Michele Benigni

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.

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.

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.

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

Abstract 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.