digestion of brain homogenate, PrPSc types from0 two 6-OPRI patients appear identical to PrPSc types observed in sporadic CJD cases of same PRNP codon 129 genotype. (B) The MRC Prion Unit designates three common PrPSc types (1–3) in sporadic CJD (Collinge et al., 1996; Wadsworth et al., 1999; Hill et al., 2003) that can be distinguished by differing fragment sizes of the three PrP glycoforms, corresponding to amino-terminally truncated protease cleavage products generated from di-, mono-, or non-glycosylated PrPSc.

Transmission of 6-OPRI disease

Table 4 reports the transmission properties of three isolates (VII.2, codon 129MV; VII.25, codon 129MM; VI.17, codon 129MM) in transgenic mice expressing human PrP with 129-valine (129VV Tg152 mice) and wild-type FVB mice (Prnp allele a). 129VV Tg152 mice lack a transmission barrier to classical (sporadic or iatrogenic) forms of CJD, regardless of the codon 129 genotype of the inoculum (Collinge et al., 1995, 1996; Hill et al., 1997), but show a marked barrier to transmission of BSE or vCJD prions that persists even on sub-passage (Hill et al., 1997; Wadsworth et al., 2004). Conversely, BSE and vCJD prions transmit disease much more efficiently to wild-type FVB mice than to 129VV Tg152 mice (Hill et al., 1997; Wadsworth et al., 2004). The transmission properties of 6-OPRI isolate VII.2, (codon 129MV, I029) closely resembled that of classical CJD isolates with the same PRNP codon 129 genotype (Hill et al., 1997), with a 100% attack rate in 129VV Tg152 mice compared with an incomplete transmission in wild-type FVB mice (Table 4). The mean incubation period of 217 ± 8 days in 129VV Tg152 mice seen with VII.2 is similar to that seen for comparable sporadic CJD isolates, 218 ± 2 days (Hill et al., 1997). VII.25 and VI.17 that showed no detectable PrPSc in 10 μl 10% brain homogenate by standard immunoblot analysis, transmitted poorly in both transgenic and wild-type mice (Table 4).

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Table 4

Primary transmission of human 6-OPRI isolates to transgenic 129VV Tg152 and wild-type FVB mice

Human inoculaa129VV Tg152 miceWild-type FVB mice
Attack ratebIncubation periodcAttack ratebIncubation periodc
VII.2 (I029)4/4d217 ± 84/5202, 498
VII.25 (I1094)5/13192 ± 42/8223, 532
VI.17 (I1095)0/111/9701
  • aVII.2 (I029), frontal cortex, PRNP 129MV with type 2 PrPSc detected in 10 μl 10% inoculum. VII.25 (I1094), frontal cortex, PRNP 129MV with no detectable PrPSc in 10 μl 10% inoculum. VI.17 (I1095), frontal cortex, PRNP 129MM with no detectable PrPSc in 10 μl 10% inoculum.

  • bAttack rate is defined as the total number of both clinically affected and sub-clinically infected mice as a proportion of the number of inoculated mice. In mice that were asymptomatic for clinical prion disease, sub-clinical prion infection was assessed by immunoblotting and/or immunohistochemical examination of brain.

  • cIncubation periods are reported for clinically affected animals in days; where n ≥ 3 the mean ± SEM is reported.

  • dAll mice showed clinical signs of prion disease and had detectable PrPSc in brain.

Gender effect

There was no sex difference for age of onset [female mean = 34.7 years, SD = 7.2 (n = 37), male mean = 35.2 years, SD = 6.6 (n = 26)]; age of death [female mean = 46.0 years, SD = 7.6 (n = 45), male mean = 43.9 years, SD = 6.7 (n = 26)]; or duration of disease [female mean = 10.2 years SD = 5.1 (n = 27), male mean = 7.8 years SD = 4.9 (n = 18)]. 34 males transmitted the mutation to their children on 26 occasions, 46 females transmitted the mutation on 60 occasions. Males transmitted the mutation to 11 men and 15 women, females transmitted the mutation to 21 men and 39 women (sex distortion not significant by χ2, P = 0.63).

Genetic modification of the 6-octapeptide repeat phenotype

The age of onset was earlier with a methionine allele at codon 129 on the normal chromosome, compared with a valine allele [129MM mean age of onset = 31.4, SD = 5.7 years (n = 30), 129MV mean age of onset = 41.7, SD = 5.3 years (n = 10), independent samples t-test P = 1.2 × 10−5]. The mean difference between 129MM and 129MV age of onset was 10.3 years (95% confidence interval of the difference 6.2–14.5). The η2 function of ANOVA (SPSS Package, SPSS) is a measure of the proportion of the total variance of a dependent variable accounted for by the effect of a categorical factor. For analysis of codon 129 and age of onset, η2 = 0.41, indicating that the genotype of codon 129 accounts for 41% of the variance in age of onset. Less data was available for age of death but a similar effect was seen [129M mean age of death = 42.4 years, SD = 5.6 (n = 19), 129MV mean age of death = 52.5, SD = 6.5 years (n = 8), independent samples t-test P = 0.001]. The mean difference between 129MM and 129 MV age of death was 10.1 years (95% confidence interval (95% CI) = 5.0–15.2). Mean duration of disease, in patients where onset and death were known was 9.0 years (SD = 5.0, n = 50). Surprisingly, there was no evidence in favour of prolonged disease duration in 129MV [129MM mean duration 11.4 years (n = 19), 129MV mean duration 8.9 years (n = 8), independent samples t-test P = 0.23]. This finding is not confounded by the correlation between age of onset and duration of disease. Codon 129 genotype was not associated with other aspects of clinical phenotype.

