Exome sequencing: an efficient diagnostic tool for complex neurodegenerative disorders.

BACKGROUND AND PURPOSE: Autosomal recessive cerebellar ataxia (ARCA) comprises a large and heterogeneous group of neurodegenerative disorders. We studied three families diagnosed with ARCA. METHODS: To determine the gene lesions responsible for their disorders, we performed high-density single-nucleotide polymorphism genotyping and exome sequencing. RESULTS: We identified a new mutation in the SACS gene and a known mutation in SPG11. Notably, we also identified a homozygous variant in APOB, a gene previously associated with ataxia. CONCLUSIONS: These findings demonstrate that exome sequencing is an efficient and direct diagnostic tool for identifying the causes of complex and genetically heterogeneous neurodegenerative diseases, early-stage disease or cases with limited clinical data.

Ataxia with isolated vitamin E deficiency is caused by mutations in the alpha-tocopherol transfer protein.

Ataxia with isolated vitamin E deficiency (AVED) is an autosomal recessive neurodegenerative disease which maps to chromosome 8q13. AVED patients have an impaired ability to incorporate alpha-tocopherol into lipoproteins secreted by the liver, a function putatively attributable to the alpha-tocopherol transfer protein (alpha-TTP). Here we report the identification of three frame-shift mutations in the alpha TTP gene. A 744delA mutation accounts for 68% of the mutant alleles in the 17 families analysed and appears to have spread in North Africa and Italy. This mutation correlates with a severe phenotype but alters only the C-terminal tenth of the protein. Two other mutations were found in single families. The finding of alpha TTP gene mutations in AVED patients substantiates the therapeutic role of vitamin E as a protective agent against neurological damage in this disease.

Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia).

Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. We previously localized the SJS1 locus to chromosome 1p34-p36.1 and found no evidence of genetic heterogeneity. Here we describe mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) in three SJS1 families. In so doing, we have identified the first human mutations in HSPG2, which underscore the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability.

The gene encoding gigaxonin, a new member of the cytoskeletal BTB/kelch repeat family, is mutated in giant axonal neuropathy.

Disorganization of the neurofilament network is a prominent feature of several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy and axonal Charcot-Marie-Tooth disease. Giant axonal neuropathy (GAN, MIM 256850), a severe, autosomal recessive sensorimotor neuropathy affecting both the peripheral nerves and the central nervous system, is characterized by neurofilament accumulation, leading to segmental distension of the axons. GAN corresponds to a generalized disorganization of the cytoskeletal intermediate filaments (IFs), to which neurofilaments belong, as abnormal aggregation of multiple tissue-specific IFs has been reported: vimentin in endothelial cells, Schwann cells and cultured skin fibroblasts, and glial fibrillary acidic protein (GFAP) in astrocytes. Keratin IFs also seem to be alterated, as most patients present characteristic curly or kinky hairs. We report here identification of the gene GAN, which encodes a novel, ubiquitously expressed protein we have named gigaxonin. We found one frameshift, four nonsense and nine missense mutations in GAN of GAN patients. Gigaxonin is composed of an amino-terminal BTB (for Broad-Complex, Tramtrack and Bric a brac) domain followed by a six kelch repeats, which are predicted to adopt a beta-propeller shape. Distantly related proteins sharing a similar domain organization have various functions associated with the cytoskeleton, predicting that gigaxonin is a novel and distinct cytoskeletal protein that may represent a general pathological target for other neurodegenerative disorders with alterations in the neurofilament network.

Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35.

Amyotrophic lateral sclerosis (ALS) usually presents as a sporadic disorder of motor neurons. However, familial forms of ALS have been described--autosomal dominant forms (ALS1, ALS3), clinically indistinguishable from the sporadic form, and autosomal recessive forms with early onset and slower progression of symptoms (ALS2). To localize the gene for one of the autosomal recessive forms of ALS, we applied linkage analysis to a large inbred family from Tunisia. A lod score maximum of Zmax = 8.2 at theta = 0.00 was obtained with marker D2S72 located on chromosome 2q33-q35. The fine mapping of this region suggested that the ALS2 locus lies in the 8 cM segment flanked by D2S155 and D2S115.

The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are neurodegenerative conditions that affect large motor neurons of the central nervous system. We have identified a familial juvenile PLS (JPLS) locus overlapping the previously identified ALS2 locus on chromosome 2q33. We report two deletion mutations in a new gene that are found both in individuals with ALS2 and those with JPLS, indicating that these conditions have a common genetic origin. The predicted sequence of the protein (alsin) may indicate a mechanism for motor-neuron degeneration, as it may include several cell-signaling motifs with known functions, including three associated with guanine-nucleotide exchange factors for GTPases (GEFs).

Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy.

Miyoshi myopathy (MM) is an adult onset, recessive inherited distal muscular dystrophy that we have mapped to human chromosome 2p13. We recently constructed a 3-Mb P1-derived artificial chromosome (PAC) contig spanning the MM candidate region. This clarified the order of genetic markers across the MM locus, provided five new polymorphic markers within it and narrowed the locus to approximately 2 Mb. Five skeletal muscle expressed sequence tags (ESTs) map in this region. We report that one of these is located in a novel, full-length 6.9-kb muscle cDNA, and we designate the corresponding protein 'dysferlin'. We describe nine mutations in the dysferlin gene in nine families; five are predicted to prevent dysferlin expression. Identical mutations in the dysferlin gene can produce more than one myopathy phenotype (MM, limb girdle dystrophy, distal myopathy with anterior tibial onset).

Microscopic polyangiitis presenting with peripheral and central neurological manifestations.

Although peripheral neuropathy is a common complication of microscopic angiitis, manifestations involving the muscle and the central nervous system have been rarely reported. We describe a 48-year-old man who rapidly developed a clinical picture of mononeuritis multiplex. A month after the appearance of the primary symptoms, he became comatose and had left hemiplegia in relation with a massive cerebral haematoma. Laboratory data revealed signs of inflammation, glomerular dysfunction with microhaematuria, and positive myeloperoxidase-antineutrophil cytoplasmic antibodies. The neuromuscular biopsy disclosed a small-vessel vasculitis, consisting with microscopic angiitis, associated with myositis and extensive axonal loss. The patient had surgical evacuation of the haematoma and received immunosuppressive therapy with good outcome. Thus, microscopic angiitis should be considered as a differential diagnosis in cases of myositis and intracerebral haemorrhage.

Stroke in the Middle-East and North Africa: A 2-year prospective observational study of intravenous thrombolysis treatment in the region. Results from the SITS-MENA Registry.

BACKGROUND AND METHODS: Intravenous thrombolysis for acute ischemic stroke in the Middle-East and North African (MENA) countries is still confined to the main urban and university hospitals. This was a prospective observational study to examine outcomes of intravenous thrombolysis-treated stroke patients in the MENA region compared to the non-MENA stroke cohort in the SITS International Registry. RESULTS: Of 32,160 patients with ischemic stroke registered using the SITS intravenous thrombolysis protocol between June 2014 and May 2016, 500 (1.6%) were recruited in MENA. Compared to non-MENA (all p < 0.001), median age in MENA was 55 versus 73 years, NIH Stroke Scale score 12 versus 9, onset-to-treatment time 138 versus 155 min and door-to-needle time 54 min versus 64 min. Hypertension was the most reported risk factor, but lower in MENA (51.7 vs. 69.7%). Diabetes was more frequent in MENA (28.5 vs. 20.8%) as well as smoking (20.8 vs. 15.9%). Hyperlipidemia was less observed in MENA (17.6 vs. 29.3%). Functional independence (mRS 0-2) at seven days or discharge was similar (53% vs. 52% in non-MENA), with mortality slightly lower in MENA (2.3% vs. 4.8%). SICH rates by SITS-MOST definition were low (<1.4%) in both groups. CONCLUSIONS: Intravenous thrombolysis patients in MENA were younger, had more severe strokes and more often diabetes. Although stroke severity was higher in MENA, short-term functional independency and mortality were not worse compared to non-MENA, which could partly be explained by younger age and shorter OTT in MENA. Decreasing the burden of stroke in this young population should be prioritized.

Mutations in the dystrophin-associated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy.

Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a progressive muscle-wasting disorder common in North Africa that segregates with microsatellite markers at chromosome 13q12. Here, it is shown that a mutation in the gene encoding the 35-kilodalton dystrophin-associated glycoprotein, gamma-sarcoglycan, is likely to be the primary genetic defect in this disorder. The human gamma-sarcoglycan gene was mapped to chromosome 13q12, and deletions that alter its reading frame were identified in three families and one of four sporadic cases of SCARMD. These mutations not only affect gamma-sarcoglycan but also disrupt the integrity of the entire sarcoglycan complex.

Mathematical modeling of T-cell activation kinetic.

