Amyotrophic lateral sclerosis causes small fiber pathology.

BACKGROUND AND PURPOSE: Our aim was to address the correlation between small fiber loss and amyotrophic lateral sclerosis (ALS) for disease onset, phenotype, genotype, duration, severity and sensory findings. METHODS: Consecutive patients referred for suspected ALS were screened. Exclusion criteria were possible ALS and previous diagnosis or known risk factors for small fiber neuropathies. A sural nerve conduction study (NCS) was bilaterally recorded. The ALS functional rating scale revised was administered and loss of functions were calculated using the Milano-Torino staging (MITOS) system. Sensory symptoms and signs were recorded. Genetic analysis was performed by the next-generation sequencing approach. Skin biopsy was performed at the distal leg and intraepidermal nerve fiber (IENF) density was quantified in three non-consecutive sections following published guidelines. Findings were referred to age- and sex-adjusted normative values. RESULTS: Fifty-seven patients including six with facial onset sensory and motor neuronopathy (FOSMN) were enrolled. Eight (15.7%) pure ALS patients and five (83%) FOSMN patients complained of sensory disturbances with different distributions. Sural NCS was normal in all except two patients. IENF density was reduced in 75.4% of pure ALS and 50% of FOSMN patients, without correlation with any disease features. IENF density was similarly reduced in bulbar (78.5%), flail limb (87.5%), pyramidal (100%), and spinal (68.2%) onset, as well as in genetic (83.3%) and sporadic (82%) ALS. There was no correlation with genotype, disease duration and severity. CONCLUSIONS: Intraepidermal nerve fiber loss is a feature of most ALS patients. It does not correlate with onset, phenotype, course and severity of the disease, and cannot be considered a clinical or prognostic biomarker.

No effect of AR polyG polymorphism on spinal and bulbar muscular atrophy phenotype.

BACKGROUND AND PURPOSE: Disease severity varies considerably among patients with Spinal and Bulbar Muscular Atrophy (SBMA). Our aim was to investigate the role of androgen receptor (AR) polymorphic repeats in SBMA phenotype. METHODS: We analyzed the length of AR polyQ and polyG tracts in 159 SBMA patients. RESULTS: No relationship between polyG size or polyG/polyQ haplotypes and clinical phenotype was found. An independent negative correlation between polyQ-length and onset of weakness was confirmed (P < 0.001). CONCLUSIONS: The negative results of our study prompt to continue the search for potential disease modifiers in SBMA outside the AR gene.

Clinical and molecular report of novel GALC mutations in Moroccan patient with Krabbe disease: case report.

BACKGROUND: Krabbe disease (KD) or globoid cell leukodystrophy is an autosomal recessive lysosomal disorder, which affects metabolic and neurologic systems. This pathology has different forms. Infantile onset is about 85% to 90% of individuals with Krabbe disease. Disorder's onset is characterized, in early childhood, by hyperirritability, psychomotor deterioration associated to episodes of fever. To date, all reported cases have been attributed to mutations in galactosylceramidase gene (GALC gene) that encodes an enzyme which degrades galactosyl-sphingolipids (galactosylceramide, psychosine), essential in myelin production. A child compounded with two new mutations in the GALC gene was detected. CASE PRESENTATION: An eleven month old male child of Moroccan origin presented to our genetic consultation with severe symptoms that included hypotonia, fever, vision loss and feeding difficulties. He was suffering from the 4th month of life. Krabbe disease was suspected. Galactocerebrosidase deficiency was confirmed by biochemical analysis. DNA sequencing revealed a novel heterozygous compound mutation in GALC gene. The child was compounded with two mutations c.860G > A; p.Cys287Tyr and c.1622G > A; p.Trp541*. CONCLUSION: These new mutations could affect GALC structure and therefore its function. The identification of these mutations and their associated phenotypes are important to predict the prognosis and to confer to families an adequate genetic counseling.