In comparison with inherited prion disease caused by point mutation, the effect of the codon 129 polymorphism in 6-OPRI disease is striking. A combination of data from our experience and available in the world literature for the common mutations P102L, D178N and E200K suggests no protective effect for codon 129 heterozygosity [Table 5, some data kindly provided by Dr Kovacs (Kovacs et al., 2002)], although for P102L this approaches significance. 6-OPRI prion disease with codon 129MM has a much earlier onset than these point mutation inherited prion diseases.

View this table:
Table 5

Comparison of 6-OPRI and point mutation prion disease

P102LD178NE200K6-OPRI
Number (homozygous/heterozygous)22/1332/1317/330/10
Homozygous age of onset (years)46.850.45831.4
Standard deviation (years)12.41112.85.7
Heterozygous age of onset (years)53.745.758.741.7
Standard deviation (years)7.611.211.75.3
P (homozygous versus heterozygous within mutation)0.0810.190.93<0.0001
P (homozygous versus other mutations)<0.0001
  • In comparison with other frequently occurring inherited prion disease, 6-OPRI is distinct in that there is marked effect of codon 129 on the age of onset. In addition, for homozygous individuals the onset is strikingly early. P-values are shown for independent t-tests. The phenotype data for P102L, D178N and E200K was combined from a literature review (Kovacs et al., 2002) and the database of inherited prion disease at the MRC Prion Unit.

The PRNP haplotype of the normal chromosome was determined in 29 individuals. 13 individuals (out of 24 with 129MM) had haplotype B on the normal chromosome. There were no differences in clinical features between B and other 129M haplotypes of the normal allele (age of onset of B haplotype = 32.0, SD = 3.7 years, age of onset non-B haplotype = 32.5, SD = 7.6 years). There was no association between clinical phenotype and genotype of the common SNP of the PRND open reading from M174T. APOE genotyping in 18 individuals did not demonstrate any association between genotype and clinical phenotype.

The heritability of age of onset was also assessed. If unlinked genetic factors determine a large portion of the residual 59% of the variance of age of onset, this might be evidenced by the correlation of first-degree relatives. Heritability (h2) has been shown for many complex traits, such as IQ and height, although the genetic factors remain largely unknown. A plot of parental Z-score for age of onset or death versus mean offspring Z-score is shown (Fig. 10). The gradient of the line of best fit for these data estimates h2/2 as only the affected parent has phenotype data, hence only half of each child's genetic inheritance. A significant correlation was not found, supporting a role for stochastic and environmental factors in the residual variance of age of onset. The considerable scatter that is frequently seen in heritability plots, even when heritability is high, does not rule out a genetic contribution from unlinked genetic loci.

Fig. 10

Heritability of age of onset. Age of onset or death has been modelled as a normal distribution for each 129 genotype and values were plotted as a Z-score (each individuals distance from the mean measured in units of the standard deviation). Where both age of onset and age of death were available the mean of both Z-scores was used. This was done to account for the known effect of codon 129 on the non-mutated chromosome 20, which is not transmitted to affected children and therefore does not contribute to heritability of age of onset. A dotted regression line is shown, but the data were not significantly correlated.

Discussion

This family has some historical importance given that the first genetic mutation causing a neurodegenerative disease was described in a patient from this pedigree VI.23 (Owen et al., 1989). A SNP and microsatellite haplotype-based analysis of the ancestry of 6-OPRI in the UK has been presented, similar to an analysis of the E200K mutation (Lee et al., 2000). We showed a large disease-associated haplotype, with infrequent recombinants, suggesting that a number of small 6-OPRI families shared a recent common ancestor with the larger family. This analysis allowed a determined genealogical survey to connect the pedigrees. Where this failed in the case of a single small family, the haplotype analysis provided a genetic justification for grouping together the clinical features of both pedigrees. The resulting collection permits a more statistically powerful analysis of clinical phenotype and its genetic modification.

This approach has allowed the collection of more case histories from a single pedigree than are known from all other OPRI families cited in the world literature combined. Some variation in disease phenotype was seen, leading to a diversity of clinical diagnosis based on the relative prominence of clinical features, but most patients presented with the manifestations of a cortical dementia. Given a retrospective review of case records, and the diversity of early cognitive features recorded by physicians, we were unable to build a clear-cut pattern of early involvement. Behavioural and psychiatric problems, probably reflecting frontal lobe involvement, were often a significant early management problem. Ataxia and relatively mild pyramidal signs typically ensue. Apraxia and myoclonus were frequently recorded. Generalized tonic–clonic seizures and extrapyramidal symptoms were however uncommon. The detailed onset and duration data will be useful for the genetic counselling of at least 84 individuals known to be at risk of inheriting this mutation.

We found that the most remarkable disease variable was age of onset, from ages 20–53 years, the variance of which in a large part (41%) was accounted for by the polymorphic codon 129 genotype. Variation in duration of disease was not accounted for by any genetic polymorphism analysed to date. A similar effect was not seen in 42 OPRI cases reported in the EUROCJD collaborative surveillance project (Kovacs et al., 2005). This discrepancy might be explained by heterogeneity of pathogenesis of long and short insertion mutations and different mutation-associated alleles at codon 129 in the EUROCJD series. Before considering the possible mechanisms of an association, the biological importance of the age of onset trait should be justified. One would expect age of onset to be determined more by genetic than environmental factors, relative to age at death, because variation in the degree of supportive care between affected individuals would be expected to have a large effect on infections and terminal decline of a patient.

