Blood miRNA expression pattern is a possible risk marker for natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis patients.

BACKGROUND: Natalizumab has shown its efficacy in reducing multiple sclerosis (MS) relapses and progression of disability; however, it has been associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML). The differential expression of microRNA (miRNA), the small non-coding RNAs that regulate gene expression, in natalizumab-treated patients has been reported and miRNA have also been described as good candidates for disease biomarkers. OBJECTIVE: To characterize the effect of natalizumab therapy on the miRNA expression pattern and to search for miRNAs that can predict PML on an individual basis. METHODS: The expression of 754 microRNAs was measured in blood samples from 19 relapsing-remitting MS patients at three time points during natalizumab therapy, using TaqMan OpenArray panels. Two patients included in this study developed PML after more than 2 years of therapy. RESULTS: We found that the expression level of three miRNAs (let-7c, miR-125a-5p and miR-642) was affected after 6 months of therapy (t6). Furthermore, we observed a differential expression of another three miRNAs (miR-320, miR-320b and miR-629) between the PML and non-PML groups after 12 months of treatment (t12); and a positive correlation was found between therapy time and the expression of miR-320. CONCLUSIONS: Natalizumab modified the expression levels of three miRNAs after a 6-month treatment. We suggest miR-320, miR-320b and miR-629 as possible biomarkers for individual PML risk assessment.

ANKRD55 and DHCR7 are novel multiple sclerosis risk loci.

Multiple sclerosis (MS) shares some risk genes with other disorders hallmarked by an autoimmune pathogenesis, most notably IL2RA and CLEC16A. We analyzed 10 single-nucleotide polymorphisms (SNPs) in nine risk genes, which recently emerged from a series of non-MS genome-wide association studies (GWAS), in a Spanish cohort comprising 2895 MS patients and 2942 controls. We identified two SNPs associated with MS. The first SNP, rs6859219, located in ANKRD55 (Chr5), was recently discovered in a meta-analysis of GWAS on rheumatoid arthritis (RA), and emerged from this study with genome-wide significance (odds ratio (OR) = 1.35; P = 2.3 × 10(-9)). The second SNP, rs12785878, is located near DHCR7 (Chr11), a genetic determinant of vitamin D insufficiency, and showed a size effect in MS similar to that recently observed in Type 1 diabetes (T1D; OR = 1.10; P = 0.009). ANKRD55 is a gene of unknown function, and is flanked proximally by the IL6ST-IL31RA gene cluster. However, rs6859219 did not show correlation with a series of haplotype-tagging SNPs covering IL6ST-IL31RA, analyzed in a subset of our dataset (D'< 0.31; r(2)< 0.011). Our results expand the number of risk genes shared between MS, RA and T1D.

Chitinase 3-like 1 plasma levels are increased in patients with progressive forms of multiple sclerosis.

BACKGROUND: Chitinase 3-like 1 (CHI3L1) is upregulated in a wide variety of inflammatory conditions. Recent studies have pointed to a role of CHI3L1 in multiple sclerosis (MS) pathogenesis. OBJECTIVE: The objective of this study was to investigate the role of plasma CHI3L1 in MS clinical course and disease activity and to evaluate the effect of interferon-beta (IFNß) treatment on protein levels. METHODS: Plasma CHI3L1 levels were determined by ELISA in 57 healthy controls (HC), 220 untreated MS patients [66 primary progressive MS patients (PPMS), 30 secondary progressive MS patients (SPMS), and 124 relapsing-remitting MS patients (RRMS), 94 during clinical remission and 30 during relapse], and 32 MS patients receiving IFNß treatment. A polymorphism of the CHI3L1 gene, rs4950928, was genotyped in 3274 MS patients and 3483 HC. RESULTS: Plasma CHI3L1 levels were significantly increased in patients with progressive forms of MS compared with RRMS patients and HC. CHI3L1 levels were similar between RRMS patients in relapse and remission. A trend towards decreased CHI3L1 levels was observed in IFNß-treated patients. Allele C of rs4950928 was significantly associated with PPMS patients and with higher plasma CHI3L1 levels. CONCLUSIONS: These findings point to a role of CHI3L1 in patients with progressive forms of MS, particularly in those with PPMS.

Validation of IRF5 as multiple sclerosis risk gene: putative role in interferon beta therapy and human herpes virus-6 infection.

