The histopathological spectrum of malignant hyperthermia and rhabdomyolysis due to RYR1 mutations.

OBJECTIVE: The histopathological features of malignant hyperthermia (MH) and non-anaesthetic (mostly exertional) rhabdomyolysis (RM) due to RYR1 mutations have only been reported in a few cases. METHODS: We performed a retrospective multi-centre cohort study focussing on the histopathological features of patients with MH or RM due to RYR1 mutations (1987-2017). All muscle biopsies were reviewed by a neuromuscular pathologist. Additional morphometric and electron microscopic analysis were performed where possible. RESULTS: Through the six participating centres we identified 50 patients from 46 families, including patients with MH (n = 31) and RM (n = 19). Overall, the biopsy of 90% of patients showed one or more myopathic features including: increased fibre size variability (n = 44), increase in the number of fibres with internal nuclei (n = 30), and type I fibre predominance (n = 13). Abnormalities on oxidative staining, generally considered to be more specifically associated with RYR1-related congenital myopathies, were observed in 52%, and included unevenness (n = 24), central cores (n = 7) and multi-minicores (n = 3). Apart from oxidative staining abnormalities more frequently observed in MH patients, the histopathological spectrum was similar between the two groups. There was no correlation between the presence of cores and the occurrence of clinically detectable weakness or presence of (likely) pathogenic variants. CONCLUSIONS: Patients with RYR1-related MH and RM exhibit a similar histopathological spectrum, ranging from mild myopathic changes to cores and other features typical of RYR1-related congenital myopathies. Suggestive histopathological features may support RYR1 involvement, also in cases where the in vitro contracture test is not informative.

Leprosy: a 'common' and curable cause of peripheral neuropathy with skin lesions.

Leprosy (or Hansen's disease) is a curable chronic infectious disease caused by the acid-fast bacillus Mycobacterium leprae. While leprosy remains one of the most common causes of neuropathy worldwide, its rarity in the UK means that many doctors are unfamiliar with the typical clinical features. This is problematic because early recognition and treatment is vital in order to minimise disease-related complications such as nerve injury. We describe a 75-year-old man who presented with multiple mononeuropathy (mononeuritis multiplex, particularly affecting the ulnar nerves) and typical granulomatous skin lesions, in whom the diagnosis was made on the basis of skin biopsy. We highlight the clinical features, investigations and treatment of the patient, and provide information about the epidemiology and pathogenesis of leprosy.

Familial amyotrophic lateral sclerosis. Molecular pathology of a patient with a SOD1 mutation.

We report the clinical, genetic, and neuropathologic findings in a patient with rapidly progressive familial amyotrophic lateral sclerosis (ALS). We detected a point mutation at codon 48 of the Cu/Zn superoxide dismutase gene (SOD1) leading to a substitution of histidine by glutamine in the copper-binding domain. The histopathologic features are consistent with those described in rapidly progressive sporadic ALS and do not support claims that sporadic and familial disease are different pathologic entities. Neurofilamentous accumulations, hyaline, and ubiquitinated inclusions were present in the motor cortex, brainstem, and anterior horn cells, but there was no evidence of abnormal SOD1 immunoreactivity. This confirms that the cytoskeletal pathology specific to ALS is secondary to an unknown biochemical disturbance caused by mutant SOD1 molecules and not its toxic accumulation.

Increased mitochondrial superoxide dismutase activity in Parkinson's disease but not amyotrophic lateral sclerosis motor cortex.

Oxidative stress may contribute to the neurodegenerative process in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Motor cortex in PD is not affected and its inclusion in studies of free radical involvement in ALS pathogenesis could help elucidate whether oxidative stress is disease specific or a more widespread phenomenon present in the neurodegeneration. We have measured cytosolic and mitochondrial isoforms of superoxide dismutase (SOD), antioxidant enzymes involved in primary defence against free radical damage, in motor cortex of six patients with sporadic form of ALS (SALS), eight with PD and eight normal control subjects. We have found no difference in the activities of cytosolic and mitochondrial SOD between SALS and control motor cortex. Mitochondrial SOD activity in PD motor cortex was, however, significantly higher than in SALS and control motor cortex whereas activity of cytosolic SOD was lower than in two other groups although the differences were not statistically significant. Our findings indicate the presence of an altered antioxidant defence system in PD but not ALS upper motor neurons, suggesting that oxidative stress may be a widespread phenomenon in PD.

