Roger Pamphlett

Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: A cross-sectional study

Summary Background We aimed to accurately estimate the frequency of a hexanucleotide repeat expansion in C9orf72 that has been associated with a large proportion of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Methods We screened 4448 patients diagnosed with ALS (El Escorial criteria) and 1425 patients with FTD (Lund-Manchester criteria) from 17 regions worldwide for the GGGGCC hexanucleotide expansion using a repeat-primed PCR assay. We assessed familial disease status on the basis of self-reported family history of similar neurodegenerative diseases at the time of sample collection. We compared haplotype data for 262 patients carrying the expansion with the known Finnish founder risk haplotype across the chromosomal locus. We calculated age-related penetrance using the Kaplan-Meier method with data for 603 individuals with the expansion. Findings In patients with sporadic ALS, we identified the repeat expansion in 236 (7·0%) of 3377 white individuals from the USA, Europe, and Australia, two (4·1%) of 49 black individuals from the USA, and six (8·3%) of 72 Hispanic individuals from the USA. The mutation was present in 217 (39·3%) of 552 white individuals with familial ALS from Europe and the USA. 59 (6·0%) of 981 white Europeans with sporadic FTD had the mutation, as did 99 (24·8%) of 400 white Europeans with familial FTD. Data for other ethnic groups were sparse, but we identified one Asian patient with familial ALS (from 20 assessed) and two with familial FTD (from three assessed) who carried the mutation. The mutation was not carried by the three Native Americans or 360 patients from Asia or the Pacific Islands with sporadic ALS who were tested, or by 41 Asian patients with sporadic FTD. All patients with the repeat expansion had (partly or fully) the founder haplotype, suggesting a one-off expansion occurring about 1500 years ago. The pathogenic expansion was non-penetrant in individuals younger than 35 years, 50% penetrant by 58 years, and almost fully penetrant by 80 years. Interpretation A common Mendelian genetic lesion in C9orf72 is implicated in many cases of sporadic and familial ALS and FTD. Testing for this pathogenic expansion should be considered in the management and genetic counselling of patients with these fatal neurodegenerative diseases. Funding Full funding sources listed at end of paper (see Acknowledgments).

Brain stem serotonin-synthesizing neurons in Alzheimer's disease: a clinicopathological correlation

SummaryThe location and number of brain stem serotonin-synthesizing neurons were analyzed in 11 patients with Alzheimers disease (AD) and 5 agematched controls using immunohistochemical techniques. In addition, the number of neuritic plaques and neurofibrillary tangles in the cortex and brain stem raphe was evaluated, as was the number of Nissl-stained raphe neurons. AD patients could be classified into two groups based on their raphe pathology; patients with such pathology (AD+) and those without (AD−). The number of large raphe neurons correlated significantly with the number of serotonin-synthesizing neurons in control material, indicating that all large neurons were serotonergic. This relationship was not apparent in AD+ patients, in whom the number of serotonin-synthesizing neurons correlated with the number of neurofibrillary tangles in the raphe of these patients. This indicates that in AD+ patients the serotonin-synthesizing neurons were selectively affected. There was no correlation between raphe and cortical pathology or raphe pathology and patient sex, age, mini-mental score or depression score, even when such scores were weighted for the interval between testing and death. There was a trend for the raphe pathology to correlate with the age of onset and duration of dementia and the Blessed dementia score in AD+ patients. Most AD+ patients with severe raphe lesions had clinical dementia only, while AD− patients had additional clinical features. The raphe lesions were more dramatic in AD+ patients with a rapid progression of symptoms.

Midbrain neuropathology in idiopathic Parkinson's disease and diffuse Lewy body disease

We have quantified midbrain cell loss in idiopathic Parkinsons disease (PD) compared with controls; six patients had PD with onset before 70 years, five patients had late onset PD (>70 years) and nine patients had diffuse Lewy body disease. The pattern of cell loss in these last two groups has not been previously described. No age associated neuronal loss was seen in controls. There was cell loss and reduced area of the pars compacta in all cases but no difference in the pattern of cell loss, which was predominantly ventral. The amount of cell loss in the dorsolateral cluster correlated with the duration of Parkinsonian symptoms, while greater cell loss in the dorsomedial cluster correlated with the presence of tremor and the absence of early dementia. These results suggest that the topography of midbrain pathology does not assist in differentiating these overlapping syndromes.