We found a strong correlation between age of onset and disease duration, with patients presenting at a more advanced age appearing to have a more aggressive disease course. The same correlation was not found between age of death and disease duration although the sample was smaller. One possible explanation is that the correlation was caused by the spurious presentation of younger patients at an earlier stage of disease. Given that a correlation between age and disease duration has been described for other prion disease categories (Pocchiari et al., 2004), with phenotypes and ages of onset distinct from 6-OPRI disease, it is reasonable to hypothesize a biological cause. It is conceivable that pathways distinct from those influencing the rate of replication of PrPSc, such as an increased sensitivity of neurons to a toxic species, or a reduced ability to degrade PrPSc in older people are relevant (Mallucci and Collinge, 2005). A comparison with vCJD is notable: distinct clinical features of this condition compared with sporadic CJD include prolonged disease duration and neuropsychiatric features. The prominence of the same clinical features in young patients with methionine homozygous 6-OPRI prion disease should lead us to consider how age might modify the phenotype of prion disease. The epidemic of kuru in the Fore people of the Eastern Highlands of Papua New Guinea is a notable contrast however, juvenile kuru being associated with a short (4–8 month) clinical duration (Hornabrook, 1976), compared with a mean of 17 months in 11 elderly patients with long incubation period kuru (Collinge et al., 2006).

One contrast with other frequently occurring inherited prion diseases is worth emphasising (Table 5). The mean age of onset of codon 129 homozygous 6-OPRI prion disease was considerably younger than for point mutations, whereas duration of disease was similar or more prolonged (data not shown for point mutations). Within the pedigree, the codon 129 allele of the non-mutated chromosome confers a 10 year difference in age of onset, but does not affect disease duration. Age of onset of point mutation prion diseases does not show a marked association with codon 129, with the exception of the F198S mutation in a small sample (Dlouhy et al., 1992). Models of prion replication hypothesize a seeding process where the initial conversion to the disease form of PrP takes place followed by a replication process and spread of the agent followed by neurotoxicity. Codon 129 is located in the region of the protein thought to be critical for the conformational change underlying the disease process and strain diversity (Collinge, 2001). According to the conformational selection model of prion transmission (Collinge, 1999; Hill et al., 2000; Hill and Collinge, 2003), M129V affects permissibility of particular strain types (Wadsworth et al., 2004). Possible sources of phenotypic heterogeneity in this kindred therefore include propagation of different prion strains in individual patients, the selection of which may be constrained by PRNP codon 129 genotype, and also differential recruitment of wild-type versus mutant PrPC into PrPSc (Wadsworth et al., 2006). The wild-type allele is known to participate in PrPSc formation for OPRI cases, but not for D178N (Chen et al., 1997). In the 6-OPRI prion disease, our data support the inference that the wild-type allele influences the early aspects of molecular pathogenesis: seeding and replication, rather than neurotoxicity. Polymorphisms of genes regulating apoptotic pathways, or factors governing pathways of degradation, such as age, (Keller et al. 2002, Grune et al. 2004) would be candidates for determinants of duration of disease.

The additional case histories obtained did not strengthen the suggestion of a premorbid personality disorder in the original analysis of the family. In particular, one family member was thought to have the typical personality disorder, but was negative for the mutation on presymptomatic genetic testing. However, it should be noted that clinical histories were obtained from medical records and the clinician may not have sought a history of abnormal premorbid personality. Many patients did have psychiatric or personality related symptoms at onset, but these were usually thought by clinicians to represent the early manifestations of dementia. The suggestion might be rigorously tested by personality assessment of family members as they come forward for presymptomatic testing. Premorbid psychiatric problems have recently been characterized in a family with early-onset inherited prion disease associated with the H187R mutation (Hall et al., 2005) again suggesting an association between age and clinical phenotype.

Whilst in the majority of cases studied neuropathologically the brains exhibited a similar pattern of PrP deposition, there was marked variability in the degree of spongiosis and astrocytosis between individual cases. There appeared to be no correlation between the severity of the spongiosis, or astrocytosis with the age of death or the duration of symptoms, however, the subgroups were very small. Similarly, no meaningful comparison could be made between genotype and neuropathological phenotype as only two cases had a confirmed 129MV genotype. The one ‘atypical’ case studied here revealed synaptic, perineuronal staining for PrP and occasional plaques in the cerebellum. There has been a recent report describing unusual plaque-like PrP immunopositivity in a patient with a 144 bp insertion (Gelpi et al., 2005). In that case the plaques were much more prominent and arranged in clusters described as ‘grape-like’; the appearances therefore appeared to differ quite considerably from those in our case.

In keeping with previous analysis, the band size and glycoform ratio of OPRI PrPSc subjected to limited proteolysis and western blotting, is similar to that found in sporadically occurring CJD and distinct from that found in point mutation inherited prion disease (Hill et al., 2006). PrPSc type may be affected by genotype as for sporadic CJD, where type 1 PrPSc is only found in methionine homozygotes, and types 2 and 3 PrPSc are found in all genotypes (Hill et al., 2003). With only two samples PrPSc-typed, no correlation with disease can be made, although PrPSc diversity is clearly a potentially important non-genetic source of variation.

