Acta Neuropathol (2014) 127:451–458 DOI 10.1007/s00401-014-1245-7
CASE REPORT
Early dipeptide repeat pathology in a frontotemporal dementia kindred with C9ORF72 mutation and intellectual disability Malcolm Proudfoot · Nick J. Gutowski · Dieter Edbauer · David A. Hilton · Mark Stephens · Julia Rankin · Ian R. A. Mackenzie
Received: 5 December 2013 / Accepted: 8 January 2014 / Published online: 21 January 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Familial cases of frontotemporal dementia (FTD) provide an opportunity to study the pathophysiology of this clinically diverse condition. The C9ORF72 mutation is the most common cause of familial FTD, recent pathological descriptions challenge existing TDP-43 based hypotheses of sporadic FTD pathogenesis. Non-ATG dependent translation of the hexanucleotide expansion into aggregating dipeptide repeat (DPR) proteins may represent a novel pathomechanism. We report detection of the DPR aggregates very early in C9ORF72 FTD development and also describe childhood intellectual disability as a clinical feature preceding dementia. The index case presented with psychiatric symptoms, progressing into typical FTD. Autopsy revealed extensive neuronal DPR aggregates but only minimal TDP-43 pathology. Her intellectually disabled elder son, also carrying the C9ORF72 mutation, died aged 26 years and at autopsy only DPR aggregates without TDP-43 were found. A second son also has intellectual
disability, his C9ORF72 status is unknown, but chromosomal microarray revealed no other cause of disability. These cases both extend the existing phenotype of C9ORF72 mutation and highlight the potential significance of DPR translation early in disease development.
M. Proudfoot (*) · N. J. Gutowski Department of Neurology, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK e-mail: [email protected]
D. A. Hilton Department of Cellular and Anatomical Pathology, Derriford Hospital, Plymouth, UK
Introduction Frontotemporal dementia (FTD) is a common neurodegenerative condition with well characterised but overlapping clinical subtypes. The previously recognised genetic causes [44] are surpassed in abundance by a mutation on chromosome 9 that links FTD and amyotrophic lateral sclerosis (ALS), ‘c9FTD/ALS’ [37, 50]. The C9ORF72 intronic hexanucleotide repeat expansion [15, 39] accounts for approximately 21 % of familial and 6 % of sporadic FTD, depending on the population sampled [32].
N. J. Gutowski University of Exeter Medical School, Exeter, UK
M. Stephens Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK
D. Edbauer German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
J. Rankin Department of Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK
D. Edbauer Adolf Butenandt Institute, Biochemistry, Ludwig-Maximilians University, Munich, Germany
I. R. A. Mackenzie Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada
D. Edbauer Munich Cluster of Systems Neurology (SyNergy), Munich, Germany
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At present, the penetrance of the C9ORF72 mutation remains uncertain; in one series it was complete by 80 years of age [19]. The minimum number of hexanucleotide repeats required for pathogenicity is also yet to be established [20], compounded by difficulties in accurate repeat sizing. There is so far no definite relationship between repeat size, age of onset and severity [4, 7]. These uncertainties complicate genetic counselling, but in keeping with other repeat expansion disorders [26], no effect is seen of normal allele repeat size [43] and a trend towards anticipation is reported [14]. In a European study, the C9ORF72 expansion appeared to arise from a single founder on an intrinsically unstable haplotype [46]. Our report adds to the expanding range of phenotypic characteristics of c9FTD/ALS. Psychotic symptoms early in the course of FTD, perhaps accompanied by repetitive behaviours [11], are suggestive of mutation in C9ORF72 [47], but reliable discrimination from sporadic cases or other monogenetic aetiologies is difficult on clinical grounds alone. MRI changes in both white and grey matter can distinguish the c9FTD/ALS pattern of atrophy from that of sporadic ALS [5], FTD [52] or FTD due to other mutations [31], but classification accuracy may be dependent upon disease stage. The pathological signature of C9ORF72 mutation is most often reported to be FTLD–TDP type B [1, 29, 38], with TDP-43 immunoreactive neuronal and glial inclusions [18, 19], as well as distinctive neuronal inclusions that stain for ubiquitin, p62 and related proteasomal proteins, but are
negative for TDP-43 [16, 17]. Independent studies have now confirmed nuclear RNA foci of sense and antisense repeats [15, 17, 33]. In addition, novel insight was provided by the remarkable discovery that dipeptide repeat (DPR) aggregates, formed via non-ATG dependent translation of the intronic GGGGCC repeats [57], co-localise with the characteristic ub/p62-positive inclusions and furthermore are not identified in a range of other neurodegenerative conditions [3, 36]. Translation of both sense and antisense transcripts, in all possible reading frames, may contribute to pathogenesis [17, 34]. Definitive distinction of c9FTD/ALS on pathological features now appears possible although reports of mixed pathological appearances [24] exist and could correspond to variability in phenotypic expression [27]. Therefore, the mechanism by which C9ORF72 mutation leads to expression of FTD and/or ALS currently remains uncertain [28]. Leading theories include any combination of (a) loss of normal gene function with haplo-insufficiency, (b) toxic gain of function via atypical translation products, or (c) interference in normal nuclear processing by RNA G-quadruplexes [16]. Description of sequential pathological changes might further illuminate the relative importance of each potential aetiological process. We describe a Caucasian kindred with FTD caused by the C9ORF72 expansion mutation, including a mother (Case 1) and her two sons (Cases 2 and 3). There is no history of consanguinity, but there is a wider family history of intellectual disability and dementia (Fig. 1). The following
Fig. 1 Circles represent female individuals and squares males. Filled symbols are those subjects with relevant symptoms of intellectual disability and/or dementia and strike-through represents deceased. The
proband is indicated by an arrow. The top number represents age of symptom onset or childhood learning difficulty (LD); the bottom number is age at death
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clinical descriptions include, in Cases 2 and 3, childhood intellectual disability followed by apparent progressive cognitive decline in early adulthood; for comparison, previous young adult cases of c9FTD/ALS include FTD onset at 18 years [45] and ALS aged 20 years [53]. Case 1 was included in a previously reported series [31] but the autopsy findings are only now reported.