Early terminal and nodal sprouting of motor axons after botulinum toxin

Axonal sprouting in distal motor axons was studied in an attempt to answer two questions: (a) is the cell body required for early axonal sprouting?, and (b) do nodal, as well as terminal, axonal sprouts arise after muscle inactivity not caused by nerve injury? Botulinum toxin (BT) was used to induce axonal sprouting without nerve trauma. Mice were injected in the right calf with a sublethal dose of BT and the soleus muscle examined ultrastructurally at times varying from 3 h to 5 days post-injection. Terminal axonal sprouts were seen 2 days after injection, and based on the time taken for BT to act and the growth rate of sprouts, axons were calculated to sprout within 24 h of muscle inactivity. This short time suggests that early axonal regrowth is initiated and controlled at the distal axon. Sprouts were seen arising from the intramuscular nodes of Ranvier from 2 days after BT injection. Unlike the terminal sprouts which elongated over time, the nodal sprouts remained short and confined by the basal lamina overlying the node, probably because without structural denervation there were no empty perineural sheaths to act as pathways to the motor endplates. The finding of terminal and nodal sprouts after botulinum toxin supports the hypothesis that muscle inactivity gives rise to a single growth factor for both terminal and nodal sprouting.

Amyotrophic Lateral Sclerosis and Exposure to Environmental Toxins: An Australian Case-Control Study

It has been suggested that environmental toxins could be risk factors for sporadic amyotrophic lateral sclerosis (SALS). We therefore analysed epidemiological data on 179 SALS cases and 179 age-, ethnicity- and sex-matched controls in Australia using self-reporting questionnaires. SALS was associated with solvent/chemical exposure (OR = 1.92, 95% CI: 1.26–2.93), overall herbicide/pesticide exposure (OR = 1.57, 95% CI: 1.03–2.41) and industrial herbicide/pesticide exposure (OR = 5.58, 95% CI: 2.07–15.06). Exposure to herbicides/pesticides showed a dose-response effect. All positive findings were more statistically significant in males. These findings support those from northern hemisphere studies, indicating that environmental toxins can be risk factors for SALS.

A large-scale international meta-analysis of paraoxonase gene polymorphisms in sporadic ALS

Background: Six candidate gene studies report a genetic association of DNA variants within the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). However, several other large studies, including five genome-wide association studies, have not duplicated this finding. Methods: We conducted a meta-analysis of 10 published studies and one unpublished study of the paraoxonase locus, encompassing 4,037 ALS cases and 4,609 controls, including genome-wide association data from 2,018 ALS cases and 2,425 controls. Results: The combined fixed effects odds ratio (OR) for rs662 (PON1 Q192R) was 1.09 (95% confidence interval [CI], 1.02–1.16, p = 0.01); the genotypic OR for RR homozygotes at Q192R was 1.25 (95% CI, 1.07–1.45, p = 0.0004); the combined OR for rs854560 (PON1 L55M) was 0.97 (95% CI, 0.86–1.10, p = 0.62); the OR for rs10487132 (PON2) was 1.08 (95% CI, 0.92–1.27, p = 0.35). Although the rs662 polymorphism reached a nominal level of significance, no polymorphism was significant after multiple testing correction. In the subanalysis of samples with genome-wide data from which population outliers were removed, rs662 had an OR of 1.06 (95% CI, 0.97–1.16, p = 0.22). Conclusions: In contrast to previous positive smaller studies, our genetic meta-analysis showed no significant association of amyotrophic lateral sclerosis (ALS) with the PON locus. This is the largest meta-analysis of a candidate gene in ALS to date and the first ALS meta-analysis to include data from whole genome association studies. The findings reinforce the need for much larger and more collaborative investigations of the genetic determinants of ALS.

Genetic susceptibility to environmental toxicants in ALS.