Cells from individuals with SOD-1 associated familial amyotrophic lateral sclerosis do not have an increased susceptibility to radiation-induced free radical production or DNA damage.

Oxidative stress may play a role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS). Superoxide dismutases (SODs) are enzymes that can influence free radical processes in irradiated cells and there is some evidence that manipulation of SODs can affect survival of cells after radiation treatments. SOD-1 associated FALS mutants may have an altered radiation response due to an enhanced generation of hydroxyl radicals or a compromised ability to neutralize free radicals. We have investigated the ability of the lymphoblastoid cell lines from FALS patients with SOD-1 gene mutations, patients with sporadic ALS and controls to handle oxidative stress induced by ionising radiation by measuring levels of intracellular reactive oxygen species and production of DNA double-strand breaks. Levels of reactive oxygen species, expressed as the slope of the relative fluorescence of a radical-reactive fluorochrome, in the cells from familial ALS patients with SOD-1 gene mutations (2.14+/-1.06 Gy(-1)) and patients with sporadic ALS (1.38+/-0.21 Gy(-1)) were not significantly different from the controls (1.54+/-0.39 Gy(-1)). No significant difference was observed in the production of DNA double-strand breaks between three groups. The ability of lymphoblastoid cells from FALS patients with SOD-1 gene mutations to scavenge radiation-induced free radicals is not compromised nor is their ability to protect DNA damage induced by ionising radiation.

Uptake of aluminum and gallium into tissues of the rat: influence of antibody against the transferrin receptor.

Transport of aluminum and gallium from blood into rat tissues following continuous i.v. infusion of metals in different chemical forms has been investigated. Tissue uptake of aluminum and gallium was similar and highly dependent on the chemical species of the metals. Aluminum and gallium accumulated in liver and spleen when infused in the chloride form. Raised citrate markedly enhanced aluminum and gallium uptake into renal cortex and bone; in contrast with gallium-transferrin, citrate increased uptake of 67Ga into renal cortex and bone by 8- and 14-fold respectively. Uptake of 67Ga with citrate into renal cortex was around 3 times smaller than that of aluminum. The antitransferrin receptor antibody OX-26 enhanced 67Ga uptake from gallium citrate into all rat tissues. 67Ga from purified gallium-transferrin was also taken into all tissues in the presence of OX-26, the effect being greatest in renal cortex and bone. No influence of antibody on aluminum transport into rat tissues was, however, observed when aluminum was infused in the citrate form. Therefore, transport of aluminum and gallium into tissues is not similar under all conditions. Transport of each metal occurs into all tissues in the presence of antitransferrin receptor antibody. The potential for such transport is much greater in the case of gallium. Transport of aluminum and gallium citrate complexes appears important especially in the renal cortex and bone.

The effect of aluminium chloride upon the transition of drugs through the blood-brain barrier into the central nervous system.

In order to determine the effect of Al3+ upon the transition of drugs through the blood-brain barrier into the central nervous system we examined its effect upon a drug that dissociates as a cation (quinidine) and drugs that dissociate as anions (acetylsalicylic acid and pentobarbital). The entry and exit of quinidine into and out of the brain in mice pre-treated with AlCl3 was inhibited. Al3+ did not compete with acetylsalicylic acid for the penetration through the blood-brain barrier but did slow down its elimination from the brain. Brain kinetics of the examined drugs showed good correlation with their central pharmacodynamic effects.

Mutations in RYR1 are a common cause of exertional myalgia and rhabdomyolysis.

Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of neuromuscular disease, ranging from various congenital myopathies to the malignant hyperthermia (MH) susceptibility trait without associated weakness. We sequenced RYR1 in 39 unrelated families with rhabdomyolysis and/or exertional myalgia, frequent presentations in the neuromuscular clinic that often remain unexplained despite extensive investigations. We identified 9 heterozygous RYR1 mutations/variants in 14 families, 5 of them (p.Lys1393Arg; p.Gly2434Arg; p.Thr4288_Ala4290dup; p.Ala4295Val; and p.Arg4737Gln) previously associated with MH. Index cases presented from 3 to 45 years with rhabdomyolysis, with or without exertional myalgia (n=12), or isolated exertional myalgia (n=2). Rhabdomyolysis was commonly triggered by exercise and heat and, less frequently, viral infections, alcohol and drugs. Most cases were normally strong and had no personal MH history. Inconsistent additional features included heat intolerance, and cold-induced muscle stiffness. Muscle biopsies showed mainly subtle changes. Familial RYR1 mutations were confirmed in relatives with similar or no symptoms. These findings suggest that RYR1 mutations may account for a substantial proportion of patients presenting with unexplained rhabdomyolysis and/or exertional myalgia. Associated clinico-pathological features may be subtle and require a high degree of suspicion. Additional family studies are paramount in order to identify potentially MH susceptible relatives.

ALSODatabase: database of SOD1 (and other) gene mutations in ALS on the Internet. European FALS Group and ALSOD Consortium.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for 20-30% of all familial cases of ALS and for 1-2% of all ALS cases. The exact number of these mutations is, however, unknown, as is the number of individuals worldwide with them who have ALS or are at risk of developing the disease. Information on the genetic and clinical data of ALS patients with the SOD1 gene mutations is, also, only available for one-third of all families and is incomplete in some of those families. Some attempts have been made to correlate the genotype with the phenotype of the patients with SOD1 gene mutations and the available evidence suggests that only a few mutations could be linked to a consistent age of onset or pattern of survival. The ALS scientific community has recognized an urgent need for collecting the genetic and clinical information on all SOD1 individuals and the ALSOD Consortium was set up with the aim of pooling all available data into a uniform centralized database. Recent advances in information technology and the wide accessibility of the Internet have provided an ideal vehicle through which data can not only be recorded but also searched, amended and updated in real time. Here, we describe the work done so far by the ALSOD Consortium and plans for the future.

Determination of aluminium in different tissues of the rat by atomic absorption spectrometry with electrothermal atomization.

Atomic absorption spectrometry with electrothermal atomization was used for the determination of aluminium in brain, liver, spleen, kidney cortex, skeletal muscle and bone of the rat following digestion by nitric acid and in serum following simple dilution and in situ oxygen ashing. The method of standard additions in the presence of a chemical modifier, ammonium dihydrogen-phosphate, was essential for bone tissues. The detection limits ranged from 3 to 58 ng per gram of wet mass of tissue and were 4-19 times lower than the observed physiological levels of aluminium. The between-day precision for serum was 8.9% at a mean concentration of 6.8 micrograms I-1 and 2.4% at a mean concentration of 125.3 micrograms I-1. Additionally, repeated analyses of National Institute of Standards and Technology Standard Reference Material 1577b Bovine Liver gave a relative standard deviation of 12.2% (mean concentration = 0.8 microgram g-1). Of the tissues studied, bone had at least ten times higher levels of aluminium than others (0.959 +/- 0.322 micrograms g-1). The aluminium concentration in cerebellum (0.073 +/- 0.043 micrograms g-1) was approximately twice that in the cerebral hemisphere (0.034 +/- 0.009 micrograms g-1).

Uptake of Ga-67 into rat cerebral hemisphere and cerebellum. Comparison with Fe-59.

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of 67Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K(in)) were four times slower than those for 59Fe. K(in) for 67Ga when infused with citrate were 0.88 +/- 0.24 and 0.94 +/- 0.39 x 10(-3) ml g-1h-1 for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, 67Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of 59Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of 67Ga into brain, particularly when infused with citrate; mean increases in uptake of 67Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified 67Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.