Materials and methods The C9ORF72 mutation was confirmed in lymphocyte DNA from Cases 1 and 2. Two repeat-primed PCR reactions were conducted. Each reaction included a separate, non-overlapping flanking primer and a repeat primer homologous to the GGGGGC repeat. The repeat primer annealed at multiple sites within the repeat to produce variable sized products. These products were in turn amplified by a third (tail) primer binding to the 3′end. Cycling conditions employed touchdown PCR from 70 to 56 °C and products were sized on an ABI 3730 genetic analyzer (Applied Bio-systems, Foster City, CA, USA). Repeat-primed PCR did not allow for sizing of the repeat, hence Southern blot was performed with two separate digests (EcoRI+BamHI and EcoRI). Post-mortem brain examinations of Cases 1 and 2 are described. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using the following antibodies : ubiquitin (DAKO anti-ubiquitin; 1:500, following microwave antigen retrieval), hyperphosphorylated tau (Innogenetics AT-8; 1:2,000 following microwave antigen retrieval), α-synuclein (Zymed anti-α-synuclein; 1:10,000, following microwave antigen retrieval), phosphorylated neurofilament (Sternberger SMI 31; 1:8,000, following protease digestion), p62 (BD Transduction anti-p62 Lck; 1:1,600 following microwave antigen retrieval), TDP43 (Proteintech anti-TDP-43; 1:12,000 following microwave antigen retrieval) A4 protein (DAKO anti-β amyloid; 1:2,000 following formic acid pretreatment) and FUS (SigmaAldrich anti-FUS; 1:200 following microwave antigen retrieval). DPR pathology was demonstrated using a recently characterised mouse monoclonal antibody that recognizes extended glycine-alanine (GA) repeats (clone 5E9; 1:500 overnight following microwave antigen retrieval) [30].
Results Case 1 Clinical findings and investigations The proband presented to psychiatric services aged 46 years with agitated depression. She had left school aged
14 years without qualifications and poor literacy. During her 4-month admission, extra-pyramidal features were noted following anti-psychotic administration. Computed tomography (CT) head and electroencephalogram (EEG) were within normal limits. Psychometric testing at that time found frontal lobe impairment with a verbal fluency below the fourth percentile. Two years on, she had become disinhibited, was unable to assist with household duties and her self-care had deteriorated. Further investigations included normal cerebrospinal fluid (CSF) examination; EEG was of low amplitude but with preserved alpha and magnetic resonance imaging (MRI) of brain revealed mild symmetric atrophy, not obviously frontotemporal in location. Cognitive assessment revealed a mini mental state examination score of 26/30, verbal IQ 71, performance IQ 74 and reading age 6–7 years. A clinical diagnosis of behavioural variant (BV) FTD was made. Genetic analysis excluded Huntington’s disease and dentatorubral-pallidoluysian atrophy and did not detect mutations in APP exons 16 and 17, PSEN1 exons 2–12, PSEN2 exons 4, 5 and 7, PRNP and CHMP2B. Following intentional overdose she was moved into residential care. Significant obsessive–compulsive symptoms were noted, with poor urinary continence and bizarre food behaviour involving mouth stuffing. More florid psychotic symptoms included delusions of control by the radio but she remained intolerant of anti-psychotics. Medical admission due to inadequate nutrition occurred at age 53 years. CSF 14-3-3 and muscle biopsy were normal. Percutaneous gastrostomy was undertaken but death from bronchopneumonia occurred shortly after. The C9ORF72 GGGGCC repeat expansion was detected in lymphocyte DNA by repeat-primed PCR and sized to at least 23 kb by Southern blot. Neuropathology General autopsy examination revealed evidence of ischaemic heart disease and bronchopneumonia. The brain weighed 1,095 g and showed a mild degree of generalised atrophy. Macroscopic examination of the brain was otherwise unremarkable and the substantia nigra was normally pigmented. Haematoxylin and eosin (H&E) stained sections from the cerebral cortex showed only very mild superficial laminar spongiosis and gliosis in the frontal lobes. There was mild diffuse gliosis of the caudate, putamen and globus pallidus. The brainstem, cerebellum and upper spinal cord appeared unremarkable. In the neocortex, the most significant pathology was the presence of numerous ubiquitin and p62-immunoreactive (ub–ir) neuronal cytoplasmic inclusions (NCI) and moderate numbers of neuronal intranuclear inclusions (NII) and dystrophic neurites (DN), throughout all cortical
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layers (Table 1). The NCI varied in size and appeared as small or large clusters of coarse granule or more smoothcontoured compact bodies. The NII were small dot-like inclusions, approximately the size of the nucleolus. The DN varied from short delicate threads to much longer, bulbous profiles. Small and large ub-ir granules were also present in the neuropil with no obvious cellular association. Small TDP-43-ir NCI and DN were present but much less common (less than 1 % the amount of ub-ir pathology), whereas DPR-ir NCI were widespread and abundant (Fig. 2). The morphology and distribution of the TDP-43-ir pathology were most consistent with FTLD–TDP type B. Rare tau-positive neurites were also present in the neocortex in the absence of any neurofibrillary tangles (NFT). In the hippocampus, there were numerous granular and compact ubiquitin/p62-ir NCI and occasional NII in dentate granule cells and numerous less compact granular NCI and occasional NII in CA3/4 pyramidal neurons. TDP-43ir NCI and NII were exceedingly rare in the dentate layer and absent in pyramidal neurons, while DPR-ir NCI were abundant in both layers. Tau-ir NFT, pre-tangles and DN were abundant in the entorhinal cortex but sparse in the hippocampal pyramidal layer (Braak stage II). The only pathology demonstrated in the midbrain was infrequent small ub-ir NCI and DN, primarily in the tectum and tegmentum. NCI and DN were also present in the striatum with more NCI demonstrated with TDP-43 than DPR IHC while the DN were most numerous in ubiquitin-stained sections. There were numerous small rounded ub-ir NCI and occasional NII in the granule cell layer of the cerebellum
(less well demonstrated with p62 IHC), but TDP-43-ir structures were not identified. Significant pathology was noted in all cortical layers of the cerebellum with DPR IHC. No pathology was demonstrated with IHC for neurofilament, A4 protein, α-synuclein or FUS (Fig. 2). Case 2 Clinical findings and investigations The elder son presented to paediatric services with significant speech and language delay. Examination did not reveal any obvious dysmorphism and there was no history of intrapartum asphyxia. His IQ at age 4 years was 39 and he required special education needs schooling. From 17 years, he was able to assist in farm labour, but developed anxious and obsessional behaviour with ritualistic checking. By 22 years he began to lose farming skills and faecal continence was problematic. EEG at that time was normal although he was noted to be unable to maintain eye closure. MRI did not show focal atrophy. Aged 25 years, he moved into residential care, but steadily deteriorated becoming withdrawn and violent. Antipsychotic medication led to bradykinesia, mouth stuffing food behaviour was also noted. Death occurred due to pulmonary embolus aged 26 years after a prolonged period of immobility. The C9ORF72 GGGGCC repeat expansion was detected in lymphocyte DNA by repeat-primed PCR. There was insufficient DNA for Southern blot.