6-OPRI is a highly transmissible disease to laboratory and transgenic mouse models, producing a fatal neurodegenerative disease and PrPSc in inoculated animals. This finding is strong evidence for the protein-only hypothesis of prion replication, as there is no reason to propose an environmental source for an infectious agent in inherited prion disease. Although it might be hypothesized that the mutation causes susceptibility to a cryptic virus or other pathogen, this suggestion is inconsistent with the complete penetrance of the 6-OPRI mutation by the seventh decade in that any such virus, which has eluded direct detection over many years of research studies, would have to be essentially ubiquitous. The transmission pattern to mouse lines is comparable to that found in cases of sporadic CJD, with a high attack rate and short incubation time in Tg152 animals, and a comparatively reduced attack rate and lengthened incubation time in FVB mice. The transmission pattern is distinct from vCJD which transmits with a higher attack rate to FVB mice (Hill et al., 1997; Wadsworth et al., 2004).

Given the widespread tissue expression of mutant PrP molecules it is important that affected individuals and those at risk of inheriting the mutation advise their doctors and dentists that precautions need to be taken with invasive procedures. In particular disposable instruments should be used where practical, and advice about decontamination taken where not. Affected or at-risk individuals should not donate blood. It is notable that a history of surgery without such precautions and blood donation was obtained from several affected and at-risk individuals.

The mechanism of OPRI mutation is unknown. One possibility is a complex unequal recombination event at PRNP leaving one chromosome deficient in repeats and the other chromosome with extra repeats. In this circumstance one might expect a disruption of the commonly observed SNP haplotypes surrounding PRNP. The SNP haplotype of the 6-OPRI pedigree provides no evidence of such a complex recombination event at the PRNP open reading frame at the time of the OPRI mutation (similar to other OPRI mutations sequenced at the MRC Prion Unit). It is likely therefore that OPRI mutation occurs by either a complex gene conversion event during meiosis, or single strand slippage in meiosis or mitosis, in each case preserving the ancestral SNP haplotype.

We are entering a new era of therapeutic intervention in the field of neurodegeneration; the natural history of 6-OPRI prion disease will be helpful to determine the effectiveness of new treatment strategies. An MRC clinical trial of quinacrine is now under way in the UK, including several patients from this family (Mallucci and Collinge, 2005). It may be advantageous that in inherited prion disease drug treatment can be initiated in presymptomatic individuals before any clinical signs of neurodegeneration.

Acknowledgments

Case reports were derived from assessments by a large number of neurology colleagues who referred patients or copied letters to the National Prion Clinic, Queen Square, London. Prof. Martin Rossor and other neurologists at the Dementia Research Centre, Queen Square, London, were responsible for the referral of a number of patients. Drs I. Bodi, S. Al-Sarraj, M. Honavar, R. Doshi, I. Janota, Mr L. Doey and the London MRC Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, London were involved in neuropathological assessment. Ray Young assisted with illustrations. Funding to pay the Open Access publication charges for this article was provided by The Medical Research Council.

Appendix

Clinical and investigation details are provided in for deceased affected individuals, some are updated from the previous paper (Poulter et al., 1992), in which case the original pedigree number is provided in parentheses. Details of living patients have been omitted to preserve anonymity. Details of many of the autopsied cases have been published separately (King et al., 2003).

Pedigree 1

IV.2

Female, born 1887, died aged 19.

Death certificate states valvular heart disease. Not included in the phenotype table.

IV.3

Female, born 1888, died aged 58.

Death certificate stated cause of death as ‘disseminated sclerosis’.

V.2

Female. Died aged 46

Was admitted to Bexley Hospital aged 43 where she remained until her death aged 46. On admission, her diagnosis was schizophrenia. Her daughter reported her to be quiet and melancholic at the time. She died of uterine carcinoma with pulmonary metastasis.

V.3

Female. Died aged 50.

Death certificate states Ia bronchopneumonia, b uremia, c arteriosclerosis. Her children were affected. Not included in phenotype table.

V.4

Female. Died aged 41

She was a patient of Hurstwood Park Hospital. Death certificate states Ia encephalitis, II uremic state.

V.10 (V.19)

Female. Died aged 40.

Death certificate states Ia bronchopneumonia, b status epilepticus, c cortical atrophy. No other details are known.

VI.3

Female. Onset aged 26, died aged 30.

She was admitted to Bexley hospital with a 2-year history of worrying over unimportant issues, difficulty writing, problems with dressing, muddled speaking, losing her way around the house. Myoclonus was identified in hospital and signs of this were confirmed on EEG which was suggestive of a progressive myoclonic epilepsy syndrome. A provisional diagnosis of Unverricht-Lundberg disease was made. She progressively deteriorated.

A post-mortem examination was carried out and the report states that she died of bronchitis and collapse of the lung due to bulbar palsy due to myoclonic epilepsy with cerebral atrophy.

VI.4

Male. Onset aged 28, died aged 38.

He was thought to have a progressive dementia from age 28 when he lost his job as a traffic controller due to problems with memory, loss of initiative, writing, speech and mental arithmetic. His banker would no longer accept his signed cheques and he became depressed. He lost his way on several occasions. There was no evidence of a premorbid personality disorder. Premorbid IQ was thought to be average but when admitted to Queen Square his verbal IQ was 78, and performance IQ 55. Receptive speech functions, spelling, constructional tasks, left–right discrimination, and memory for recent events were particularly poor. Air encephalogram showed mild cortical atrophy. He was discharged to long-stay in-patient care. He had a sinus operation aged 20.

On examination aged 30 he was only capable of simple communication with evidence of global dementia, dysarthria, and repeated smacking movements of the lips. There was reduced arm swing. Reflexes were brisk and the left plantar response was extensor. He had a prominent grasp reflex with the right hand. Evidence of cerebellar ataxia was notably absent. EEG was very low voltage with a small alpha rhythm.

He remained under long-term hospital care, unable either to speak or obey simple commands. A very limited post-mortem examination identified the cause of death as bilateral bronchopneumonia.