In recent reports, IRF5 polymorphisms showed significant association with multiple sclerosis (MS) susceptibility in three studied populations and Irf5-deficient mice exhibited an increased susceptibility to viral infection, linked to a significant decrease in the induction of serum type I interferon (IFN). In the present study, we evaluated the association of two IRF5 polymorphisms with MS predisposition and we also addressed whether these polymorphisms were associated with active replication of human herpes virus-6 (HHV-6) observed in a subgroup of MS patients, and/or with response to IFN-ß therapy. A total of 1494 MS patients and 1506 ethnically matched controls were genotyped for rs4728142 and rs3807306 with TaqMan pre-designed assays. One hundred and six patients were classified as responders to IFN-ß therapy (no relapses/increases in EDSS over the 2-year follow-up) and 112 as non-responders (at least two relapses or an increase in expanded disability status scale (EDSS) of at least one point during the same period). The combined analysis of available datasets yielded an effect size on MS with odds ratio (OR)(Mantel-Haenszel)=1.14 (P<0.002) for the IRF5 polymorphisms rs4728142 and rs3807306. Additionally, trends for association were observed between rs3807306T and infection with HHV-6 [p=0.05, OR (95% CI)=1.56 (1.00-2.44)] and response to IFN-ß therapy [P=0.09, OR (95% CI)=1.39 (0.95-2.05)].

Replication of top markers of a genome-wide association study in multiple sclerosis in Spain.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with presumed autoimmune origin, triggered by genetic and environmental risk factors. A recent genome-wide association study conducted on MS identified new biallelic markers outside the HLA (human leucocyte antigen) region involved in disease susceptibility: rs1109670 (DDEF2); rs1458175 (PDZRN4); rs1529316 and rs2049306 (CSMD1); rs16914086 (TBC1D2); rs1755289 (SH3GL2); rs1841770 (ZIC1); rs651477 (EN1); rs7607490 (TRIB2); rs397020 (C20orf46); rs908821 (SLC25A36); rs7672826 (MGC45800) and rs9523762 (GPC5). We aimed at replicating these top association signals in a Spanish cohort of 2863 MS patients and 2930 sex- and age-matched controls. Only rs9523762 mapping in the GPC5 gene was significantly associated (G allele, P=1.6 × 10(-5); odds ratio (95% confidence interval)=1.23 (1.12-1.36)), supporting a role for this proteoglycan in MS predisposition. The independent replication of association signals to validate data generated by genome-wide association scans is a first step in the effort to improve patient care.

The autoimmune disease-associated KIF5A, CD226 and SH2B3 gene variants confer susceptibility for multiple sclerosis.

Genome-wide association studies (GWAS) have revealed that different diseases share susceptibility variants. Twelve single-nucleotide polymorphisms (SNPs) previously associated with different immune-mediated diseases in GWAS were genotyped in a Caucasian Spanish population of 2864 multiple sclerosis (MS) patients and 2930 controls. Three SNPs were found to be associated with MS: rs1678542 in KIF5A (P=0.001, odds ratio (OR)=1.13, 95% confidence interval (CI)=1.05-1.23); rs3184504 in SH2B3 (P=0.00001, OR=1.19, 95% CI=1.10-1.27) and rs763361 in CD226 (P=0.00007, OR=1.16, 95%CI=1.08-1.25). These variants have previously been associated with rheumatoid arthritis and type 1 diabetes. The SH2B3 polymorphism has additionally been associated with systemic lupus erythematosus. Our results, in addition to validating some of these loci as risk factors for MS, are consistent with shared genetic mechanisms underlying different immune-mediated diseases. These data may help to shape the contribution of each pathway to different disorders.

Cognitive/personality pattern and triplet expansion size in adult myotonic dystrophy type 1 (DM1): CTG repeats, cognition and personality in DM1.

BACKGROUND: Although central nervous system (CNS) involvement in adult myotonic dystrophy type 1 (DM1) was described long ago, the large number of variables affecting the cognitive and personality profile have made it difficult to determine the effect of DM1 on the brain. The aim of this study was to define the cognitive and personality patterns in adult DM1 patients, and to analyse the relationship between these clinical patterns and their association with the underlying molecular defect. METHOD: We examined 121 adult DM1 patients with confirmed molecular CTG repeat expansion and 54 control subjects using comprehensive neuropsychological tests and personality assessments with the Millon Clinical Multiaxial Inventory (MCMI)-II. We used a multiple linear regression model to assess the effect of each variable on cognition and personality adjusted to the remainders. RESULTS: Patients performed significantly worse than controls in tests measuring executive function (principally cognitive inflexibility) and visuoconstructive ability. In the personality profile, some paranoid and aggressive traits were predominant. Furthermore, there was a significant negative correlation between the CTG expansion size and many of the neuropsychological and personality measures. The molecular defect also correlated with patients' daytime somnolence. CONCLUSIONS: Besides muscular symptomatology, there is significant CTG-dependent involvement of the CNS in adult DM1 patients. Our data indicate that the cognitive impairment predominantly affects the fronto-parietal lobe.