Table 1 Summary of neuropathological immunohistochemistry Case 1
Frontal
HC-dentate CA3/4
Striatum
Cerebellum
Case 2
ubiq
p62
TDP
DPR
ubiq
p62
TDP
DPR
NCI++++ NII+++ DN+++ NCI++++ NII++ NCI++++ NII++ DN++ NCI++ NII+ DN++++ NCI++++ NII+
NCI++++ NII++ DN++ NCI++++ NII++ NCI+++ NII++ DN− NCI++ NII+ DN++ NCI++ NII+
NCI+ NII− DN++ NCI+ NII+ NCI− NII− DN− NCI+++ NII− DN+++ NCI− NII−
NCI++++ NII++ DN+ NCI++++ NII+++ NCI++++ NII+++ DN++ NCI++ NII− DN+ NCI++++ NII++
NCI+++ NII++ DN++ NCI++++ NII++ NCI+++ NII+ DN+ NCI++ NII− DN+++ NCI++ NII+
NCI++++ NII++ DN++ NCI++++ NII+ NCI++++ NII+ DN− NCI+ NII− DN++ NCI+ NII−
NCI− NII− DN− NCI− NII− NCI− NII− DN− NCI− NII− DN− NCI− NII−
NCI++++ NII++ DN+ NCI++++ NII++ NCI++++ NII++ DN++ NCI++ NII− DN− NCI+++ NII++
DN++
DN++
DN−
DN++++
DN+
DN+
DN−
DN+
Semi-quantitative comparison of immunohistochemistry found at autopsy in mother (Case 1) and elder son (Case 2) NCI neuronal cytoplasmic inclusions, NII neuronal intranuclear inclusions, DN dystrophic neurites − none,+ rare, ++ mild, +++ moderate, ++++ severe
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Fig. 2 Immunohistochemical findings case 1 (proband, a–e) and case 2 (son, f–h). The proband had very rare TDP-43-immunoreactive neuronal cytoplasmic inclusions (NCI) in the frontal neocortex (arrow, a) and hippocampal dentate granule layer (arrow, b). In contrast, immunohistochemistry using an antibody against GA dipeptide repeats (DPR) demonstrated much more abundant DPR-immunore-
active NCI in the frontal cortex (c), hippocampus (d) and cerebellar granular layer (e). No TDP-43-immunoreactive pathology was found in case 2; however, abundant DPR-immunoreactive NCI were present in the frontal cortex (f), hippocampus (g) and cerebellum (h). Scale bar 50 microns
Neuropathology
valproate. His family described him as irritable and nervous but neurological examination was normal. Investigations included serial MRI brain in 2005 (aged 23 years), 2008 and 2012. Subtle evidence of progressive widening of the sylvian fissures and enlargement of the temporal horns was noted. EEG was normal in 2005 but failure of full eye closure was noted with accompanying alpha attenuation. Genetic investigations included exclusion of fragile X syndrome and normal chromosomal micro array analysis. Neuropsychometry was performed aged 23 years. His Wechsler Abbreviated Score of Intelligence verbal IQ was 73, non-verbal 67. On the Schonell Graded Word Reading Test, he averaged 11 years. Assessment of memory on the Repeatable Battery for the Assessment of Neuropsychological Status scored 69 on immediate memory with a delayed memory score of 91. Subsequent review at age 27 years included comparable neuropsychometry. No relative weakness in short-term memory was noted this time on the Wechsler memory scale III. On the Delis–Kaplan Executive Function System trail making task, he attained a scaled score of 1 (average = 10). This was accompanied by a Behavioural Assessment of the Dysexecutive Syndrome “zoo map” open-ended test at which he was severely impaired indicating poor executive function but overall there was no clear decline from the previous assessment. By this time, however, frontal release signs were noted including palmomental and pout reflex accompanied by excessive gaze aversion. A further EEG aged 29 years showed long runs of frontotemporal slow waves without epileptiform activity.
General autopsy revealed extensive pulmonary embolism. The brain weighed 1,426 g and had a normal macroscopic appearance. H&E stained sections did not reveal any abnormalities, and in particular there was no obvious neuronal loss or gliosis in the cortex. The ub/p62-ir pathology was similar to that present in Case 1 but slightly less abundant in all neuroanatomical regions examined (Table 1). Numerous ub/p62-ir NCI and occasional NII and DN were present in the neocortex, numerous NCI and occasional NII in the hippocampal granule and pyramidal neurons and moderate numbers of large swollen DN and occasional NCI in the striatum. Moderate numbers of NCI and DN were present in the cerebellar granular layer. Importantly, no TDP-43-ir NCI or DN were identified in any region, in contrast to a widespread anatomical distribution of DPR-ir structures which correlated well with the ub/p62 staining (Fig. 2). No pathology was demonstrated with IHC for tau, A4, neurofilament, α-synuclein or FUS. Case 3 The younger son presented to an educational psychologist aged 3 years with global cognitive delay. He remained in mainstream education albeit with a statement of special education needs. After graduation from agricultural college, he began work on the family farm and was able to drive a tractor. He was seen by adult neurology services aged 22 years following generalised seizures and was treated with sodium
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He remains in the care of his family who describe him as very set in routines and lacking motivation. He and his family currently do not wish to pursue his C9ORF72 carrier status.