VI.5

Female. Died aged 38.

Death certificate states Ia bronchopneumonia, b Huntington's Chorea Dementia.

VI.16 (VI.18) (129MV haplotype A)

Male. Onset aged 43, died aged 54.

Some details have been published. He was described as having an aggressive personality from school age. He developed a progressive loss of short-term memory aged 43. He was unable to work by 46 years of age. Aged 47 his verbal IQ was estimated as 74, performance IQ 76 with an estimated optimal IQ of 94. PET scanning showed cerebellar hypometabolism.

Reassessment, aged 50, showed little deterioration. He had developed bilateral pout reflexes, and a positive glabellar tap. Aged 54 there was evidence of continuing gradual deterioration. He remained short-tempered, and had problems with simple tasks such as doing up shoelaces, tie, or shaving. MMSE at this time was 19/30. His had an impaired tandem gait, and brisk reflexes throughout although plantar responses were flexor. MRI brain performed at age 51 showed widening of the cortical sulci, particularly in the parietal lobes, althought the cerebellum was also atrophic. Multiple white matter lesions were seen which were thought to be ischaemic in nature. The hippocampi appeared normal.

His condition deteriorated rapidly over one month and he developed difficulty swallowing before dying aged 54. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VI.18

Female. Onset aged 36, died aged 45

At onset she had difficulty with a number of shop assistant jobs, with both learning new tasks and getting on with her colleagues, but there was no evidence of a premorbid personality disorder. Aged 36 she began to have speech problems with staccato and stuttering. She would become irritable at losing things, and would shout spontaneously. She became unsteady and would fall to her left.

Aged 37 she had a neuropsychological assessment: Performance IQ was <41, verbal IQ was 58 with global decline in cognitive function. Acalculia was particularly severe. She had attended a grammar school. By age 40, her son had to look after her. When a psychiatrist visited her at home she threw papers at him, shouted and pulled her hair. She was unable to understand any spoken language and did not speak. On examination, she showed increased limb tone, brisk reflexes and cerebellar ataxia. She had a generalized tonic–clonic seizure and was then treated with phenytoin.

By the age of 44 she had deteriorated further, fell often, was disorientated and unable to feed without help. She was incontinent of urine and faeces.

CT brain showed generalized cerebral and cerebellar atrophy. EEG, aged 37, showed no abnormality.

VI.19

Female. Onset aged 47, died aged 52.

She was thought to have an abnormal ‘emotional state’ when seen on a number of occasions by orthopaedic surgeons for back pain when in her late 40s. Dementia of the Alzheimer's type was diagnosed aged 51, following an attempted drug overdose after the break up of her third marriage. When seen by a consultant psychiatrist at The Maudsley Hospital, aged 52, she scored 4/30 on MMSE, with disorientation of time and place, registering three object on the third attempt only, and acalculia. She was to be admitted for further assessment but this did not happen.

Of note in her past medical history was a lobectomy of the left lung for tuberculosis.

Aged 52, she was admitted with a history of vomiting and collapse caused by pneumonia and gastrointestinal bleeding. She died suddenly in hospital. Post-mortem examination showed a pulmonary embolism and cortical atrophy. The brain was not examined histologically.

VI.20 (129MV haplotype A)

Male. Onset aged 46, died aged 55.

He became forgetful aged 46. He also noticed unsteadiness and a tendency to fall backwards. His daughter reported a mild degree of personality change, depression, and difficulty looking after himself at home. MMSE was 16/30. He had broken pursuit eye movements, a wide-based gait with loss of arm swing and evidence of heel/toe ataxia. There were no other extrapyramidal features. Myoclonic jerks were observed in his hands and arms. There was evidence of dyspraxia, frontal lobe dysfunction and primitive reflexes. MRI brain showed generalized cerebral and cerebellar atrophy. On neuropsychological assessment, verbal IQ was 77 and performance IQ 70, with evidence of significant widespread cognitive decline.

At review later that year he had been getting lost, becoming prone to aggressive outbursts and lacking in insight.

VI.22 (VI.22) (129MM haplotype D)

Male. Onset aged 49, died aged 51.

He developed definite signs of neurodegenerative disease in 1995, aged 49. His employers had begun to have concerns about his performance at work. Later his wife noticed he was very short-tempered and had difficulty dressing. He began to get lost in familiar surroundings. He developed spasticity and global cognitive function declined rapidly; he died of bronchopneumonia in 1997. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VI.23 (VI.25) (129MM haplotype B)

Female. Onset aged 33, died aged 52.

Some details have been published. She presented aged 37 years with a 4-year history of progressive intellectual deterioration. She had become unable to take simple decisions or write and lacked insight into her illness. On examination, she had a mild dysarthria and expressive dysphasia. There was marked dyscalculia and severe impairment of short-term memory. There was a dressing and a constructional apraxia. She was unable to follow complex commands. She had a flat effect and was unconcerned about her ability to perform simple tasks. Limb tone and power were normal. Tendon reflexes were normal and plantar responses flexor. No choreiform movements were observed. Sensory examination was normal. Gait was normal and coordination grossly intact.

EEG demonstrated a slow dominant rhythm with minor excess of theta activity but no specific abnormality. Visual evoked potentials were normal. A CT scan showed generalized cerebral atrophy.

On review two months later, involuntary movements were noted. The diagnosis given was Huntington's disease. Aged 45 she was unable to speak or understand speech. She could not hold a spoon or cup and could only walk supported by two nurses. There was frequent facial grimacing, jerking of the head, and a tendency to bring both hands up to the face. Myoclonic jerks were present. There was a general increase in tone in the limbs with brisk reflexes. Plantar responses were extensor.