Somatic mosaicism in a case of apparently sporadic Creutzfeldt-Jakob disease carrying a de novo D178N mutation in the PRNP gene.

Transmissible spongiform encephalopathies (TSEs) are a group of rare fatal neurodegenerative disorders. Creutzfeldt-Jakob disease (CJD) represents the most common form of TSE and can be classified into sporadic, genetic, iatrogenic and variant forms. Genetic cases are related to prion protein gene mutations but they only account for 10-20% of cases. Here we report an apparently sporadic CJD case with negative family history carrying a mutation at codon 178 of prion protein gene. This mutation is a de novo mutation as the parents of the case do not show it. Furthermore the presence of three different alleles (wild type 129M-178D and 129V-178D and mutated 129V-178N), confirmed by different methods, indicates that this de novo mutation is a post-zygotic mutation that produces somatic mosaicism. The proportion of mutated cells in peripheral blood cells and in brain tissue was similar and was estimated at approximately 97%, suggesting that the mutation occurred at an early stage of embryogenesis. Neuropathological examination disclosed spongiform change mainly involving the caudate and putamen, and the cerebral cortex, together with proteinase K-resistant PrP globular deposits in the cerebrum and cerebellum. PrP typing was characterized by a lower band of 21 kDa. This is the first case of mosaicism described in prion diseases and illustrates a potential etiology for apparently sporadic neurodegenerative diseases. In light of this case, genetic counseling for inherited and sporadic forms of transmissible encephalopathies should take into account this possibility for genetic screening procedures.

MiR-219a-5p Enriched Extracellular Vesicles Induce OPC Differentiation and EAE Improvement More Efficiently Than Liposomes and Polymeric Nanoparticles.

Remyelination is a key aspect in multiple sclerosis pathology and a special effort is being made to promote it. However, there is still no available treatment to regenerate myelin and several strategies are being scrutinized. Myelination is naturally performed by oligodendrocytes and microRNAs have been postulated as a promising tool to induce oligodendrocyte precursor cell differentiation and therefore remyelination. Herein, DSPC liposomes and PLGA nanoparticles were studied for miR-219a-5p encapsulation, release and remyelination promotion. In parallel, they were compared with biologically engineered extracellular vesicles overexpressing miR-219a-5p. Interestingly, extracellular vesicles showed the highest oligodendrocyte precursor cell differentiation levels and were more effective than liposomes and polymeric nanoparticles crossing the blood-brain barrier. Finally, extracellular vesicles were able to improve EAE animal model clinical evolution. Our results indicate that the use of extracellular vesicles as miR-219a-5p delivery system can be a feasible and promising strategy to induce remyelination in multiple sclerosis patients.

Characterization of novel CAPN3 isoforms in white blood cells: an alternative approach for limb-girdle muscular dystrophy 2A diagnosis.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

Molecular characterization of putative modulatory factors in two Spanish families with A1555G deafness.

The aim of this work is to characterize possible modifying factors in 2 large families carrying the A1555G mitochondrial mutation. The heteroplasmy of the mutation, the presence of aminoglycosides, the cosegregation with other mitochondrial mutations, the proposed linkage in chromosome 8 and the association with TRMU and MTO1 genes were studied. None of the mentioned modifying factors were related with the phenotype presentation of A1555G mutation. However, TRMU G28T single nucleotide polymorphism is present in 1 of the studied families.

Minimizing creatine kinase variability in rats for neuromuscular research purposes.