Discussion The discovery of a kindred with C9ORF72 mutation, abundant DPR pathology but little or no TDP-43 pathology, reinforces the pathogenic role of DPR inclusions. This kindred furthermore demonstrates clinical features well recognised within the FTD/ALS spectrum attributed to C9ORF72 mutation [23], but in addition the discovery of at least two cases with intellectual disability may further broaden the existing phenotypic range. Previous reports have noted a long prodromal period of psychotic symptoms and personality change [49], perhaps stretching back decades, which inevitably complicates attempts to quantify disease duration in genetic carriers. Subtle neuropsychological deficits have been described in other pre-symptomatic dementia mutation carriers before. PSEN-1 carriers show impairment in executive, memory and visuo-spatial function if tested within 10 years of expected age of onset [41] and executive dysfunction has been demonstrated many decades before predicted disease onset in carriers (mean age 31 years) of the MAPT mutation [18]. Significant intellectual disability has not previously been reported with C9ORF72 expansion and engenders consideration as to what age DPR aggregation becomes detectable in C9ORF72 patients. Could an inter-generational expansion of C9ORF72 hexanucleotide repeat size may underpin the intellectual disability and apparent early age of onset in our proband’s children? Other DNA repeat disorders, such as myotonic dystrophy, demonstrate anticipation such that more severe and earlier onset of disease [21] follows a dramatic increase in repeat tract size. Affected children of C9ORF72 carriers have shown a trend towards development of ALS at an earlier age, manifesting symptoms on average 7 years earlier than their parents in one study [12], but to date there is no evidence to suggest that this is caused by an inter-generational increase in repeat tract length. An alternative explanation for the observed childhood features of our cases would be the co-occurrence of an additional disorder. Such developmental vulnerability could unmask earlier expression of a subsequent FTD phenotype [42]. There were, however, no features such as dysmorphism or structural brain abnormalities to suggest a second diagnosis and genetic investigations (fragile X testing and chromosome analysis by array CGH) gave normal results. Nevertheless, and despite the absence of any pathological evidence to suggest concurrent mutations, the possibility of
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an as yet undiagnosed, possibly X linked, disorder resulting in intellectual disability remains. In addition, synergistic mutations in other genes implicated in FTD have not been fully excluded. Co-occurrence of more than one causative gene has been found above chance in familial ALS [8] and furthermore additive mutations may contribute to an earlier age of symptom onset [6, 40]. Conversely, a case with concurrent C9ORF72 repeat expansion and p.Ala239Thr MAPT variant did not exhibit early or aggressive disease onset despite mixed pathological findings [24]. The present report therefore demonstrates segregation of intellectual disability and subsequent FTD, with autosomal dominant inheritance of the C9ORF72 expansion. The autopsy findings provide significant new details of early pathological changes in the development of this disorder. The dominant abnormality throughout the cortex of Case 1 was that of NCI, NII and DN that stained for both ubiquitin and p62. TDP-ir pathology was present in a pattern consistent with the FTLD–TDP subtype which is most often reported in cases with the C9ORF72 mutation (type B), and distinct from the limited TDP pathology that often occurs as a coincidental finding in the setting of other neurodegenerative diseases (usually type A) [2]. However, the degree of this TDP-ir pathology was so limited as to be initially overlooked. In contrast, the consistent finding of abundant and widespread DPR pathology seems to represent a highly sensitive and specific pathological marker for the presence of the C9ORF72 mutation. The pathological examination of Case 2 uniquely captures an early phase in the evolution of c9FTD/ALS (Table 1), interrupted in this instance by death from pulmonary embolus. Although only one previous report of FTLD–UPS within C9ORF72 mutation carriers [19] exists, the total absence of TDP-43 aggregation again questions the primacy of this protein in the initiation of FTD pathogenesis. This finding is at variance with a recent study showing that phenotype and neurodegeneration correlated strongly with TDP-43, but not DPR, pathology [30]; however, it would still seem highly likely that DPR protein formation and pathological deposition precedes that of TDP-43 accumulation. Abnormalities of TDP-43 causing neurodegeneration are of course well described in both human TDP-43 mutation carriers [48] and in animal models [51, 54, 55] but the exact function of the protein is actively under research. The question remains whether the severe intellectual disability of this case could be a developmental consequence of the extensive DPR-ir pattern in clinically relevant anatomical locations, despite the lack of macroscopic abnormalities. Assuming instead that the ub/p62/DPR-ir intra-neuronal inclusions described do not fully account for Case 2’s cognitive impairment, the pathological changes could then be considered to reflect very early, perhaps even preclinical
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FTD. These findings do not contradict proposed staging classifications for TDP-43 “proteinopathies”, which now include cases with C9ORF72 repeat expansions [10], since pre-symptomatic pathological changes were already anticipated, if not previously demonstrated. It may yet be the case that TDP-43 metabolism is at some critical point interrupted as a consequence of the C9ORF72 mutation, but aetiological descriptions will presumably remain speculative until more detail of the gene’s physiological function is discovered. The full components of the ub/p62-ir intra-neural inclusions continue to be described with various protein species already identified [9, 13, 35] in addition to sense and antisense DPR transcripts [34]. Previous proposed mechanisms of FTD pathogenesis are not necessarily excluded by the presence of DPR protein and RNA foci, and indeed RNA foci do not co-localise with TDP-43 aggregates [25]. The relationship between C9ORF72 mutation and TDP-43 cytoplasmic mislocalisation therefore remains opaque, but animal models currently in development may elucidate this further [56] and detection of DPR formation will remain a crucial investigative step in determining the cellular processes that culminate in FTD. In conclusion, this report describes pathological evidence of DPR protein deposition in a genetically confirmed case of C9ORF72 mutation, prior to the development of obvious TDP-43 pathology. The clinical features may represent an unusual extended prodrome of childhood learning disability, thus potentially expanding the existing FTD phenotype. Following submission of our manuscript, another paper was published that includes description of a patient with the C9ORF72 mutation, abnormal development and early learning disability [22], further supporting our findings of this expanded phenotype. Acknowledgments MP is supported by the Guarantors of Brain. DE is supported by the Helmholtz Young Investigator program (HZNG-607) and by a grant of the Centres of Excellence in Neurodegeneration Research (CoEN). IM is supported by Canadian Institutes of Health Research (74580) and the Pacific Alzheimer’s Research Foundation (C06-01).