Environmental toxicants such as heavy metals, pesticides, and chemicals appear to be risk factors for sporadic amyotrophic lateral sclerosis (SALS). An impaired ability to break down these toxicants because of differences in detoxification genes could underlie some cases of this disease. We therefore examined the frequencies of single nucleotide polymorphisms (SNPs) in 186 SALS patients and 186 controls at the allele, genotype, and haplotype levels for the metallothionein (MT) family of genes, metal transcription factor‐1 (MTF‐1), and glutathione synthetase (GSS). Exposure to heavy metals, solvents/chemicals, and pesticides/herbicides was assessed by questionnaire, and gene‐toxicant interactions were analyzed. An intronic SNP upstream of MT‐Ie differed in SALS patients and controls at the allele and genotype levels. Haplotypes covering MT‐I isoforms also differed between the two groups. Alleles and genotypes of one MTF‐1 SNP differed in female SALS patients. One GSS haplotype interacted with both metals and solvents/chemicals to increase the risk of the disease. Differences in genes involved in handling toxicants, and interactions between toxicants and these genes, appear to be present in some patients with SALS. This suggests that impaired detoxification mechanisms play a role in SALS.

A genome-wide analysis of brain DNA methylation identifies new candidate genes for sporadic amyotrophic lateral sclerosis

Genetic variants may underlie sporadic amyotrophic lateral sclerosis (SALS), but in only a few percent of patients have causative mutations been found. This is possibly because SALS is more often due to a variation in DNA methylation, an epigenetic phenomenon involved in gene silencing. Methylation across the whole genome was examined in brain DNA of 10 SALS patients and 10 neurologically-normal controls. Methylated DNA was immunoprecipitated and interrogated by Affymetrix GeneChip Human Tiling 2.0R Arrays. Methylation levels were compared between SALS patients and controls at each region of methylation across the genome. SALS patients had either hypo- or hyper-methylation at 38 methylation sites (p ≤ 0.01). Of these, 23 were associated with genes and three with CpG islands. Pathway analysis showed that genes with different methylation in SALS were particularly involved in calcium homeostasis, neurotransmission and oxidative stress. In conclusion, a number of genes, either unsuspected in SALS or in potential cell death pathways, showed altered methylation in SALS brains. The possibility of epigenetic therapy for SALS should encourage confirmation of these initial results in a future larger whole-genome DNA methylation study.

Blood levels of toxic and essential metals in motor neuron disease

Toxic and essential metals have been implicated in the pathogenesis of sporadic motor neuron disease (SMND), but attempts to measure blood levels of these metals have led to contradictory results. We, therefore, measured blood levels of various metals using paired SMND/controls. In 20 subjects with SMND (15 males, five females, mean age 56.8 years) and 20 partner controls (15 females, five males, mean age 55.0 years) cadmium, lead, mercury, copper, zinc and selenium levels were measured in blood, plasma and red cells with inductively coupled plasma mass spectrometry and manganese levels with atomic absorption spectrophotometry. Results were analysed using non-parametric tests. Hypo-osmotic red blood cellfragility was estimated in six SMND/control pairs to see if hemolysis could account for increased metal levels. The plasma cadmium level was significantly raised in SMND cases (P = 0.005), but with considerable overlap between SMND and controls. No other metal levels were significantly different, though plasma lead in SMND had a tendency to be higher than controls. No difference in red cell fragility was found between groups. In conclusion, plasma levels of cadmium were raised in this SMND group, but the biological significance of this is uncertain. The measurement of metals in the blood of SMND cases seems unwarrranted for routine diagnostic testing.

Consensus neuropathological diagnosis of common dementia syndromes: testing and standardising the use of multiple diagnostic criteria

Abstract. The aim of this study was to assess the variation between neuropathologists in the diagnosis of common dementia syndromes when multiple published protocols are applied. Fourteen out of 18 Australian neuropathologists participated in diagnosing 20 cases (16 cases of dementia, 4 age-matched controls) using consensus diagnostic methods. Diagnostic criteria, clinical synopses and slides from multiple brain regions were sent to participants who were asked for case diagnoses. Diagnostic sensitivity, specificity, predictive value, accuracy and variability were determined using percentage agreement and kappa statistics. Using CERAD criteria, there was a high inter-rater agreement for cases with probable and definite Alzheimers disease but low agreement for cases with possible Alzheimers disease. Braak staging and the application of criteria for dementia with Lewy bodies also resulted in high inter-rater agreement. There was poor agreement for the diagnosis of frontotemporal dementia and for identifying small vessel disease. Participants rarely diagnosed more than one disease in any case. To improve efficiency when applying multiple diagnostic criteria, several simplifications were proposed and tested on 5 of the original 20 cases. Inter-rater reliability for the diagnosis of Alzheimers disease and dementia with Lewy bodies significantly improved. Further development of simple and accurate methods to identify small vessel lesions and diagnose frontotemporal dementia is warranted.