Preliminary insights into RNA in CSF of pediatric SMA patients after 6 months of nusinersen.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare autosomal-recessive neurodegenerative disorder caused by mutations in survival motor neuron 1 (SMN1) gene, and consequent loss of function of SMN protein, which results in progressive loss of lower motor neurons, and muscular wasting. Antisense oligonucleotide (ASO) nusinersen (Spinraza®) modulates the pre-mRNA splicing of the SMN2 gene, allowing rebalance of biologically active SMN. It is administered intrathecally via lumbar puncture after removing an equal amount of cerebrospinal fluid (CSF). Its effect was proven beneficial and approved since 2017 for SMA treatment. Given the direct effect of nusinersen on RNA metabolism, the aim of this project was to evaluate cell-free RNA (cfRNA) in CSF of SMA patients under ASOs treatment for biomarker discovery. METHODS: By RNA-sequencing approach, RNA obtained from CSF of pediatric SMA type 2 and 3 patients was processed after 6 months of nusinersen treatment, at fifth intrathecal injection (T6), and compared to baseline (T0). RESULTS: We observed the deregulation of cfRNAs in patients at T6 and we were able to classify these RNAs into disease specific, treatment specific and treatment dependent. Moreover, we subdivided patients into "homogeneous" and "heterogeneous" according to their gene expression pattern. The "heterogeneous" group showed peculiar activation of genes coding for ribosomal components, meaning that in these patients a different molecular effect of nusinersen is observable, even if this specific molecular response was not referable to a clinical pattern. CONCLUSIONS: This study provides preliminary insights into modulation of gene expression dependent on nusinersen treatment and lays the foundation for biomarkers discovery.

Novel optineurin mutations in patients with familial and sporadic amyotrophic lateral sclerosis.

BACKGROUND: Optineurin (OPTN), a causative gene of hereditary primary open-angle glaucoma, has been recently associated with amyotrophic lateral sclerosis (ALS) with mainly autosomal recessive, but also dominant, traits. To further define the contribution of OPTN gene in ALS, we performed a mutational screening in a large cohort of Italian patients. METHODS: A group of 274 ALS patients, including 161 familial (FALS) and 113 sporadic (SALS) cases, were screened for OPTN mutations by direct sequencing of its coding sequence. All patients fulfilled the El Escorial criteria for probable or definite ALS and were negative for mutations in SOD1, ANG, TARDBP and FUS/TLS genes. RESULTS: The genetic analysis revealed six novel variants in both FALS and SALS patients, all occurring in an heterozygous state. We identified three missense (c.844A→C p.T282P, c.941A→T p.Q314L, c.1670A→C p.K557T), one nonsense (c.67G→T p.G23X) and two intronic mutations (c.552+1delG, c.1401+4A→G). The intronic c.552+1delG variant determined a splicing defect as demonstrated by mRNA analysis. All mutations were absent in 280 Italian controls and over 6800 worldwide glaucoma patients and controls screened so far. The clinical phenotype of OPTN-mutated patients was heterogeneous for both age of onset and disease duration but characterised by lower-limb onset and prevalence of upper motor neuron signs. CONCLUSION: In this cohort, OPTN mutations were present both in FALS (2/161), accounting for 1.2% cases, and in SALS patients (4/113), thereby extending the spectrum of OPTN mutations associated with ALS. The study further supports the possible pathological role of optineurin protein in motor neuron disease.

Predictive genetic tests in neurodegenerative disorders: a methodological approach integrating psychological counseling for at-risk individuals and referring clinicians.

The identification of the molecular basis of numerous hereditary neurological disorders allowed the feasibility of predictive genetic tests for at-risk family members. In agreement with international guidelines, we tested a protocol for a predictive test to optimize cooperation among specialists, well-being of participants, and organization of clinical activities. The psychiatrist/psychologist did not meet the at-risk subjects, but cooperated with the team, integrating psychological support for participants and clinicians. We enrolled 60 subjects at risk for Huntington disease, and 32 at risk for spinocerebellar ataxias. Seventy-two subjects (78%) continued the visit program; 55 (60%) received the genetic result, and 38 subjects (41%) completed the program. Participation and outcome were similar in both groups. Mean psychological scores were all below significant levels; however, the need for psychological support was recognized for 5 mutation carriers and a non-carrier. Our data provide a methodological example of a simple and safe procedure for a predictive test, and indicate that the clinical conference represents a good setting to handle psychosocial impact associated with disclosure of genetic results in hereditary late-onset disorders.

High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis.

Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)-43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP-43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues.

Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.

Friedreich's ataxia (FRDA) is an autosomal recessive, degenerative disease that involves the central and peripheral nervous systems and the heart. A gene, X25, was identified in the critical region for the FRDA locus on chromosome 9q13. This gene encodes a 210-amino acid protein, frataxin, that has homologs in distant species such as Caenorhabditis elegans and yeast. A few FRDA patients were found to have point mutations in X25, but the majority were homozygous for an unstable GAA trinucleotide expansion in the first X25 intron.

Early white matter involvement in an infant carrying a novel mutation in ACOX1.