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CASE REPORT
Early dipeptide repeat pathology in a frontotemporal dementia kindred with C9ORF72 mutation and intellectual disability Malcolm Proudfoot · Nick J. Gutowski · Dieter Edbauer · David A. Hilton · Mark Stephens · Julia Rankin · Ian R. A. Mackenzie
Received: 5 December 2013 / Accepted: 8 January 2014 / Published online: 21 January 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Familial cases of frontotemporal dementia (FTD) provide an opportunity to study the pathophysiology of this clinically diverse condition. The C9ORF72 mutation is the most common cause of familial FTD, recent pathological descriptions challenge existing TDP-43 based hypotheses of sporadic FTD pathogenesis. Non-ATG dependent translation of the hexanucleotide expansion into aggregating dipeptide repeat (DPR) proteins may represent a novel pathomechanism. We report detection of the DPR aggregates very early in C9ORF72 FTD development and also describe childhood intellectual disability as a clinical feature preceding dementia. The index case presented with psychiatric symptoms, progressing into typical FTD. Autopsy revealed extensive neuronal DPR aggregates but only minimal TDP-43 pathology. Her intellectually disabled elder son, also carrying the C9ORF72 mutation, died aged 26 years and at autopsy only DPR aggregates without TDP-43 were found. A second son also has intellectual
disability, his C9ORF72 status is unknown, but chromosomal microarray revealed no other cause of disability. These cases both extend the existing phenotype of C9ORF72 mutation and highlight the potential significance of DPR translation early in disease development.
M. Proudfoot (*) · N. J. Gutowski Department of Neurology, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK e-mail: [email protected]
D. A. Hilton Department of Cellular and Anatomical Pathology, Derriford Hospital, Plymouth, UK
Introduction Frontotemporal dementia (FTD) is a common neurodegenerative condition with well characterised but overlapping clinical subtypes. The previously recognised genetic causes [44] are surpassed in abundance by a mutation on chromosome 9 that links FTD and amyotrophic lateral sclerosis (ALS), ‘c9FTD/ALS’ [37, 50]. The C9ORF72 intronic hexanucleotide repeat expansion [15, 39] accounts for approximately 21 % of familial and 6 % of sporadic FTD, depending on the population sampled [32].
N. J. Gutowski University of Exeter Medical School, Exeter, UK
M. Stephens Institute of Medical Genetics, University Hospital of Wales, Cardiff, UK
D. Edbauer German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
J. Rankin Department of Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK
D. Edbauer Adolf Butenandt Institute, Biochemistry, Ludwig-Maximilians University, Munich, Germany
I. R. A. Mackenzie Department of Pathology, University of British Columbia and Vancouver General Hospital, Vancouver, Canada
D. Edbauer Munich Cluster of Systems Neurology (SyNergy), Munich, Germany
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At present, the penetrance of the C9ORF72 mutation remains uncertain; in one series it was complete by 80 years of age [19]. The minimum number of hexanucleotide repeats required for pathogenicity is also yet to be established [20], compounded by difficulties in accurate repeat sizing. There is so far no definite relationship between repeat size, age of onset and severity [4, 7]. These uncertainties complicate genetic counselling, but in keeping with other repeat expansion disorders [26], no effect is seen of normal allele repeat size [43] and a trend towards anticipation is reported [14]. In a European study, the C9ORF72 expansion appeared to arise from a single founder on an intrinsically unstable haplotype [46]. Our report adds to the expanding range of phenotypic characteristics of c9FTD/ALS. Psychotic symptoms early in the course of FTD, perhaps accompanied by repetitive behaviours [11], are suggestive of mutation in C9ORF72 [47], but reliable discrimination from sporadic cases or other monogenetic aetiologies is difficult on clinical grounds alone. MRI changes in both white and grey matter can distinguish the c9FTD/ALS pattern of atrophy from that of sporadic ALS [5], FTD [52] or FTD due to other mutations [31], but classification accuracy may be dependent upon disease stage. The pathological signature of C9ORF72 mutation is most often reported to be FTLD–TDP type B [1, 29, 38], with TDP-43 immunoreactive neuronal and glial inclusions [18, 19], as well as distinctive neuronal inclusions that stain for ubiquitin, p62 and related proteasomal proteins, but are
negative for TDP-43 [16, 17]. Independent studies have now confirmed nuclear RNA foci of sense and antisense repeats [15, 17, 33]. In addition, novel insight was provided by the remarkable discovery that dipeptide repeat (DPR) aggregates, formed via non-ATG dependent translation of the intronic GGGGCC repeats [57], co-localise with the characteristic ub/p62-positive inclusions and furthermore are not identified in a range of other neurodegenerative conditions [3, 36]. Translation of both sense and antisense transcripts, in all possible reading frames, may contribute to pathogenesis [17, 34]. Definitive distinction of c9FTD/ALS on pathological features now appears possible although reports of mixed pathological appearances [24] exist and could correspond to variability in phenotypic expression [27]. Therefore, the mechanism by which C9ORF72 mutation leads to expression of FTD and/or ALS currently remains uncertain [28]. Leading theories include any combination of (a) loss of normal gene function with haplo-insufficiency, (b) toxic gain of function via atypical translation products, or (c) interference in normal nuclear processing by RNA G-quadruplexes [16]. Description of sequential pathological changes might further illuminate the relative importance of each potential aetiological process. We describe a Caucasian kindred with FTD caused by the C9ORF72 expansion mutation, including a mother (Case 1) and her two sons (Cases 2 and 3). There is no history of consanguinity, but there is a wider family history of intellectual disability and dementia (Fig. 1). The following
Fig. 1 Circles represent female individuals and squares males. Filled symbols are those subjects with relevant symptoms of intellectual disability and/or dementia and strike-through represents deceased. The
proband is indicated by an arrow. The top number represents age of symptom onset or childhood learning difficulty (LD); the bottom number is age at death
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clinical descriptions include, in Cases 2 and 3, childhood intellectual disability followed by apparent progressive cognitive decline in early adulthood; for comparison, previous young adult cases of c9FTD/ALS include FTD onset at 18 years [45] and ALS aged 20 years [53]. Case 1 was included in a previously reported series [31] but the autopsy findings are only now reported.