T-cell activation is a crucial step in mounting of the immune response. The dynamics of T-cell receptor (TCR) specific recognition of peptide presented by major histocompatibility complex (MHC) molecule decides the fate of the T cell. Several biochemical interactions interfere resulting in a highly complex mechanism that would be difficult to understand without computer help. The aim of the present study was to define a mathematical model in order to approach the kinetics of monoclonal T-cell-specific activation. The reaction scheme was first described and the model was tested using experimental parameters from the published data. Simulations were concordant with experimental data showing proportional decrease of membrane TCR and production of interleukin-2 (IL-2). Agonist and antagonist peptides induce different levels of intracellular signal that could make the yes or no decision for entry to cell cycle. Different conditions (peptide concentrations, initial TCR density and exogenous IL-2 levels) can be tested. Several parameters are missing for parameters estimation and adjustment before it could be adapted for a polyclonal T-cell reaction model. However, the model should be of interest in setting experiments, simulation of clinical responses and optimization of preventive or therapeutic immunotherapy.

L-2-hydroxyglutaric aciduria: clinical and molecular study in three Tunisian families. Identification of a new mutation and inter-familial phenotype variability.

We report clinical and molecular studies in three unrelated Tunisian families containing seven patients with L2HGA. Although the age of onset is similar in all these patients at nearly 6 years, they progressively developed peculiar clinical phenotypes different from family to family. The three patients of family 1 showed mental retardation, epilepsy, cerebellar ataxia and pyramidal and pseudobulbar syndromes. The two patients of family 2 showed mental retardation and parkinsonism especially extrapyramidal stiffness, dystonia and myoclonus. The two patients of family 3 showed an intermediate phenotype; they share some clinical signs of the patients of family 1 (epilepsy, pyramidal and extrapyramidal syndromes) and some clinical signs of the patients of family 2 (extrapyramidal stiffness and dystonia). Molecular study identified a novel homozygous c.185C>A, p.A62D mutation on the L2HGDH gene in families 1 and 3 and the already known homozygous c.241A>G, p.K81E mutation in family 2. We suppose that the type of mutation in the L2HGDH gene does not play a complete role in the inter-familial phenotype variability. Disturbance of other unknown metabolic pathways related to L2HGA may contribute to this phenomenon.

A founding LRRK2 haplotype shared by Tunisian, US, European and Middle Eastern families with Parkinson's disease.

The Leucine-rich repeat kinase 2 (LRRK2) gene has been identified as a disease susceptibility gene for Parkinson's disease (PD), with G2019 (6055G>A) being the most frequent mutation. This mutation was present in 42% (38/91) of Tunisian families and 2% (1/39) of US families we have studied. A founding haplotype was identified in our data and it is shared by families from Tunisia, US, European and Middle Eastern countries. The most recent common founder of the mutation was dated to 2600 (95% CI: 1950-3850) years ago although additional studies are warranted to ensure an accurate age estimate for this mutation.

Osteoma of the calvaria in L-2-hydroxyglutaric aciduria.

L-2-Hydroxyglutaric aciduria (L-2-OHGA) is a rare autosomal recessive neurometabolic disease linked to chromosome 14q21.1 and is caused by mutations in the gene that most likely encodes L: -2-hydroxyglutarate dehydrogenase, which normally catalyses L: -2-hydroxyglutarate to alpha-ketoglutarate. It is characterized by progressive mental deterioration, pyramidal and cerebellar syndromes, macrocephaly and marked polycystic white-matter degeneration mainly involving frontal lobes. Brain tumours of variable nature have frequently been observed in L-2-OHGA. We report a patient affected by this disease who at the age of 20 years developed a bone tumour involving the right frontal region of the calvaria. He had first presented at the age of 10 years with psychomotor delay, clumsy gait and moderate mental impairment. Examination showed macrocephaly, cerebellar ataxia and quadripyramidal syndrome. Brain MRI showed low signal intensities on T1-weighted images and high signal intensities on T2-weighted images in cerebral subcortical white matter. Serum and urinary amino acid assay was normal. Urinary 2-hydroxyglutaric acid was 1418 mmol/mol creatinine (controls <25). Analysis of the L-2-hydroxyglutarate dehydrogenase gene revealed a homozygous mutation in exon 2 (A320G). At the age of 20 years, an osteoma of the right frontal bone was diagnosed. This finding reinforces the opinion concerning the association of L-2-OHGA and tumorigenesis and prompted us to verify the possible responsibility of some overproduced substances in this disease for the development of tumours and to look for any correlation between the type of mutation in the L-2-OHGA gene and the tumorigenic potential observed in some patients affected by this disease.

Reversible metronidazole-induced encephalopathy.

