Exposure to environmental toxins and the risk of sporadic motor neuron disease: an expanded Australian case-control study.

BACKGROUND AND PURPOSE: It remains unclear what role environmental toxins play in sporadic motor neuron disease (SMND) and its most common subtype, amyotrophic lateral sclerosis (SALS). Most previous studies of this issue have contained only small numbers of SMND cases. We sought to re-examine possible associations between toxins and SMND in a large Australian case-control study. METHODS: Questionnaire data were available from 787 patients with SMND (614 with SALS) and 778 non-related controls. Individuals were asked whether they had been exposed to metals or chemicals/solvents at work or to herbicides/pesticides. Chi-square tests with odds ratios and 95% confidence intervals were calculated for responses, and significance levels were corrected for multiple testing. RESULTS: Men were more likely to acquire SALS if they worked with metals (OR = 1.95, 95% CI = 1.24-3.07) or chemicals/solvents (OR = 1.96, 95% CI = 1.46-2.61) or if they had been exposed to herbicides or pesticides (OR = 1.77, 95% CI = 1.30-2.39). Women who had worked with chemicals or solvents also appeared to be at increased risk of acquiring SALS (OR = 1.71, 95% CI = 1.22-2.40). CONCLUSIONS: These results support previous reports that exposures to metals or chemicals are associated with SMND. A suggested protocol for future multinational studies of environmental toxins and SMND is presented.

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 METHODS: We conducted a meta-analysis of 10 published studies and one unpublished study of the paraoxonase locus, encompassing 4,037 ALS cases and 4,609 controls, including genome-wide association data from 2,018 ALS cases and 2,425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.

Low yield in screening patients with sporadic motor neuron disease for Kennedy disease.

The diagnostic yield of testing for Kennedy disease in patients diagnosed with sporadic motor neuron disease (MND) is unclear. We measured the CAG repeat lengths in the androgen receptor gene of patients with progressive limb weakness who had either upper and lower motor signs (n = 130), or lower motor neuron signs alone (n = 30). Only one patient with a long history of lower motor weakness had a repeat length in the Kennedy disease range. Testing for Kennedy disease is unlikely to benefit MND patients with upper motor neuron signs or those with a short history of lower motor signs.

Ubiquitin-positive inclusions and progression of pathology in frontotemporal dementia and motor neurone disease identifies a group with mainly early pathology.

Frontotemporal lobar degeneration (FTLD) with tau-negative, ubiquitin-positive inclusions has been a topic of major interest in recent years, with this group now accounting for the majority of tau-negative cases of frontotemporal degeneration. The severity of neurodegeneration in FTLD is dependent on the stage of disease and is substantial even in the earliest stages. Elucidating the pathogenesis of FTLD requires evaluation of changes during the earliest possible stage of disease. However, the long survival of most frontotemporal dementia cases means that cases with early neuropathology are not frequently encountered. Cases of FTLD with the shortest survival are those with coexisting motor neurone disease (FTLD + MND), making these the ideal group for studying early FTLD pathology. It is not clear, however, what the pathological contribution of MND is in these cases. This study evaluates the pathology of 20 cases of FTLD (11 with no clinical signs of MND and nine with FTLD + MND) as well as 10 cases of MND without dementia. Our findings indicate that the deposition of ubiquitin does not play a key role in the neurodegenerative process in FTLD, and that the severity of neurodegeneration in FTLD is similar in cases with and without clinical MND.

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease.

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.

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

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 Alzheimer's disease but low agreement for cases with possible Alzheimer's 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 Alzheimer's 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.

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.

Mercury vapor uptake into the nervous system of developing mice.