Materials and methods The C9ORF72 mutation was confirmed in lymphocyte DNA from Cases 1 and 2. Two repeat-primed PCR reactions were conducted. Each reaction included a separate, non-overlapping flanking primer and a repeat primer homologous to the GGGGGC repeat. The repeat primer annealed at multiple sites within the repeat to produce variable sized products. These products were in turn amplified by a third (tail) primer binding to the 3′end. Cycling conditions employed touchdown PCR from 70 to 56 °C and products were sized on an ABI 3730 genetic analyzer (Applied Bio-systems, Foster City, CA, USA). Repeat-primed PCR did not allow for sizing of the repeat, hence Southern blot was performed with two separate digests (EcoRI+BamHI and EcoRI). Post-mortem brain examinations of Cases 1 and 2 are described. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using the following antibodies : ubiquitin (DAKO anti-ubiquitin; 1:500, following microwave antigen retrieval), hyperphosphorylated tau (Innogenetics AT-8; 1:2,000 following microwave antigen retrieval), α-synuclein (Zymed anti-α-synuclein; 1:10,000, following microwave antigen retrieval), phosphorylated neurofilament (Sternberger SMI 31; 1:8,000, following protease digestion), p62 (BD Transduction anti-p62 Lck; 1:1,600 following microwave antigen retrieval), TDP43 (Proteintech anti-TDP-43; 1:12,000 following microwave antigen retrieval) A4 protein (DAKO anti-β amyloid; 1:2,000 following formic acid pretreatment) and FUS (SigmaAldrich anti-FUS; 1:200 following microwave antigen retrieval). DPR pathology was demonstrated using a recently characterised mouse monoclonal antibody that recognizes extended glycine-alanine (GA) repeats (clone 5E9; 1:500 overnight following microwave antigen retrieval) [30].
Results Case 1 Clinical findings and investigations The proband presented to psychiatric services aged 46 years with agitated depression. She had left school aged
14 years without qualifications and poor literacy. During her 4-month admission, extra-pyramidal features were noted following anti-psychotic administration. Computed tomography (CT) head and electroencephalogram (EEG) were within normal limits. Psychometric testing at that time found frontal lobe impairment with a verbal fluency below the fourth percentile. Two years on, she had become disinhibited, was unable to assist with household duties and her self-care had deteriorated. Further investigations included normal cerebrospinal fluid (CSF) examination; EEG was of low amplitude but with preserved alpha and magnetic resonance imaging (MRI) of brain revealed mild symmetric atrophy, not obviously frontotemporal in location. Cognitive assessment revealed a mini mental state examination score of 26/30, verbal IQ 71, performance IQ 74 and reading age 6–7 years. A clinical diagnosis of behavioural variant (BV) FTD was made. Genetic analysis excluded Huntington’s disease and dentatorubral-pallidoluysian atrophy and did not detect mutations in APP exons 16 and 17, PSEN1 exons 2–12, PSEN2 exons 4, 5 and 7, PRNP and CHMP2B. Following intentional overdose she was moved into residential care. Significant obsessive–compulsive symptoms were noted, with poor urinary continence and bizarre food behaviour involving mouth stuffing. More florid psychotic symptoms included delusions of control by the radio but she remained intolerant of anti-psychotics. Medical admission due to inadequate nutrition occurred at age 53 years. CSF 14-3-3 and muscle biopsy were normal. Percutaneous gastrostomy was undertaken but death from bronchopneumonia occurred shortly after. The C9ORF72 GGGGCC repeat expansion was detected in lymphocyte DNA by repeat-primed PCR and sized to at least 23 kb by Southern blot. Neuropathology General autopsy examination revealed evidence of ischaemic heart disease and bronchopneumonia. The brain weighed 1,095 g and showed a mild degree of generalised atrophy. Macroscopic examination of the brain was otherwise unremarkable and the substantia nigra was normally pigmented. Haematoxylin and eosin (H&E) stained sections from the cerebral cortex showed only very mild superficial laminar spongiosis and gliosis in the frontal lobes. There was mild diffuse gliosis of the caudate, putamen and globus pallidus. The brainstem, cerebellum and upper spinal cord appeared unremarkable. In the neocortex, the most significant pathology was the presence of numerous ubiquitin and p62-immunoreactive (ub–ir) neuronal cytoplasmic inclusions (NCI) and moderate numbers of neuronal intranuclear inclusions (NII) and dystrophic neurites (DN), throughout all cortical
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layers (Table 1). The NCI varied in size and appeared as small or large clusters of coarse granule or more smoothcontoured compact bodies. The NII were small dot-like inclusions, approximately the size of the nucleolus. The DN varied from short delicate threads to much longer, bulbous profiles. Small and large ub-ir granules were also present in the neuropil with no obvious cellular association. Small TDP-43-ir NCI and DN were present but much less common (less than 1 % the amount of ub-ir pathology), whereas DPR-ir NCI were widespread and abundant (Fig. 2). The morphology and distribution of the TDP-43-ir pathology were most consistent with FTLD–TDP type B. Rare tau-positive neurites were also present in the neocortex in the absence of any neurofibrillary tangles (NFT). In the hippocampus, there were numerous granular and compact ubiquitin/p62-ir NCI and occasional NII in dentate granule cells and numerous less compact granular NCI and occasional NII in CA3/4 pyramidal neurons. TDP-43ir NCI and NII were exceedingly rare in the dentate layer and absent in pyramidal neurons, while DPR-ir NCI were abundant in both layers. Tau-ir NFT, pre-tangles and DN were abundant in the entorhinal cortex but sparse in the hippocampal pyramidal layer (Braak stage II). The only pathology demonstrated in the midbrain was infrequent small ub-ir NCI and DN, primarily in the tectum and tegmentum. NCI and DN were also present in the striatum with more NCI demonstrated with TDP-43 than DPR IHC while the DN were most numerous in ubiquitin-stained sections. There were numerous small rounded ub-ir NCI and occasional NII in the granule cell layer of the cerebellum
(less well demonstrated with p62 IHC), but TDP-43-ir structures were not identified. Significant pathology was noted in all cortical layers of the cerebellum with DPR IHC. No pathology was demonstrated with IHC for neurofilament, A4 protein, α-synuclein or FUS (Fig. 2). Case 2 Clinical findings and investigations The elder son presented to paediatric services with significant speech and language delay. Examination did not reveal any obvious dysmorphism and there was no history of intrapartum asphyxia. His IQ at age 4 years was 39 and he required special education needs schooling. From 17 years, he was able to assist in farm labour, but developed anxious and obsessional behaviour with ritualistic checking. By 22 years he began to lose farming skills and faecal continence was problematic. EEG at that time was normal although he was noted to be unable to maintain eye closure. MRI did not show focal atrophy. Aged 25 years, he moved into residential care, but steadily deteriorated becoming withdrawn and violent. Antipsychotic medication led to bradykinesia, mouth stuffing food behaviour was also noted. Death occurred due to pulmonary embolus aged 26 years after a prolonged period of immobility. The C9ORF72 GGGGCC repeat expansion was detected in lymphocyte DNA by repeat-primed PCR. There was insufficient DNA for Southern blot.