She regularly was admitted to a nursing home for respite care over the last nine years of her life. She died aged 52. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum. PrPSc type was 3.

VII.1 (VII.2)

Female. Onset aged 34, died aged 43.

Details have previously been presented but are included here because of her atypical immunocytochemical findings at autopsy. She presented aged 38 with a 3–4 year history of falls due to myoclonic jerking. A diagnosis of myoclonic epilepsy was made supported by the EEG finding of bilateral atypical spike-wave activity. A few years later her personality was affected and memory impaired. On examination her gait was wide-based and arm swing reduced on the left. She died aged 43 of bronchopneumonia. Autopsy was performed. The immunohistochemistry revealed plaque-like and perineuronal immunopositivity for PrP in the molecular and granule cell layer of the cerebellum. They are discussed in the results section.

VII.2 (VII.3) (129MV)

Female. Onset aged 40, died aged 53.

She presented with a gradual deterioration in self-care and her performance at work as a typesetter. At presentation, verbal IQ was 75 and performance IQ was 75, with word retrieval difficulties, apraxia, poor auditory attention span, spelling, arithmetic and perceptual skills.

CT brain showed early atrophic changes in both cerebral hemispheres. She gradually deteriorated, aged 51 she scored 5/30 on the MMSE. Myoclonus was observed and she had a number of generalized tonic–clonic seizures. She died aged 53. PrPSc type was 2.

VII.3 (VII.4) (129MV)

Female. Onset aged 47, died aged 65.

VII.5 (129MM haplotype B)

Male. Onset aged 32, died aged 44.

His mother first reported simple misspellings in letters from America in 1994 when her son was aged 32. Since that time, memory and behaviour have gradually deteriorated, becoming more socially withdrawn and less patient. Memory impairment has become such that he would not drive and took only a few familiar routes out of the house in case he got lost. Speech had become hesitant. There was no premorbid personality disorder.

On examination aged 35 he was disinhibited and disorientated in time and place. MMSE 10/30. His gait was broad based and he was unable to heel–toe walk. Reflexes were brisk but plantar responses were flexor. On neuropsychological assessment, verbal IQ was 65 and performance IQ 67. There was marked impairment of memory, word retrieval, attention and early visual perception loss. MRI brain showed generalized atrophy. EEG was normal.

Follow-up examination aged 36 showed a slow deterioration with a MMSE of 8/30.

Aged 42 he was looked after at home with frequent care givers, unable to usefully communicate. He died aged 44 of pneumonia.

VII.6 (129MM haplotype B)

Male. Onset aged 32, died aged 42.

Aged 32 he began to have problems with balance. Aged 34 he became short-tempered and aggressive. He found himself repeating things but otherwise language function was normal. MMSE was 25/30 (largely due to dyscalculia). There was mild impairment of tandem gait but little else on neurological examination. At the time of assessment the clinician was not confident that there were signs of disease. MRI showed mild diffuse brain atrophy including the cerebellum. EEG showed non-specific abnormalities. Neuropsychological assessment confirmed a mild cognitive decline, particularly in the visual domain.

On review, aggressive episodes were becoming more of a problem with anxiety and agitation, and he became involved in a legal dispute. Thioridazine was considered. He would get lost when travelling on the underground. His tandem gait was impaired. Psychiatric problems became more prominent and he expressed suicidal ideation. Aged 39, MMSE was 19/30. Repeat detailed neuropsychology showed severe memory impairment, below the 5th percentile, particularly in the visual domain. He died suddenly.

VII.7 (129MM haplotype B)

Female, born 1962. Onset aged 31, died aged 40.

Her illness began aged 31 initially referred to neurologists because of odd behaviour. She needed help dressing and her husband had to put a knife and fork into her hands to eat. Subsequently there was a global decline in cognitive function over 2–3 years, becoming unable to look after her young child, have no concept of time passing, difficulty writing, doing housework and becoming clumsy. On examination, she would alternate between laughter and extreme irritability. In conversation, she displayed word finding difficulties and perseveration. She was unable to name any words beginning with ‘C’. She was unable to perform simple arithmetic or copy simple actions. Reading and spelling were poor. Memory, measured by digit span and recall of recent events, was abnormal. Visual memory and right hemisphere skills were also impaired. Her gait was broad based and myoclonic jerks were evident on action. MRI showed mild generalized atrophy. EEG showed mild non-specific abnormalities.

Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.8 (VII.7) (129MM)

Female. Onset aged 23, died aged 39.

Little is known of the progress of this patient other than that her cognition continued to decline gradually. She was diagnosed in life with Huntington's chorea. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.9 (VII.8) (129MM haplotype D)

Female. Onset aged 20, died aged 35.

She had an episode of severe post-natal depression aged 20, but otherwise there was no evidence of a premorbid personality disorder. Three years later it became obvious that she was deteriorating intellectually. This involved refusing to use the telephone, difficulty writing, becoming forgetful and unreliable with money. She was unsteady and had word finding difficulties. On examination aged 27 she was disorientated in place and time, had a poor concentration span, word finding difficulties, dysphasia, dysgraphia, dyspraxia and acalculia. She had a mild slurring dysarthria. Coordination was normal in the limbs but tone was increased, reflexes brisk and plantars were extensor. Insight was limited. Inappropriate behaviour manifested as spontaneous laughter.

When admitted to hospital aged 32 she had deteriorated to the extent that she was severely demented and totally dependent on care givers at her nursing home. On examination, she had an unsteady gait, with some extrapyramidal features. She had bilateral grasp reflexes, utilisation behaviour, apraxia, and bilateral pyramidal signs with extensor plantars. Axial and stretch sensitive myoclonus was observed.