The localisation of mercury in the developing nervous system following mercury vapor (Hg(0)) exposure is not clear. We therefore looked for mercury in the mouse nervous system following fetal or neonatal exposure to Hg(0). Mice were exposed to 50 or 500 microg/m(3) Hg(0) for 4 h a day for 5 days in late pregnancy, and pups sacrificed on postnatal day (P)1 or P40. Neonatal mice were exposed to 500 microg/m(3) Hg(0) for 2 h between P1 and P23, and were sacrificed 2 days later or at P40. Paraffin sections of the nervous system were stained with autometallography to detect inorganic mercury. No mercury was seen in the nervous system of pups after fetal exposure to the 50 microg/m(3) Hg(0) dose rate. After fetal exposure to the 500 microg/m(3) Hg(0) dose rate, mercury was seen in nervous system blood vessels and sensory ganglia. No mercury was seen in the nervous system after neonatal exposure to 500 microg/m(3) Hg(0) for 2 h between P1 and P10. From this exposure at P11 onwards, mercury was detected in motor neurons. The lack of stainable mercury in early developing central neurons suggests that the fetal and neonatal nervous systems are somehow protected from Hg(0) uptake.

Severe infantile axonal neuropathy with respiratory failure.

We describe 5 infants (4 male, 1 female) with a severe intractable form of motor-sensory axonal neuropathy. All became ventilator-dependent, 4 have since died and 1 remains static. Diaphragmatic paralysis was an early feature with generalized neuropathy evolving rapidly. Nerve conduction studies and biopsies were consistent with axonal disease. This disorder could be a new condition or part of the spectrum of inherited neuropathies of the axonal degenerative type. It may be that there is a "switching-off" in the infant's Schwann cell-axonal interactions in utero or in the early postnatal period, resulting in severe progressive deterioration and then a static period without recovery.

Bismuth autometallography: protocol, specificity, and differentiation.

We provide a detailed protocol of the autometallographic bismuth technique and evaluate the specificity of the technique. We show by the multi-element technique "proton-induced X-ray microanalysis" (PIXE) that the autometallographic grains contain silver, bismuth, and sulfur, proving that autometallography can be used for specific tracing of bismuth bound as bismuth sulfide clusters in tissue sections from Bi-exposed animals or humans. In sections from animals exposed concurrently to selenium and bismuth, the autometallographic grains also contain selenium. This demonstrates that, if present in excess in the organisms, selenium will bind to exogenous bismuth, creating bismuth selenide clusters. As a further possible control for specificity and as a tool for differentiating among autometallographically detectable metals in sections containing more than one, we describe how bismuth sulfide clusters can be removed from Epon-embedded tissue sections by potassium cyanide.

Uptake of bismuth in motor neurons of mice after single oral doses of bismuth compounds.

Bismuth, a component of many gastrointestinal medications, is a heavy metal little studied as regards nervous system uptake. We were interested to see if low doses of intragastric bismuth entered the nervous system, and if dietary selenium influenced the amount of bismuth detected. Mice were given 40 to 1200 mg/kg of bismuth subnitrate (BSN), bismuth subsalicylate (BSS), colloidal bismuth subcitrate (CBS), or ranitidine bismuth citrate (RBC) intragastrically. Mice on low- or high-selenium diets were given 4 to 32 mg/kg of bismuth from RBC. One week later, sections of nervous tissue were stained with autometallography to detect bismuth grains (Bi(AMG)). Bismuth was found in neurons with axons outside of the nervous system, in particular motor neurons, and in cells outside the blood-brain barrier. The lowest bismuth dose which resulted in Bi(AMG) in motor neurons was 696 mg/kg from BSN, 57 mg/kg from BSS, 29 mg/kg from CBS, and 26 mg/kg from RBC. No bismuth was seen in motor neurons of mice on the low-selenium diet. Intragastric doses of bismuth therefore enter mouse motor neurons, and the amount detectable varies with dietary selenium.

Tissue uptake of bismuth from shotgun pellets.

Shotgun pellets containing bismuth have been suggested to be less environmentally toxic than those containing other metals. We sought to find if bismuth from shotgun pellets embedded within an animal enters the tissues of that animal. Five bismuth-containing shotgun pellets were placed intraperitoneally into adult mice. Four or 9 weeks later the tissue distribution of bismuth was examined histologically using silver lactate autometallography. Bismuth was seen in the nervous system of the mice, either in cells with processes outside the nervous system or in cells not protected by the blood-brain barrier. Bismuth was also seen in the kidney, liver, spleen, and lung. The amount of bismuth within tissues varied widely between animals at both time intervals. Bismuth from shotgun pellets enters the tissues of mice, with some mice taking up more bismuth than others. Some animals wounded with bismuth pellets are therefore likely to accumulate large amounts of potentially toxic bismuth in their tissues.

