Identification of miRNAs as Potential Biomarkers in Cerebrospinal Fluid from Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disorder. Since no diagnostic laboratory test exists, the identification of specific biomarkers could be fundamental in clinical practice. microRNAs (miRNAs) are considered promising biomarkers for neurodegenerative diseases. The aim of the study was to identify a CSF miRNA set that could differentiate ALS from non-ALS condition. miRNA profiling in CSF from ALS patients (n = 24; eight with C9orf72 expansion) and unaffected control subjects (n = 24) by quantitative reverse transcription PCR identified fourteen deregulated miRNAs. Validation experiments confirmed eight miRNAs as significantly deregulated in ALS. No significant differences were observed between ALS patients with or without C9orf72 expansion. The receiver operator characteristic (ROC) curve analyses revealed the highest diagnostic accuracy for the upregulated miR181a-5p and the downregulated miR21-5p and miR15b-5p. The miR181a-5p/miR21-5p and miR181a-5p/miR15b-5p ratios detected ALS with 90 and 85 % sensitivity and 87 and 91 % specificity, respectively, confirming the application potential as disease biomarkers. These deregulated miRNAs are implicated in apoptotic way and provide insight into processes responsible for motor neuron degeneration.

Lack of relationship between the P413L chromogranin B variant and a SALS Italian cohort.

Chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). Particularly, a variation c.1238C>T (p.Pro413Leu) in the chromogranin B gene, CHGB, has been associated with an earlier age at onset in both familial and sporadic ALS in French/French-Canadian populations studied. The aim of our study was to evaluate the P413L chromogranin variation in Italian patients with sporadic ALS. The study included 366 Italian patients with sporadic ALS and 382 control subjects. Genotyping of the polymorphism P413L in the CHGB gene was performed and the clinical characteristics of patients were analyzed in relation to their genotype. Our study on a cohort of Italian patients with SALS and controls failed to confirm an increased frequency of the 413L variant in SALS patients. Furthermore, we did not confirm the previous observation of a difference of age at onset between T-allele carriers and non-carriers (median age of onset 58.5 vs. 60.2years of age, respectively). Our findings do not support the 413L variant as a risk factor for sporadic ALS in the Italian population.

Genotyping of macrophage migration inhibitory factor (MIF) CATT5₋8 repeat polymorphism by denaturing high-performance liquid chromatography (DHPLC).

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed in many different cell types and implicated in the pathogenesis of numerous acute and chronic inflammatory diseases. Variable Number of Tandem Repeat (VNTR) CATT5-8 at position -794 in the promoter of the MIF gene has been associated with several human pathological conditions. Different methods for genotyping the CATT tetranucleotide repeats have been described. Here, we report, for the first time, the complete characterization of the CATT5-8 repeat polymorphism using exclusively the denaturing high-performance liquid chromatography (DHPLC) technique under partially denaturing conditions. This approach, based on a step-by-step DHPLC protocol, allowed the accurate determination of all the homozygous and heterozygous genotypes in 350 DNA samples from control subjects. The results were validated by comparison to DNA sequencing, and the DHPLC approach was accurate, sensitive, and highly reproducible. Data from the current study demonstrate that this method of analysis by DHPLC may represent a powerful and sensitive alternative tool for a rapid and efficient genotyping of short tandem repeats presenting a limited number of alleles.

Lack of mutations of the telomerase RNA component in familial papillary thyroid cancer with short telomeres.

BACKGROUND: The occurrence of familial papillary thyroid cancer (FPTC) is well established but no susceptibility genes for this disease have been discovered. Our group has recently demonstrated that patients with FPTC have shorter telomeres, not associated with mutations in telomerase reverse transcriptase, gene than patients with sporadic papillary thyroid cancer (SPTC), healthy subjects (HS), and unaffected family members (UFMs). Several diseases, however, have short telomeres associated with mutations in the telomerase RNA component (TERC) gene or in the shelterin complex (POT1, RAP1, TIN2, TPP1, TRF1, and TRF2) genes. The objective of the present study was to verify whether short telomeres observed in FPTC patients were related to mutations in TERC or shelterin genes. METHODS: Sixty-six patients with FPTC, 46 UFMs, 111 patients with SPTC, and 153 HS were analyzed by polymerase chain reaction followed by denaturing high performance liquid chromatography analysis and direct sequencing for the presence of TERC or shelterin gene mutations. When present, single-nucleotide polymorphisms were tested by χ(2) analysis at the genotypic, allelic, and haplotypic levels. RESULTS: The entire sequence of the TERC gene was analyzed with particular attention to known mutations known to be associated with short telomeres. All samples appeared to be homozygous wild type for A-771G, C-99G, G305A, G322A, C323T, C408G, G450A, T467C, G508A, A514G, G623A, and C727G substitutions and for the 378Δ→3' deletion in the TERC gene. In addition, upon analysis of all samples for shelterin proteins, we observed a significant decrease in POT1 and RAP1 protein expression in the blood of FPTC patients compared with SPTC subjects. However, no mutations or polymorphisms were found when in the coding sequences of both genes. CONCLUSIONS: To our knowledge this is the first study of TERC mutations or alterations in the shelterin complex in relation to FPTC. Shorter telomeres observed in FPTC are not linked to mutations or polymorphisms in TERC, POT1, or RAP1 genes.