Table 1 Summary of neuropathological immunohistochemistry Case 1
Frontal
HC-dentate CA3/4
Striatum
Cerebellum
Case 2
ubiq
p62
TDP
DPR
ubiq
p62
TDP
DPR
NCI++++ NII+++ DN+++ NCI++++ NII++ NCI++++ NII++ DN++ NCI++ NII+ DN++++ NCI++++ NII+
NCI++++ NII++ DN++ NCI++++ NII++ NCI+++ NII++ DN− NCI++ NII+ DN++ NCI++ NII+
NCI+ NII− DN++ NCI+ NII+ NCI− NII− DN− NCI+++ NII− DN+++ NCI− NII−
NCI++++ NII++ DN+ NCI++++ NII+++ NCI++++ NII+++ DN++ NCI++ NII− DN+ NCI++++ NII++
NCI+++ NII++ DN++ NCI++++ NII++ NCI+++ NII+ DN+ NCI++ NII− DN+++ NCI++ NII+
NCI++++ NII++ DN++ NCI++++ NII+ NCI++++ NII+ DN− NCI+ NII− DN++ NCI+ NII−
NCI− NII− DN− NCI− NII− NCI− NII− DN− NCI− NII− DN− NCI− NII−
NCI++++ NII++ DN+ NCI++++ NII++ NCI++++ NII++ DN++ NCI++ NII− DN− NCI+++ NII++
DN++
DN++
DN−
DN++++
DN+
DN+
DN−
DN+
Semi-quantitative comparison of immunohistochemistry found at autopsy in mother (Case 1) and elder son (Case 2) NCI neuronal cytoplasmic inclusions, NII neuronal intranuclear inclusions, DN dystrophic neurites − none,+ rare, ++ mild, +++ moderate, ++++ severe
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Fig. 2 Immunohistochemical findings case 1 (proband, a–e) and case 2 (son, f–h). The proband had very rare TDP-43-immunoreactive neuronal cytoplasmic inclusions (NCI) in the frontal neocortex (arrow, a) and hippocampal dentate granule layer (arrow, b). In contrast, immunohistochemistry using an antibody against GA dipeptide repeats (DPR) demonstrated much more abundant DPR-immunore-
active NCI in the frontal cortex (c), hippocampus (d) and cerebellar granular layer (e). No TDP-43-immunoreactive pathology was found in case 2; however, abundant DPR-immunoreactive NCI were present in the frontal cortex (f), hippocampus (g) and cerebellum (h). Scale bar 50 microns
Neuropathology
valproate. His family described him as irritable and nervous but neurological examination was normal. Investigations included serial MRI brain in 2005 (aged 23 years), 2008 and 2012. Subtle evidence of progressive widening of the sylvian fissures and enlargement of the temporal horns was noted. EEG was normal in 2005 but failure of full eye closure was noted with accompanying alpha attenuation. Genetic investigations included exclusion of fragile X syndrome and normal chromosomal micro array analysis. Neuropsychometry was performed aged 23 years. His Wechsler Abbreviated Score of Intelligence verbal IQ was 73, non-verbal 67. On the Schonell Graded Word Reading Test, he averaged 11 years. Assessment of memory on the Repeatable Battery for the Assessment of Neuropsychological Status scored 69 on immediate memory with a delayed memory score of 91. Subsequent review at age 27 years included comparable neuropsychometry. No relative weakness in short-term memory was noted this time on the Wechsler memory scale III. On the Delis–Kaplan Executive Function System trail making task, he attained a scaled score of 1 (average = 10). This was accompanied by a Behavioural Assessment of the Dysexecutive Syndrome “zoo map” open-ended test at which he was severely impaired indicating poor executive function but overall there was no clear decline from the previous assessment. By this time, however, frontal release signs were noted including palmomental and pout reflex accompanied by excessive gaze aversion. A further EEG aged 29 years showed long runs of frontotemporal slow waves without epileptiform activity.
General autopsy revealed extensive pulmonary embolism. The brain weighed 1,426 g and had a normal macroscopic appearance. H&E stained sections did not reveal any abnormalities, and in particular there was no obvious neuronal loss or gliosis in the cortex. The ub/p62-ir pathology was similar to that present in Case 1 but slightly less abundant in all neuroanatomical regions examined (Table 1). Numerous ub/p62-ir NCI and occasional NII and DN were present in the neocortex, numerous NCI and occasional NII in the hippocampal granule and pyramidal neurons and moderate numbers of large swollen DN and occasional NCI in the striatum. Moderate numbers of NCI and DN were present in the cerebellar granular layer. Importantly, no TDP-43-ir NCI or DN were identified in any region, in contrast to a widespread anatomical distribution of DPR-ir structures which correlated well with the ub/p62 staining (Fig. 2). No pathology was demonstrated with IHC for tau, A4, neurofilament, α-synuclein or FUS. Case 3 The younger son presented to an educational psychologist aged 3 years with global cognitive delay. He remained in mainstream education albeit with a statement of special education needs. After graduation from agricultural college, he began work on the family farm and was able to drive a tractor. He was seen by adult neurology services aged 22 years following generalised seizures and was treated with sodium
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He remains in the care of his family who describe him as very set in routines and lacking motivation. He and his family currently do not wish to pursue his C9ORF72 carrier status.