Histochemical tracing of bismuth in testis from rats exposed intraperitoneally to bismuth subnitrate.

The histochemical silver amplification technique autometallography (AMG), was used to trace bismuth in the testis of Wistar rats injected intraperitoneally with bismuth subnitrate. In the seminiferous tubules, bismuth was located in lysosomes of Sertoli cells closely associated with heads of spermatids in the late stages of the spermatogenesis, i.e. shortly before the release of Step 19 spermatids in Stage XIII. No bismuth-specific AMG silver grains were detected in the spermatogenic cell line. However, tails of free sperm cells located in the tubular lumen showed autometallographic grains in close contact to the nine outer microtubule doublets in the axonema. Leydig cells concentrated huge amounts of AMG-bismuth in their lysosomes. Furthermore, parallel exposure to selenium significantly increased the amount of histochemically traceable bismuth in the rat testis.

Vertebral artery compression resulting from head movement: a possible cause of the sudden infant death syndrome.

OBJECTIVE: Vertebral artery compression causing brainstem ischemia has been suggested to underlie the sudden infant death syndrome. Vertebral artery distortion from neck movements has been demonstrated by angiography in infants, but direct evidence for arterial compression is lacking. In an attempt to demonstrate vertebral artery compression from head movement, we examined at postmortem the vertebral arteries of infants after neck extension or rotation. METHODS: The C1-C7 spinal column, together with a 2-cm rim of skull base, was removed from 20 infants dying from sudden infant death syndrome or other causes. In 5 cases the neck was extended, in 9 cases it was rotated 90 degrees to the right, and in 6 cases the neck was held in the neutral position. The neck was maintained in these positions during formalin fixation, and serial sections of selected blocks were examined microscopically. RESULTS: In 3 of 5 extended cases, bilateral vertebral artery compression was seen between the occipital bone and C1. In 3 of 9 rotated cases, the left vertebral artery was compressed adjacent to C1 before the artery entered the transverse foramen. No vertebral artery compression was seen in the necks held in the neutral position. CONCLUSIONS: The vertebral arteries of some infants can be compressed by neck movement. This could induce lethal brainstem ischemia in infants with inadequate collateral blood flow or with poor compensatory arterial dilatation, and may underlie some cases of sudden infant death syndrome.

Prolonged survival and pulmonary metastasis after local cure of glioblastoma multiforme.

A 59-year-old woman underwent a frontal lobectomy and irradiation for a glioblastoma multiforme, with further craniotomies to drain postoperative subdural and extradural infections. She survived for 5 years after the initial operation, but died as a result of pulmonary metastatic tumors. At post-mortem examination no residual intracranial tumor could be found. The pulmonary tumors were metastases from the glioblastoma. Prolonged survival, extraneural metastasis and curative local treatment are each uncommon in glioblastoma, but all occurred in this patient.

Mercury in human spinal motor neurons.

Inorganic mercury has been proposed as a neurotoxin that could cause sporadic motor neuron disease (SMND). We were therefore interested to see if mercury could be detected in the upper and lower motor neurons of SMND patients, and if mercury accumulated within motor neurons during life. Paraffin sections of formalin-fixed spinal cord (22 control adults, 20 SMND adults, 25 infants) and frontal primary motor cortex (9 control adults, 18 SMND adults, 20 infants) were stained with silver nitrate autometallography to detect ionic mercury. Mercury was found in the spinal motor neurons of 36% of adult control cases and 45% of adult SMND cases, with no significant difference between groups. No mercury was seen in infant spinal motor neurons, or in any adult or infant corticomotoneurons. In conclusion, many humans appear to accumulate mercury in their spinal motor neurons by the time they are adults, but mercury does not appear to play a major role in the loss of upper or lower motor neurons in SMND.