Rat serum or plasma creatine kinase (CK) activity is widely used to evaluate myopathic processes, to test the myotoxicity of different drugs, or to analyse the benefits of emerging gene therapies in some neuromuscular disorders. However, great variability is found in this determination. The aim of this study has been to control some factors of variation in order to reduce variability and increase the reproducibility of analytical data. 8-10-week-old Wistar-Han rats were used. The study consisted of four sequential phases. Phase I aimed to analyse the effect of ether and isoflurane as anaesthetic drugs. The objective of Phase II was to evaluate bleeding rats via retro-orbital sinus vs. tail vein. Phases III and IV were designed as two separate, repeated measure experiments on two factors: habituation to laboratory handling procedures in Phase III and gender in Phase IV. The repeated factor was the storage temperature of blood sample prior to centrifugation. Ether did not significantly increased the CK value. Using isoflurane, getting rats accustomed to laboratory handling procedures and whole blood refrigeration prior to centrifugation and serum separation resulted in statistically significant reduction in CK value and variability. Male rats showed significantly higher values than female rats. In the light of our findings, CK value and variability in rats may be minimized by choosing tail vein as site of bleeding, getting rats accustomed to laboratory handling procedures and maintaining whole blood refrigerated until centrifugation and serum separation.

Models for Studying Myelination, Demyelination and Remyelination.

One of the most widely studied demyelinating diseases is multiple sclerosis, which is characterised by the appearance of demyelinating plaques, followed by myelin regeneration. Nevertheless, with disease progression, remyelination tends to fail, increasing the characteristic neurodegeneration of the disease. It is essential to understand the mechanisms that operate in the processes of myelination, demyelination and remyelination to develop treatments that promote the production of new myelin, thereby protecting the central nervous system. A huge variety of models have been developed to help improve our understanding of these processes. Nevertheless, no single model allows us to study all the processes involved in remyelination and usually more than one is needed to provide a full picture of related mechanisms. In this review, we summarise the most commonly used models for studying myelination, demyelination and remyelination and we analyse them critically to outline the most suitable ways of using them.

Apolipoprotein E epsilon4 allele in familial and sporadic Parkinson's disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease after Alzheimer's disease (AD). Common risk factors for both diseases have been explored to study potential etiologic interactions between these two neurodegenerative disorders. The APOE epsilon4 allele, previously associated with AD, has also been associated with risk of PD and with the presence of some clinical features in PD patients. However, the role of APOE epsilon4 allele in risk of PD remains unclear. We studied the distribution of APOE alleles in 276 unrelated familial and sporadic PD patients and in 212 controls. Patients and controls were classified by ethnicity. No genetic heterogeneity between Basques and people from other regions of Spain was found. No significant differences in APOE allele distribution between PD patients and controls were found; however, lower epsilon4 allele frequency was observed when the sporadic PD group was analyzed separately. By contrast, an increase in epsilon4 allele frequency was found in familial PD patients with cognitive decline. We conclude that the APOE epsilon4 allele may be associated with the risk of developing PD in isolated cases and that it is linked to the presence of cognitive decline in familial PD in our sample.

LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene.

We present here the clinical, molecular and biochemical findings from 238 limb-girdle muscular dystrophy type 2A (LGMD2A) patients, representing approximately 50% (238 out of 484) of the suspected calpainopathy cases referred for the molecular study of the calpain 3 (CAPN3) gene. The mean age at onset of LGMD2A patients was approximately 14 years, and the first symptoms occurred between 6 and 18 years of age in 71% of patients. The mean age at which the patients became wheelchair bound was 32.2 years, with 84% requiring the use of a wheelchair between the age of 21 and 40 years. There was no correlation between the age at onset and the time at which the patient became wheelchair bound, nor between the sex of the patient and the risk of becoming wheelchair bound. Of the cases where the CAPN3 gene was not affected, approximately 20% were diagnosed as LGMD2I muscular dystrophy, while facioscapulohumeral muscular dystrophy (FSHD) was uncommon in this sample. We identified 105 different mutations in the CAPN3 gene of which 50 have not been described previously. These were distributed throughout the coding region of the gene, although some exons remained free of mutations. The most frequent mutation was 2362AG-->TCATCT (exon 22), which was present in 30.7% of the chromosomes analysed (146 chromosomes). Other recurrent mutations described were N50S, 550DeltaA, G222R, IVS6-1G-->A, A483D, IVS17+1G-->T, 2069-2070DeltaAC, R748Q and R748X, each of which was found in >5 chromosomes. The type of mutation in the CAPN3 gene does not appear to be a risk factor for becoming dependent on a wheelchair at a determined age. However, in the cases with two null mutations, there were significantly fewer patients that were able to walk than in the group of patients with at least one missense mutation. Despite the fact that the results of phenotyping and western blot might be biased due to multiple referral centres, producing a diagnosis on the basis of the classical phenotype is neither sufficiently sensitive (86.7%) nor specific (69.3%), although western blot proved to be even less sensitive (52.5%) yet more specific (87.8%). In this case LGMD2I was a relevant cause of false-positive diagnoses. Considering both the clinical phenotype and the biochemical information together, the probability of correctly diagnosing a calpainopathy is very high (90.8%). However, if one of the analyses is lacking, the probability varies from 78.3 to 73.7% depending on the information available. When both tests are negative, the probability that the sample comes from a patient with LGMD2A was 12.2%.

Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA.

BACKGROUND: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form. OBJECTIVES: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS. RESULTS: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls). CONCLUSIONS: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Mitochondrial polymporphisms in Parkinson's Disease.

The mtDNA polymorphisms A4336G, A10398G and T4216C have been associated with PD. While A4336G is thought to be a genetic risk factor, A10398G appears to be a protective factor and T4216C is only weakly associated with the disease. In this work we analyzed the association between these three genetic polymorphisms and PD in a Spanish-PD population. The samples were classified by ethnic origin in Basques or other origin. Our analysis confirm the association between A4336G and PD. Our results with A10398G polymorphism highlight the importance of performing the association studies in ethnically homogeneous populations.

Cognitive function in facioscapulohumeral dystrophy correlates with the molecular defect.

Previous studies based on case descriptions and neuroradiological findings have suggested central nervous system (CNS) involvement in facioscapulohumeral dystrophy. The aim of this work is to explore the relationship between cognitive/personality pattern and the underlying molecular defect for this muscular dystrophy. We performed a wide-ranging neuropsychological assessment of 34 molecularly confirmed facioscapulohumeral dystrophy patients and 49 control subjects, all of whom also received the Millon-II Multiaxial Clinical Inventory (MCMI-II). Patients and controls show mild learning-level differences in the neuropsychological profile, and only the hysteriform scale is statistically higher in patients than controls. The patients' intelligence quotient (IQ) is related to the size of the deleted fragment but not to the degree of muscular impairment. The results of this study indicate a cut-off point and two distinct cognitive profiles in facioscapulohumeral dystrophy, depending on the patients' molecular defect: patients with a fragment size > 24 kb show a relatively normal cognitive pattern, whereas those with a fragment size < or = 24 kb show a significantly reduced IQ and difficulties with verbal function and visuo-constructive tasks. This work provides more evidence for the involvement of the CNS in facioscapulohumeral dystrophy and suggests that the fragment size should be taken into account in the clinical management of facioscapulohumeral dystrophy as it has a predictive value on the cognitive phenotype.

Association between synapsin III gene promoter SNPs and multiple sclerosis in Basque patients.

BACKGROUND: Synapsins are a family of neuron-specific phosphoproteins, one of whose subunits is encoded by the SYN3 gene. This gene is located close to one of the multiple sclerosis susceptibility regions (in 22q13.1). Two single-nucleotide polymorphisms (SNPs) (rs133945 and rs133946) in the promoter region of this gene have been proposed as factors protecting against MS. This relationship is not clear because another report failed to found such association. OBJECTIVES: In an attempt to clarify this association, the frequency of these SNPs was analyzed in a population of 221 Spanish MS patients with a cluster of 72 Basque patients and in 373 controls with a cluster of 138 controls of a Basque origin. METHODS: The SNis analysis was performed by 9 PCR. RESULTS: According to our findings, these SNPs are differently distributed in the two populations. This significant bias should therefore be taken into account in association studies. Our data suggest that the C/C genotype in rs133946 and the G/G genotype in rs133945 could be protecting factors against MS in the Basque population.

Identification of (1H)-pyrroles as histone deacetylase inhibitors with antitumoral activity.

Histone deacetylases (HDACs) play a key role in the regulation of gene expression and chromatin structure, and drugs targeting these enzymes might have an important impact in the treatment of human cancer. Herein, we report the characterization of (1H)-pyrroles as a new subfamily of HDAC inhibitors obtained by computational modeling of class-I human HDACs. From a functional standpoint, (1H)-pyrroles are powerful inductors of acetylation of histones H3 and H4, and restore the expression of growth-inhibitory genes. From a cellular view, these compounds cause a marked decrease in the viability of cancer cells in vitro and in vivo, associated with a cell-cycle arrest at G2/M and an inhibition of angiogenesis. Thus, (1H)-pyrroles emerge as a novel group of HDAC inhibitors with promising antitumoral features.