We report the neuroimaging findings of a case of reversible metronidazole-induced encephalopathy. Magnetic resonance imaging (MRI) demonstrated lesions in highly suggestive locations. Follow-up imaging performed 1 month after cessation of metronidazole therapy demonstrated resolution of imaging findings.

Genetic heterogeneity within a consanguineous family involving the LGMD 2D and the LGMD 2C genes.

The sarcoglycanopathies are a group of autosomal recessive limb girdle muscular dystrophies (AR-LGMD 2) characterised by mutations in gene encoding one of the sarcoglycan subunits. Mutations in SGCA, SGCB, SGCG and SGCD genes are associated with LGMD 2D, 2E, 2C and 2F, respectively. We report three Tunisian patients belonging to the same consanguineous family sharing similar LGMD 2 phenotype but heterogeneous sarcoglycans immunohistochemical patterns. Linkage analysis suggests linkage with the LGMD 2D locus for the two siblings and with LGMD 2C locus for the third patient. Mutation analysis revealed two distinct mutations. A del521T homozygous mutation in exon 6 of the SGCG gene (LGMD 2C), widely distributed in Tunisian patients, was found in one patient, whereas a 157G>A homozygous mutation in exon 2 of the SGCA gene (LGMD 2D) was found in the two siblings. The presence of two distinct genetic forms, LGMD 2C and LGMD 2D in a consanguineous family raises the problem of the complexity of genetic counselling in inbred populations.

[A comparative study of clinical and paramedical aspects of multiple sclerosis in Tunisia]. / Etude comparative des aspects cliniques et paracliniques de la sclérose en plaques en Tunisie.

INTRODUCTION: Tunisia is considered as a low zone of prevalence for multiple sclerosis (MS). Consequently, only very few studies have taken an interest in this disorder in North Africa. The objective of this study was to compare the clinical and paraclinical parameters and the outcome of the disease in patients affected with MS in Tunisia during two periods (1974-1978 and 1996-2000) and to determine the incidence of the disease and the impact of the use of diagnostic criteria, including the MRI. PATIENTS AND METHODS: We report the results of a retrospective study concerning patients classified with MS and followed in the National Institute of Neurology in Tunis between 1974 and 2000, with 1058 records examined. We divided the patients into two groups belonging to two periods: Group I (1974-1978, 125 patients classified according to the McAlpine criteria) and Group II (1996-2000, 247 patients classified according to Poser's criteria. We compared the clinical and paraclinical parameters of the two groups. RESULTS: The incidence of the disease was evaluated at 1.3 per 100,000 individuals, placing Tunisia in the middle zone of prevalence. There was no significant difference in the mean age of onset (32.4+/-10.1 years) between the two groups. A slight male preponderance was observed in Group I (M/F sex ratio=1.25). The clinical outcome factors were age of onset after 40 years, pyramidal signs as the first symptom, and the progressive forms of the disease. Although Group II had an earlier diagnosis of the disease and a more systematic treatment of relapses, the functional outcome was similar between the two groups. CONCLUSION: MS in Tunisia has the same clinical characteristics and disease outcome as in other countries. The use of MRI allowed earlier diagnosis but did not increase the overall proportion of definite MS.

[Cardiac involvement in Emery-Dreifuss muscular dystrophy: a case report]. / L'atteinte cardiaque dans la dystrophie musculaire d'Emery-Dreifuss. A propos d'un cas.

Emery Dreifuss muscular dystrophy (EDMD) is an uncommon hereditary myopathy characterized by 3 symptoms: slow progressive muscular atrophy, muscular contractures and cardiac disease which affect prognosis. We report a 22 year-old patient with EDMD which shows the typical features of the associated dilated cardiomyopathy, ventricular arrhythmia, atrio-ventricular block, atrial standstill then atrial paralysis.

Allelic heterogeneity of GNE gene mutation in two Tunisian families with autosomal recessive inclusion body myopathy.

Autosomal recessive hereditary inclusion body myopathy (AR-HIBM), with sparing of the quadriceps, is characterized by adult-onset, with weakness and atrophy of distal lower limb muscles, and typical histopathological findings in muscle biopsy. AR hIBM is associated with mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene on chromosome 9p12-13 . We report two unrelated Tunisian families with clinical and pathological features of AR HIBM. One distinct homozygous GNE missense mutation, M712T, previously reported in Middle Eastern Jewish patients, and a newly identified one, L379H, were found in one patient from each family. We conclude that AR HIBM in Tunisia shows an allelic genetic heterogeneity.