No association of MTHFR c.677C>T variant with sporadic ALS in an Italian population.

The c.677C>T polymorphism in the 5,10-methylenetetrahydrofolate reductase gene (MTHFR) has been recently associated with susceptibility to sporadic amyotrophic lateral sclerosis (ALS). We have investigated this association in 450 ALS patients and 700 control subjects from Italy. No significant association was observed at the genotype and allelic level, either for the c.677C>T variant alone or in combination with PON1 polymorphisms. Our negative results suggest that the MTHFR c.677C>T polymorphism is not a risk factor for ALS in the Italian population.

Telomere length in neoplastic and nonneoplastic tissues of patients with familial and sporadic papillary thyroid cancer.

INTRODUCTION: Many studies have found an association between altered telomere length (TL), both attrition or elongation, and cancer phenotype. Recently, we have reported that patients with the familial form of papillary thyroid cancer (FPTC) have short telomeres in blood leucocytes. AIM: To evaluate relative TL (RTL) at somatic level in neoplastic and nonneoplastic tissues of patients with FPTC (n = 30) and sporadic PTC (n = 46). METHODS: RTL was measured by quantitative PCR in neoplastic thyroid tissues, in the corresponding nontumor thyroid tissues (normal contralateral thyroid), and in other extrathyroidal tissues (lymph nodes, muscles, or buccal mucosa). RTL was also measured in adenomas and hyperplastic nodules. In a subset of samples, telomerase expression was measured by quantitative PCR. RESULTS: Mean ± SD RTL of FPTC patients was short in neoplastic thyroid tissues (0.87 ± 0.2) with no difference from the normal contralateral thyroid tissues (0.85 ± 0.11) and extrathyroidal tissues (0.85 ± 0.31). On the contrary, in patients with sporadic PTC, the mean ± SD RTL in the neoplastic tissues (1.73 ± 0.63) was significantly shorter than that found in normal contralateral tissues (2.58 ± 0.89) and extrathyroidal tissues (2.5 ± 0.86). For all tissue samples (cancer, normal thyroid, and nonthyroidal tissues) the mean ± SD RTL of familial cases was shorter (P < 0.0001) than that found in tissues from sporadic PTC. RTL of FPTC was also lower (P < 0.0001) than that of 23 follicular adenomas (1.6 ± 0.7) and 24 hyperplastic nodules (2.2 ± 0.9). CONCLUSIONS: Our results demonstrate that short telomeres are a consistent feature of PTC, which in familial cases, is not restricted to the tumor tissue. This finding suggests that FPTC has a distinct, heritable, genetic background.

Lack of association of PON polymorphisms with sporadic ALS in an Italian population.

Paraoxonase (PON) gene polymorphisms have been associated with susceptibility to sporadic amyotrophic lateral sclerosis (ALS). We have investigated the role of the previously associated single nucleotide polymorphisms rs854560, rs662, and rs6954345 in 350 ALS patients and 376 matched controls from Italy. No significant association was observed at genotype and haplotype level. Our data suggest that PON polymorphisms are not involved in ALS pathogenesis in an Italian population.

Severe familial ALS with a novel exon 4 mutation (L106F) in the SOD1 gene.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease associated with a positive familial history in 5-10% of ALS cases. Mutations in the superoxide dismutase-1 (SOD1) gene have been found in 12%-23% of patients diagnosed with familial ALS. Here we report a novel mutation in exon 4 of SOD1 gene in a 55-year-old ALS patient belonging to a large Italian family with ALS first clinically described in 1968. In the family the clinical presentation was characterized by relatively early age of onset, spinal onset with proximal distribution weakness, bulbar involvement and a rapid disease course. Molecular analysis showed a heterozygous mutation at codon 106 resulting in a substitution of phenylalanine for leucine in the SOD1 protein (L106F). In analogy with the previously reported L106V mutation, we propose that the L106F causes a relevant destabilization of the protein chain around the mutation site, able to affect the SOD1 monomer and dimer structures suggesting a pathogenic role for this novel mutation.

A novel exon 1 mutation (G10R) in the SOD1 gene in a patient with familial ALS.

Mutations in the superoxide dismutase-1 (SOD1) gene have been found in 12-23% of patients with a diagnosis of ALS. Although the mechanism by which mutant SOD1 causes neural death remains elusive, several lines of evidence suggest that ALS is a protein-folding disease. Here we report a novel missense mutation in exon 1 of the SOD1 gene in a 68-year-old female with familial ALS characterized by spinal onset with upper and lower motor neuron signs and early neuroimaging evidence of corticospinal tract involvement. Molecular analysis identified a heterozygous mutation in codon 10, with substitution of a highly conserved glycine with arginine (G10R). Modelling of the mutant SOD1 showed a strong destabilization of the protein secondary structure that could influence the strength of the dimer interface. This property can result in a failure of the protein to fold and generation of toxic intracellular aggregates, suggesting a pathogenic role for the mutation.