We describe the clinical findings and MRI features observed in a child who presented a two-step disease course: he was hypotonic at birth and soon afterwards developed seizures, which were partially responsive to treatment; he subsequently showed developmental delay and a progressive neurological deterioration with the onset of severe seizures at around three years of age. Head MRI at age 20 days was unremarkable, whereas at 25 months it showed bilateral hyperintensity of the deep cerebellar nuclei; five months later, the signal hyperintensity was also present in the cerebellar white matter and ventral pontine fibre tracts. Molecular analysis revealed a novel ACOX1 mutation, predicting a largely truncated protein. The white matter involvement, which followed an ascending trajectory from cerebellar and brainstem structures to the cerebral hemispheres, seemed to originate from the perinuclear white matter of the deep cerebellar nuclei.

Evidence for two distinct mitochondrial malic enzymes in human skeletal muscle: purification and properties of the NAD(P)+-dependent enzyme.

Human muscle mitochondria reduced either NADP+ or NAD+ in the presence of L-malate and Mn2+ or Mg2+. After polyacrylamide slab gel electrophoresis and agarose gel isoelectrofocusing, two bands were seen in mitochondrial extract, one strictly NADP+-dependent and the other reacting with either NAD+ or NADP+. The two mitochondrial malic enzymes were separated by DEAE-Sepharose chromatography. The NAD+/NADP+-dependent enzyme was purified 1600-fold with a final yield of 34% and a final specific activity of 32.9 units/mg of protein by employing affinity chromatography on Agarose-ATP. SDS electrophoresis revealed a single band having an apparent Mr = 64,000. Estimates of the native apparent molecular weight upon gel filtration yielded a value of 140,300. Kinetic characterization showed that succinate and ATP were activator and inhibitor, respectively. In the absence of succinate the Km values for malate, NAD+ and NADP+ were 3.7, 0.13 and 0.78 mM, respectively; in the presence of succinate the Km value for malate was 1.9 mM. ATP was found to be an inhibitor competitive with malate, with a Ki (ATP) of 0.2 mM. This is the first report to show that mammalian skeletal muscle mitochondria contains two distinct malic enzymes, one active with either NAD+ or NADP+ and the other active only with NADP+.

Multiple founder effects in spinal and bulbar muscular atrophy (SBMA, Kennedy disease) around the world.

SBMA (spinal and bulbar muscular atrophy), also called Kennedy disease, is an X-chromosomal recessive adult-onset neurodegenerative disorder caused by death of the spinal and bulbar motor neurones and dorsal root ganglia. Patients may also show signs of partial androgen insensitivity. SBMA is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene on the X-chromosome. Our previous study suggested that all the Nordic patients with SBMA originated from an ancient Nordic founder mutation, but the new intragenic SNP marker ARd12 revealed that the Danish patients derive their disease chromosome from another ancestor. In search of relationships between patients from different countries, we haplotyped altogether 123 SBMA families from different parts of the world for two intragenic markers and 16 microsatellites spanning 25 cM around the AR gene. The fact that different SBMA founder haplotypes were found in patients from around the world implies that the CAG repeat expansion mutation has not been a unique event. No expansion-prone haplotype could be detected. Trinucleotide diseases often show correlation between the repeat length and the severity and earlier onset of the disease. The longer the repeat, the more severe the symptoms are and the onset of the disease is earlier. A negative correlation between the CAG repeat length and the age of onset was found in the 95 SBMA patients with defined ages at onset.

Sensitivity of FRDA lymphoblasts to salts of transition metal ions.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease resulting from decreased expression of the nuclear-encoded mitochondrial protein, frataxin. FRDA patients have characteristic iron deposits and dysfunction of mitochondrial enzymes in the heart. Inactivation of the frataxin homologue in yeast causes dysregulation of both mitochondrial iron levels and iron export. Previously, we have observed sensitivity of FRDA fibroblasts to FeCl3 and hydrogen peroxide, results consistent with the hypothesis that FRDA cells may experience increased Fenton chemistry. To determine whether the sensitivity of FRDA cells to transition metal ions is a general or specific property, we have compared the sensitivity of lymphoblasts from FRDA patients and healthy controls to the transition metal salts CoCl2, CuSO4 FeCl3 FeSO4, MnCl2, and ZnCl2. FRDA lymphoblasts were significantly more sensitive to FeCl3 and MnCl2 than control cells. However, there were no significant differences observed in sensitivity to CoCl2, CuSO4, FeSO4 and ZnCl2 in the concentration ranges studied. Thus, the sensitivity of FRDA lymphoblasts exposed to transition metals appears to be specific, and could be relevant to the pathophysiological mechanism, which is discussed.

Psychiatric symptoms do not correlate with cognitive decline, motor symptoms, or CAG repeat length in Huntington's disease.

