Judith Hartley

Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS)

Background Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. Methodology/Principal Findings Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. Conclusions/Significance We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.

Novel FUS/TLS Mutations and Pathology in Familial and Sporadic Amyotrophic Lateral Sclerosis

OBJECTIVE To determine the frequency of and clinicopathologic phenotypes associated with FUS/TLS mutations in a large cohort of amyotrophic lateral sclerosis (ALS) cases from the north of England. DESIGN Genetic screening project with neuropathologic examination of postmortem tissue in selected cases. The clinical details of selected cases are also presented. SETTING Neurology departments of 2 university teaching hospitals in the north of England. PARTICIPANTS The 15 exons of FUS/TLS were sequenced in an initial cohort of 42 familial ALS (FALS) and 117 sporadic ALS (SALS) cases. Exons 14 and 15 were subsequently screened in a larger cohort of 431 SALS cases. Regions mutated in ALS cases were also screened in 293 controls. MAIN OUTCOME MEASURE Evaluation of gene-sequencing chromatographs and detailed histopathologic analysis of the central nervous system. RESULTS Four heterozygous mutations, 1 of which is novel, were identified in 6 patients with ALS (4 with FALS and 2 with SALS). Two of the substitutions were not found to be present in controls, and neuropathology in these cases revealed neuronal and/or glial cytoplasmic inclusions positive for the FUS/TLS protein. One of these cases is also the first reported SALS case with an FUS/TLS mutation. The other 2 substitutions identified were also identified in control cases. Neuropathology in these cases revealed typical SALS pathology, suggesting that they are likely to represent benign polymorphisms. CONCLUSIONS FUS/TLS mutations represented approximately 5% of FALS cases screened. A FUS/TLS mutation was also identified in a single SALS case. Subsequent screening of this region in a larger cohort of SALS cases, however, did not reveal any additional mutations.

Broad clinical phenotypes associated with TAR-DNA binding protein ( TARDBP ) mutations in amyotrophic lateral sclerosis

The finding of TDP-43 as a major component of ubiquitinated protein inclusions in amyotrophic lateral sclerosis (ALS) has led to the identification of 30 mutations in the transactive response-DNA binding protein (TARDBP) gene, encoding TDP-43. All but one are in exon 6, which encodes the glycine-rich domain. The aim of this study was to determine the frequency of TARDBP mutations in a large cohort of motor neurone disease patients from Northern England (42 non-superoxide dismutase 1 (SOD1) familial ALS (FALS), nine ALS-frontotemporal dementia, 474 sporadic ALS (SALS), 45 progressive muscular atrophy cases). We identified four mutations, two of which were novel, in two familial (FALS) and two sporadic (SALS) cases, giving a frequency of TARDBP mutations in non-SOD1 FALS of 5% and SALS of 0.4%. Analysis of clinical data identified that patients had typical ALS, with limb or bulbar onset, and showed considerable variation in age of onset and rapidity of disease course. However, all cases had an absence of clinically overt cognitive dysfunction.

C9ORF72 expansions, parkinsonism, and Parkinson disease A clinicopathologic study

Objective: To determine the histopathologic bases for the observed incidence of parkinsonism in families with C9ORF72 expansions, which typically cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia. Methods: DNA was extracted from 377 brains with the histopathologic diagnosis of idiopathic Parkinson disease or related disorders and analyzed for C9ORF72 expansions. α-Synuclein and p62 immunohistochemistry of the substantia nigra (SN) was undertaken in brains of 17 ALS cases with (C9ORF72+) and 51 without (C9ORF72−) the C9ORF72 expansion. Results: Only 1 of 338 cases with pathologically confirmed idiopathic Parkinson disease had a C9ORF72 expansion. Similarly, only 1 of 17 C9ORF72+ brains displayed features suggestive of α-synucleinopathy. In contrast, p62-positive, TDP-43–negative neuronal cytoplasmic inclusions within the SN were considerably more frequent in C9ORF72+ brain tissue than in the C9ORF72− brains (p = 0.005). Furthermore, there was a more marked loss of dopaminergic neurons in the SN of C9ORF72+ ALS brains than C9ORF72− ALS brains (p = 0.029). Conclusions: SN involvement is common in C9ORF72+ ALS but can be clearly distinguished from Parkinson disease–related mechanisms by the presence of p62-positive inclusions and the absence of α-synuclein–positive Lewy bodies or Lewy neurites.

Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis

BackgroundVascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS.MethodsWe have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor.ResultsNo mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS.ConclusionMechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.

MUTATIONS IN VAPB ARE NOT ASSOCIATED WITH SPORADIC ALS

ALS is the third most common adult-onset neurodegenerative disorder. The majority of cases are sporadic (SALS), whereas 5 to 10% are familial (FALS). There are 13 known loci, with causative genes identified at seven, including superoxide dismutase 1 ( SOD1 ) and angiogenin ( ANG ).1,2 Recently, mutations in vesicle-associated membrane protein ( V AMP) a ssociated p rotein B ( VAPB ) have been associated with autosomal dominant, adult-onset ALS8.3 VAPB is ubiquitously expressed, and evidence suggests that it plays a role in vesicle trafficking.4 As VAPB was the only gene, other than SOD1 , known to cause adult-onset, autosomal dominant ALS, we undertook a mutation screening strategy to establish the contribution of VAPB mutations in a large cohort of ALS patients. Although it is appreciated that mutations would be most likely to be found in familial cases, mutations in SOD1 and more recently ANG have been reported in SALS. Therefore, both FALS and SALS cases were screened for mutations in VAPB . ### Methods. DNA was extracted from 301 cases of ALS (23 FALS in whom …

Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation

Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases.