Discussion The discovery of a kindred with C9ORF72 mutation, abundant DPR pathology but little or no TDP-43 pathology, reinforces the pathogenic role of DPR inclusions. This kindred furthermore demonstrates clinical features well recognised within the FTD/ALS spectrum attributed to C9ORF72 mutation [23], but in addition the discovery of at least two cases with intellectual disability may further broaden the existing phenotypic range. Previous reports have noted a long prodromal period of psychotic symptoms and personality change [49], perhaps stretching back decades, which inevitably complicates attempts to quantify disease duration in genetic carriers. Subtle neuropsychological deficits have been described in other pre-symptomatic dementia mutation carriers before. PSEN-1 carriers show impairment in executive, memory and visuo-spatial function if tested within 10 years of expected age of onset [41] and executive dysfunction has been demonstrated many decades before predicted disease onset in carriers (mean age 31 years) of the MAPT mutation [18]. Significant intellectual disability has not previously been reported with C9ORF72 expansion and engenders consideration as to what age DPR aggregation becomes detectable in C9ORF72 patients. Could an inter-generational expansion of C9ORF72 hexanucleotide repeat size may underpin the intellectual disability and apparent early age of onset in our proband’s children? Other DNA repeat disorders, such as myotonic dystrophy, demonstrate anticipation such that more severe and earlier onset of disease [21] follows a dramatic increase in repeat tract size. Affected children of C9ORF72 carriers have shown a trend towards development of ALS at an earlier age, manifesting symptoms on average 7 years earlier than their parents in one study [12], but to date there is no evidence to suggest that this is caused by an inter-generational increase in repeat tract length. An alternative explanation for the observed childhood features of our cases would be the co-occurrence of an additional disorder. Such developmental vulnerability could unmask earlier expression of a subsequent FTD phenotype [42]. There were, however, no features such as dysmorphism or structural brain abnormalities to suggest a second diagnosis and genetic investigations (fragile X testing and chromosome analysis by array CGH) gave normal results. Nevertheless, and despite the absence of any pathological evidence to suggest concurrent mutations, the possibility of
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an as yet undiagnosed, possibly X linked, disorder resulting in intellectual disability remains. In addition, synergistic mutations in other genes implicated in FTD have not been fully excluded. Co-occurrence of more than one causative gene has been found above chance in familial ALS [8] and furthermore additive mutations may contribute to an earlier age of symptom onset [6, 40]. Conversely, a case with concurrent C9ORF72 repeat expansion and p.Ala239Thr MAPT variant did not exhibit early or aggressive disease onset despite mixed pathological findings [24]. The present report therefore demonstrates segregation of intellectual disability and subsequent FTD, with autosomal dominant inheritance of the C9ORF72 expansion. The autopsy findings provide significant new details of early pathological changes in the development of this disorder. The dominant abnormality throughout the cortex of Case 1 was that of NCI, NII and DN that stained for both ubiquitin and p62. TDP-ir pathology was present in a pattern consistent with the FTLD–TDP subtype which is most often reported in cases with the C9ORF72 mutation (type B), and distinct from the limited TDP pathology that often occurs as a coincidental finding in the setting of other neurodegenerative diseases (usually type A) [2]. However, the degree of this TDP-ir pathology was so limited as to be initially overlooked. In contrast, the consistent finding of abundant and widespread DPR pathology seems to represent a highly sensitive and specific pathological marker for the presence of the C9ORF72 mutation. The pathological examination of Case 2 uniquely captures an early phase in the evolution of c9FTD/ALS (Table 1), interrupted in this instance by death from pulmonary embolus. Although only one previous report of FTLD–UPS within C9ORF72 mutation carriers [19] exists, the total absence of TDP-43 aggregation again questions the primacy of this protein in the initiation of FTD pathogenesis. This finding is at variance with a recent study showing that phenotype and neurodegeneration correlated strongly with TDP-43, but not DPR, pathology [30]; however, it would still seem highly likely that DPR protein formation and pathological deposition precedes that of TDP-43 accumulation. Abnormalities of TDP-43 causing neurodegeneration are of course well described in both human TDP-43 mutation carriers [48] and in animal models [51, 54, 55] but the exact function of the protein is actively under research. The question remains whether the severe intellectual disability of this case could be a developmental consequence of the extensive DPR-ir pattern in clinically relevant anatomical locations, despite the lack of macroscopic abnormalities. Assuming instead that the ub/p62/DPR-ir intra-neuronal inclusions described do not fully account for Case 2’s cognitive impairment, the pathological changes could then be considered to reflect very early, perhaps even preclinical
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FTD. These findings do not contradict proposed staging classifications for TDP-43 “proteinopathies”, which now include cases with C9ORF72 repeat expansions [10], since pre-symptomatic pathological changes were already anticipated, if not previously demonstrated. It may yet be the case that TDP-43 metabolism is at some critical point interrupted as a consequence of the C9ORF72 mutation, but aetiological descriptions will presumably remain speculative until more detail of the gene’s physiological function is discovered. The full components of the ub/p62-ir intra-neural inclusions continue to be described with various protein species already identified [9, 13, 35] in addition to sense and antisense DPR transcripts [34]. Previous proposed mechanisms of FTD pathogenesis are not necessarily excluded by the presence of DPR protein and RNA foci, and indeed RNA foci do not co-localise with TDP-43 aggregates [25]. The relationship between C9ORF72 mutation and TDP-43 cytoplasmic mislocalisation therefore remains opaque, but animal models currently in development may elucidate this further [56] and detection of DPR formation will remain a crucial investigative step in determining the cellular processes that culminate in FTD. In conclusion, this report describes pathological evidence of DPR protein deposition in a genetically confirmed case of C9ORF72 mutation, prior to the development of obvious TDP-43 pathology. The clinical features may represent an unusual extended prodrome of childhood learning disability, thus potentially expanding the existing FTD phenotype. Following submission of our manuscript, another paper was published that includes description of a patient with the C9ORF72 mutation, abnormal development and early learning disability [22], further supporting our findings of this expanded phenotype. Acknowledgments MP is supported by the Guarantors of Brain. DE is supported by the Helmholtz Young Investigator program (HZNG-607) and by a grant of the Centres of Excellence in Neurodegeneration Research (CoEN). IM is supported by Canadian Institutes of Health Research (74580) and the Pacific Alzheimer’s Research Foundation (C06-01).
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