OBJECTIVES: To investigate the hypothesis that psychiatric disturbances in Huntington's disease are related to degree of cognitive or motor compromise and to determine correlations between CAG repeat length within the gene for Huntington's disease and disease severity. DESIGN: Consecutive series of patients with Huntington's disease. SETTING: Neurological specialty hospital. PATIENTS: Seventeen men and 12 women from 24 families. MAIN OUTCOME MEASURES: The Hamilton Psychiatric and Anxiety Rating Scales and Brief Psychiatric Rating Scale were used to assess psychiatric disturbances; Folstein's Quantified Neurological Examination to evaluate motor status; and the Mini-Mental State Examination, Raven Progressive Matrices), Phonemic Verbal Fluency Test, Short Tale Test, Visual Search Test, and Benton's Visual Orientation Line Test to evaluate cognitive function. The length of the CAG repeat sequence in the Huntington's gene was determined by quantitative polymerase chain reaction. RESULTS: Cognitive test scores correlated significantly with each other; of these, results of the Visual Search and Short Tale tests correlated significantly with the Folstein's Quantified Neurological Examination score (P = .05 and P = .03, respectively). Results of the Folstein's Quantified Neurological Examination also correlated with the illness duration and the length of the CAG repeat. Although psychiatric scores correlated significantly among themselves (P < .01), neither cognitive compromise, motor deterioration, nor CAG length were related to the extent of psychiatric compromise. Patients who were depressed when they were examined tended to have a history of psychiatric disorders. CONCLUSIONS: The lack of correlation between disease severity and psychiatric disturbances indicates that psychiatric disorders progress nonlinearly, possibly because of differential degeneration of the striatal-cortical circuits; the possibility that psychiatric disorders are prevalent in certain families with a member who has Huntington's disease is being further investigated. The lack of correlation between CAG length and cognitive and psychiatric variables needs further investigation.

Very late onset Friedreich's ataxia without cardiomyopathy is associated with limited GAA expansion in the X25 gene.

Molecular analysis of spinocerebellar ataxias revealed a pathologic GAA expansion in the gene encoding frataxin in six adult patients from three families. These patients, carrying expanded alleles in the low-range size, had an exceptionally late onset and lacked cardiomyopathy, pointing to phenotypic variability of Friedreich's ataxia. Both mitotic and gametic instability of the expanded triplet repeat were present in these families.

Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.

A 7-month-old boy died in a demented state after a clinical history characterized by generalized seizures, psychomotor deterioration, and fumaric aciduria. We found a marked deficiency of both mitochondrial and cytosolic fumarases in skeletal muscle, brain, cerebellum, heart, kidney, liver, and cultured fibroblasts. Fumarase activities were 30 to 50% compared with controls in both mitochondria and cytosol from cultured fibroblasts of the parents. Antifumarase cross-reacting material was present in negligible amounts in the patient's tissues. Our data indicate that this disease is an autosomal recessive encephalopathy, due to a single mutation affecting the gene encoding both forms of the enzyme.

Pathogenic effect of an intermediate-size SCA-6 allele (CAG)(19) in a homozygous patient.

SCA6 is caused by CAG expansion in the alpha 1A voltage-dependent calcium channel subunit gene. The authors studied an Italian family in which one patient carried a fully expanded SCA6 allele with 26-CAG repeats, whereas the other affected family member was homozygous for an intermediate-size allele of 19-CAG repeats. Three family members, heterozygous for the intermediate allele, were clinically unaffected. The findings demonstrate a dose-dependent pathogenic effect of an intermediate CAG expansion in the SCA6 gene.

Dominantly inherited mitochondrial myopathy with multiple deletions of mitochondrial DNA: clinical, morphologic, and biochemical studies.

We studied a large family with a dominantly inherited mitochondrial myopathy characterized by progressive external ophthalmoplegia, dysphagia, cataract, lactic acidosis, exercise intolerance, and early death. Morphologic studies of muscle biopsies suggested mitochondrial heteroplasmy and revealed ragged-red fibers and decreased histochemical reactions for cytochrome c oxidase and succinate dehydrogenase. Biochemistry showed a partial defect of cytochrome c oxidase and a mild generalized reduction of other mitochondrial enzymes requiring mitochondrial DNA-encoded subunits. Southern blot analysis and PCR amplification showed mitochondrial DNA deletions in muscle of all affected members, but not in lymphocytes or fibroblasts, suggesting a tissue-specific distribution. Deletions were multiple and seemed to increase with time and to correlate with the severity of the disease.