She was admitted to long-term in-patient care, and during this time had at least one generalized tonic–clonic convulsion. CT head showed generalized cerebral (particularly occipital) and cerebellar atrophy. EEG showed minor, non-specific slow wave abnormalities.

VII.13 (VII.14) (129MM haplotype B)

Female. Onset aged 32, died aged 53.

VII.14 (VII.15) (129MM haplotype B)

Male. Onset aged 31, died aged 45.

In 1991, his mother described an 8- or 9-year history of progressive behavioural change, difficulty dressing, shaving and finding words. He was unable to do mental arithmetic and was unsteady on his feet. On examination he was dysphasic and dyspraxic. He had an intermittent tremor of the hands. There was no myoclonus. CT showed cerebellar and cerebral atrophy. The EEG was low amplitude only.

He remained a hospital in-patient for 4 years before his death in 1997. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.15 (129MM haplotype B)

Female. Onset aged 36, died aged 46.

She had learning disability as a child, but no premorbid personality disorder. Aged forty she had personality change and emotional lability with uncharacteristically poor personal hygeine. She progressively declined both cognitively and emotionally. There were no seizures or myoclonus, or evidence of cerebellar ataxia. Although she had dental extractions when symptomatic she had no other surgery and was not a blood donor. Pyramidal signs were evident as brisk reflexes and ankle clonus. She had a marked extrapyramidal response to thioridazine, with severe rigidity, and tortion dystonia of the spine.

MRI showed diffuse cerebral and cerebellar atrophy. EEG was flat and featureless. EMG and nerve conduction studies showed borderline central motor conduction time, but normal peripheral conduction.

When reviewed aged 43 she had been unable to walk for twelve months. Speech was minimal and repetitive, she frequently screamed and was unable to write. There were no EEG correlates of her screaming episodes. Help was required for dressing and feeding. She was doubly incontinent. On examination she had widespread rigidity and a rest tremor.

She continued to be cared for at home and her clinical condition remained largely unchanged until she died aged 46.

VII.17 (129MV)

Male. Onset and death aged 42.

The patient presented with a tremor affecting his hands, worse on the right side. This was initially put down to anxiety related to stress at work. Over the next 8 months his tremor progressed to the point where he had difficulty manipulating cutlery and holding cups of drink. At the same time he developed gait problems with unsteadiness and falls. He had slurring of his speech and was admitted to hospital for investigation. His symptoms continued to progress rapidly and he developed swallowing problems, double incontinence and myoclonus.

On examination only three weeks before his death, he was unable to be assessed by the mini-mental state examination. His speech was hypophonic and slurred. He had broken pursuit movements with nystagmus in the horizontal plane. He had a marked involuntary resting tremor with superimposed myoclonus. Power was preserved in the limbs with brisk reflexes and extensor plantar responses. There was evidence of poor coordination and marked apraxia. There were no sensory signs.

A decision was taken not to institute assisted feeding and to manage him palliatively. He died shortly afterwards, although the exact cause of death was unclear.

VII.21 (VII.47) (129MM haplotype B)

Female. Onset aged 23, died aged 38.

From early childhood, she had blackouts presumed to be faints. She developed marked behavioural abnormalities and self-harm from age 7, thought to be in response to social difficulties. She also took overdoses, but had no formal psychiatric input until age 26 when, following the death of her father, she developed depression and violent mood swings, leading to admission to a psychiatric hospital. Dementia was diagnosed at this time.

Aged 25 she had a clearly different attack from her faints, possibly a generalized tonic–clonic seizure preceded by a run of generalized myoclonus. On assessment she had a vIQ of 79 and pIQ of 77. Performance on reading tests suggested an average premorbid intellingence.

Her behaviour became difficult to manage with aggressive outbursts related to low mood, irritability and frustration with speech difficulties. The presumed epilepsy was treated with valproate, on which she became drowsy, subsequently carbamazepine and phenytoin.

Aged 29 she had signs of mild ataxia, and saccadic interruption of pursuit eye movements.

Information is available from a full neurological assessment aged 33: she was found to have had a severe short-term memory loss, acalculia, apraxia of gait, cerebellar ataxia, myoclonus (particularly on action) and occasional choreiform or dystonic movements. Tendon reflexes were normal.

Two years later, a receptive and expressive dysphasia was noted. She was disorientated in time, unable to count to five, and had severe word retention difficulties. Thioridazine 25 mg tds did not seem to help control her behaviour. Eventually her mood stabilized on dothiepin.

She had a sterilization operation.

Aged 33 she was unable to give any useful history. Notable on examination was her marked apraxia. Gait was only mildly unsteady. Myoclonus was observed but mild. Reflexes were brisk but plantars were flexor. vIQ was 63, pIQ was 52. Cognition was globally impaired.

Aged 35 she had become doubly incontinent and had a scoliosis due to dystonia.

CT head scan in 1991 showed cerebral and cerebellar atrophy. EEG was normal aged 25, but showed excess theta aged 33. EMG showed no evidence of anterior horn cell disease. CSF examination for routine constituents on two occasions was unremarkable. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.25 (VII62) (129MM haplotype B)

Male. Onset aged 35, died aged 44.

Full details are given in the previous publication. He died in 1993 following a 9-year illness characterized by dementia, cerebellar ataxia with personality changes. An autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.27 (129MM haplotype B)

Male. Onset aged 31, died aged 39.