Mitochondrial DNA haplogroups and amyotrophic lateral sclerosis

Sir: Mitochondrial dysfunction has been implicated in the pathophysiology of sporadic amyotrophic lateral sclerosis (ALS). One group [1] (but not others [2]) reported deficient mitochondrial respiration in ALS cybrids when compared to controls sharing the same nuclear background but containing different mtDNA, suggesting that inherited genetic variants of mtDNA contribute to the pathophysiology of ALS. In keeping with this, mtDNA haplogroup I was significantly more common in 222 Italian ALS patients than in 151 control subjects [3]. We sought to confirm these findings in a larger UK cohort. We studied three groups. Group 1: 102 neuropathologically confirmed cases of ALS and 179 controls confirmed at autopsy. Group 2: 402 cases of definite or probable ALS cases according to El Escorial criteria and 344 sequential live births from the same geographic region. Group 3: 493 UK population controls (UK MRC 1958 cohort). There was no difference in the overall mtDNA haplogroup distribution between the neuropathologically proven cases of ALS and controls (P=0.0629, Table 1). Fisher’s exact test for the individual haplogroups revealed a higher frequency of haplogroup V in the ALS cases compared to controls (P=0.02), which did not reach statistical significance after Bonferroni correction for the multiple haplogroups under comparison (P=0.22). Whilst this study was adequately powered to exclude a major haplogroup effect, there was limited power to detect a disease association with the less common haplogroups, including haplogroup I. We therefore compared a larger independent group of ALS cases to controls (group 2). Again, there was no significant difference in the overall haplogroup distribution. Fisher’s exact test did not reveal a significant association between haplogroup V (P=0.84) and haplogroup I (P=0.63), but the frequency of haplogroup W was significantly less in the ALS cases than in controls (P=0.04). The frequency of haplogroup W in the group 2 controls was greater than other published UK values, raising the possibility that the apparent association was actually due to a spuriously high frequency of W. Comparison of the entire ALS cohort (n=504) to the 493 UK population controls revealed an almost identical haplogroup distribution (P= 0.8444), including the frequency of haplogroup W (P=0.77). After pooling all of the data from the ALS cases (n=504) and all of the controls (n=1016), there was no significant difference in the frequency of haplogroup W (P=0.54). We found no evidence that mtDNA haplogroups contribute to the risk of developing ALS. Although we cannot exclude a weak association with one of the rarer haplogroups, or a monogenic form of ALS due to a pathogenic mtDNA mutation in some families, this is unlikely to have a major impact on sporadic cases of the Neurogenetics (2007) 8:65–67 DOI 10.1007/s10048-006-0066-9

Neurodegeneration caused by intronic expansions of C9ORF72 is a clinically heterogeneous but pathologically distinct disease

Abstract Background Crossover between neurodegenerative diseases is described but poorly understood. Hexanucleotide repeat expansions of C9ORF72 are a major cause of motorneuron disease (MND)/frontotemporal dementia but we and others have observed clinical heterogeneity within C9ORF72 -positive probands and their families, including a high incidence of parkinsonism. We aimed to determine whether C9ORF72 expansions are an upstream cause of clinically and pathologically distinct neurodegenerative diseases. This would have significant implications for neurodegeneration research. Methods In cohorts of patients with clinical parkinsonism (n=518), multiple sclerosis (MS) (n=215), and MND (n=563) we screened DNA for the C9ORF72 expansion, reviewed clinical histories, and undertook pathological evaluation of brain tissue where available. Findings We identified the C9ORF72 expansion in one patient with clinical parkinsonism (0·2%), 23 patients with MND (13·7%), and none of the patients with MS. The C9ORF72 positive parkinsonian patient had a family history of MND and displayed pathology consistent with MND with C9ORF72 expansion in addition to α-synucleinopathy. Two further patients with MND were identified with α-synucleinopathy: one with the C9ORF72 expansion, the other without. Of five MND patients who initially presented with MS, four (80%) were positive for the C9ORF72 expansion. C9ORF72 -MND is more rapidly progressive in the presence of preceding MS. Pathological examination of MND patients with C9ORF72 expansions revealed p62 positive, TDP-43 negative neuronal cytoplasmic inclusions in frontal cortex, hippocampus, and substantia nigra, which were relatively absent in MND patients without C9ORF72 expansions. Interpretation C9ORF72 expansions are not a major cause of either classic Parkinsons disease with α-synucleinopathy or MS. MS appears to increase the penetrance of the C9ORF72 expansion and exaggerate the severity. We suggest that p62 positive, TDP-43 negative neuronal cytoplasmic inclusions within the substantia nigra account for the association between C9ORF72 expansions and parkinsonism. Moreover we suggest that the distribution of these inclusions determines the clinical heterogeneity of C9ORF72 disease. Funding UK Medical Research Council.