Mutations in the lysyl oxidase gene are not associated with amyotrophic lateral sclerosis.

BACKGROUND: There is an urgent need to identify genes involved in familial ALS (FALS), as mutations in the CuZn superoxide dismutase (SOD1) gene can account for 20% of FALS cases. The mechanisms by which the many mutations in the SOD1 gene lead to motoneuron degeneration are unknown, although current experimental evidence supports a toxic gain of function, possibly through copper-induced cytotoxicity. Copper is an integral component of a number of enzymes as well as SOD1. Since abnormalities in connective tissue cross-linking have been reported in ALS patients, an enzyme of possible relevance is lysyl oxidase (LOX), a copper-containing enzyme which catalyses the crosslinking of collagens and elastin. The aim of this study was to investigate the hypothesis that allelic variants or mutants of LOX gene result in altered function of LOX in ALS patients. METHODS: The coding regions of the LOX gene were screened for polymorphism and mutations in a cohort of sporadic and familial ALS patients. RESULTS: A novel polymorphism, Pro159Gln, was identified in eight individuals with sporadic ALS (5.0%) and five controls (3.6%). The previously identified Arg158Gln polymorphism was also detected in ALS patients and controls. These polymorphisms were genotyped in 192 ALS patients, including 31 unrelated familial cases and 138 controls, and no association was found between any of these polymorphisms and amyotrophic lateral sclerosis or its phenotype. CONCLUSION: Mutations in the LOX gene are unlikely to be directly causative of ALS.

Uptake of 26-Al and 67-Ga into brain and other tissues of normal and hypotransferrinaemic mice.

Aluminium uptake from blood into tissues of control and homozygous hypotransferrinaemic (hpx/hpx) mice, following continuous intravenous infusion of 26Al and 67Ga, has been compared with that of gallium, a proposed tracer for aluminium. 26Al uptake into tissues of control (hpx/+ and +/+) mice occurred in the order (expressed as a space): bone 464.7 ml 100 g-1; renal cortex 102.9 ml 100 g-1; liver 13.0 ml 100 g-1; spleen 8.4 ml 100 g-1 and brain 0.8 ml 100 g-1. 67Ga uptakes were similar in liver, spleen and brain, but smaller in the renal cortex and bone, at one-third and one-fifth of the values for 26Al, respectively. In the hypotransferrinaemic mice, uptake of 67Ga into all tissues was increased, especially in renal cortex (ninefold) and bone (twentyfold) as compared with the controls. Increases in 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem of the hypotransferrinaemic mice were 3.8, 4.2 and 2.8 fold, respectively. 26Al uptake into tissues of the hypotransferrinaemic mice was similar to control values except in bone where it was three times greater. Pre-treatment of control animals with the anti-transferrin receptor antibody, RI7 208, enhanced 67Ga uptake in all tissues, the effect being greatest in renal cortex (tenfold) and bone (ninefold). 67Ga uptakes into cerebral hemisphere, cerebellum and brain stem in the mice pre-treated with RI7 208 were 6.4, 6 and 10 times greater than in untreated mice, respectively. No influence of antibody on 26AI uptake into mouse tissues was observed except in spleen where it was three times greater than in untreated mice. Hence, transport of aluminium and gallium into mouse tissues is not similar under all conditions. Non-transferrin mediated transport of each metal can occur into all tissues, especially in renal cortex and bone, where gallium may be a suitable marker for aluminium.

Mutations in all five exons of SOD-1 may cause ALS.

Eight of 38 patients (21%) with familial and 5 of 175 patients (3%) with sporadic amyotrophic lateral sclerosis (ALS) had missense mutations in the SOD-1 gene. Two novel mutations were identified. One in exon 4 substituting leucine with phenylalanine (L84F) in a familial patient and the second in exon 3 at substituting glycine with serine (G72S) in an "apparently" sporadic patient. Over 60 point mutations have now been described in all five exons of SOD-1, involving 43 of the 153 residues. Hypotheses about the toxic role of mutant SOD-1 in the pathogenesis of ALS must account for this molecular diversity.