He became forgetful around 1993 and had difficulties with his job as a cabinet-maker. He had difficulty expressing himself and would get words muddled up. Clumsiness and gait disturbance developed two years later. He became aggressive and this was controlled with stelazine. In 1996, it was difficult to hold a conversation with him and his speech was slurred. He needed help with simple household tasks, dressing and shaving. Neuropsychological assessment in March 1996 showed a verbal IQ of 66, performance IQ 58 with poor memory and generalized cognitive decline. By October 1997, this had deteriorated to a verbal IQ of 57, and performance IQ of 56. He developed urinary and faecal incontinence, facial twitching and myoclonus. On examination aged 38, he had marked cognitive impairment, gait and limb ataxia and eyelid apraxia.

He died in 1999 of a left lower lobe pneumonia. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VII.28 (129MM haplotype B)

Male. Onset aged 30, died aged 44.

Aged 33, three years following onset, a progressive intellectual deterioration was diagnosed on neurological assessment. He had lost ability to distinguish right from left, had reduced reading and writing ability. There were also behavioural disturbances such as persuading his children to steal. His wife was concerned about him exposing himself in public. On examination, his speech was slurred.

He died aged 44. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

VIII.1 (129MM)

Male. Onset aged 27, died aged 33.

His onset was dated in retrospect by his stepmother who noted abnormal behaviour: odd dressing, lack of hygiene and flushing food down the toilet. Neurological presentation was aged 31 with falls and jerkiness with witnessed myoclonus. He had a prominent hand tremor. The condition progressed rapidly with slurring dysarthria, poor handwriting and lack of insight. Other examination findings included perseveration, finger-nose ataxia, and a broad based gait. MMSE was 5/30. Reflexes were brisk.

In 2004 detailed cognitive assessment showed marked generalized cognitive impairment with anterior regions most severely affected and visuoperceptual and visuospatial skills preserved. MRI showed cerebral and cerebellar atrophy. EEG showed cerebral dysfunction but no periodic or epileptiform activity. Later that year he was so dysarthric his voice was barely intelligible and he had developed urinary incontinence. His behaviour became dangerous and he required sectioning.

He died of pneumonia.

Unaffected family member.

He lost temper easily, and had generalized seizures with injuries about 1–2× per month, although none had been witnessed. He was frequently involved with the police, with antisocial behaviour from school age. Heavy alcohol intake. Came forward for presymptomatic testing but found to be negative for the 6-OPRI mutation.

Pedigree 2

2I.1

Male. Died aged 51, in hospital related to ‘shock of burns’. Uncertain affected status. His wife was unaffected.

2II.1

Female. Died in an asylum aged <50 years. Not included in phenotype table.

2II.2

Male. Died aged 56, death certificate states, Ia secondary sarcomatosis, b fibrosarcoma of bowel. Not known to be affected himself but has an affected sister and children. Not included in phenotype table.

2III.1 (129MV)

Female. Onset aged 38, died aged 49

She had a history of pre-senile dementia of 11–12 years duration. She died of bronchopneumonia. Autopsy was performed, but only a very limited number of blocks were available for examination. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

2III.2

Female. Onset aged 42, died aged 57.

She was first examined aged 44 in a remote farmhouse. She had been deteriorating mentally for at least 2 years and when seen she was not capable of any useful housework. She had not ventured outside for many months. On neurological examination myoclonus was observed, limb reflexes were brisk though plantars were flexor. On neuropsychological assessment, premorbid IQ was thought to be 98, estimated with the Schonell Reading Test. She fell into the demented group on the Kendrick Battery for the detection of dementia and showed marked impairment of the Inglis Paired Associate Learning Test. CT head showed generalized cerebral atrophy.

She was admitted to long-stay in-patient hospital care. During this time, she had two generalized tonic–clonic seizures and aggressive outbursts.

2III.3

Female, born 1947. Onset aged 32, died aged 39.

She presented with rapidly progressive memory loss aged 32. At presentation, she was unable to do any housework and neglected herself. On examination, she was unable to name the Prime Minister or Monarch, and was unable to subtract 7s from 100, or retain any sentences. Copying and naming was not impaired. She had a dressing apraxia. Neurological examination showed poor tandem gait and brisk limb reflexes with extensor plantar responses. Full scale IQ was 75 (verbal IQ was 71 and performance IQ was 85), noting that she appeared to be an intelligent child, passing her 11+ examinations and the Schonell Reading Test suggested an average premorbid IQ. CT head showed cerebral atrophy. EEG showed no focal abnormalities but generalized slowing.

Aged 35 a repeat neuropsychological examination was almost impossible due to marked progression of her disease. She was tearful and perseverated her speech.

She died suddenly aged 39. A limited post-mortem examination was performed, demonstrating an acute subarachnoid haemorrhage. Very limited neuropathological examination of the brain at that time showed cortical atrophy and subcortical gliosis but no vasculopathy. The cerebellum was not commented upon.

2IV.1 (129MM haplotype E).

Female. Onset aged 37, died aged 44.

She presented with headaches followed shortly by progressive cognitive impairment. Initially this manifested as an inability to learn new tasks at work but progressed such that she was unable to do any housework or cook meals. She would get lost when driving in her car. Later walking became unsteady and she has difficulty getting up stairs. On examination, she had a marked apraxia, and there was evidence of cerebellar ataxia. MMSE 11/30. There was dyscalculia, dysgraphia and dyslexia. EEG was normal. Verbal IQ was 53 performance IQ was 56 reflecting a severe degree of intellectual deterioration. MRI mild diffuse atrophy. Autopsy was performed. On immunohistochemistry there was PrP deposition in the form of patches in the molecular layer of the cerebellum.

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