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Biomedical subjects

Massimo Pandolfo

Publications and source records attributed to Massimo Pandolfo.

At least 19 recordsLinked to original sources
PubMed2006

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue.

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.

Aged↗
PubMed2006

Genetic analysis of candidate genes modifying the age-at-onset in Huntington's disease.

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington's disease (HD) and determines 42-73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.

Acyltransferases↗
PubMed2006

Study of the adenosinergic system in the brain of HPRT knockout mouse (Lesch-Nyhan disease).

BACKGROUND: Lesch-Nyhan disease (LND), an X-linked genetic disease caused by complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), is characterized by hyperuricemia and psychiatric disturbance, mainly self-aggressiveness. Literature dates support the hypothesis that dopaminergic deficit and serotonergic excess in the circuit of basal ganglia are responsible for the aggressive behavior. Altered adenosine transport across the membrane of HPRT-deficient lymphocytes has been reported, suggesting adenosine involvement in LND. METHODS: The expression of several genes related to the adenosinergic system (ADORA1A, ADORA2A, ADORA2B) were studied in the brain of the murine model of LND by real time PCR. Nucleotide levels and enzyme activities possibly involved in adenosine release were also measured. RESULTS: Studies performed by real time PCR showed 95% increase in ADORA1A expression, 15% decrease in ADORA2A expression, and no change in ADORA2B expression in knockout mice compared to controls. No significant differences were found in the level of nucleotides or enzyme activities between control and mutant mice. CONCLUSIONS: Our results suggest that adenosine neurotransmission might be involved in the specific neurobehavioral features of LND by increased expression of adenosine A1 receptors.

Adenosine↗
PubMed2006

Gene expression profiling in frataxin deficient mice: microarray evidence for significant expression changes without detectable neurodegeneration.

Friedreich's ataxia (FRDA) is caused by reduction of frataxin levels to 5-35%. To better understand the biochemical sequelae of frataxin reduction, in absence of the confounding effects of neurodegeneration, we studied the gene expression profile of a mouse model expressing 25-36% of the normal frataxin levels, and not showing a detectable phenotype or neurodegenerative features. Despite having no overt phenotype, a clear microarray gene expression phenotype was observed. This phenotype followed the known regional susceptibility in this disease, most changes occurring in the spinal cord. Additionally, gene ontology analysis identified a clear mitochondrial component, consistent with previous findings. We were able to confirm a subset of changes in fibroblast cell lines from patients. The identification of a core set of genes changing early in the FRDA pathogenesis can be a useful tool in both clarifying the disease process and in evaluating new therapeutic strategies.

Animals↗
PubMed2006

Eight novel mutations in SPG4 in a large sample of patients with hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases. OBJECTIVE: To search for disease-causing mutations in a large series of Italian patients with HSP. DESIGN: Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13. SETTING: Molecular testing facility in Italy. PATIENTS: Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP. MAIN OUTCOME MEASURES: Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13. RESULTS: We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene. CONCLUSIONS: The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP.

Adenosine Triphosphatases↗
PubMed2006

Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation.

OBJECTIVE: To report the clinical manifestations and functional aspects of Tuberous Sclerosis Complex (TSC), resulting from Codon 905 mutations in TSC2 gene. METHODS: We performed a detailed study of the TSC phenotype and genotype in a large French-Canadian kindred (Family A). Subsequently, clinical and molecular data on 18 additional TSC families with missense mutations at the same codon of TSC2 were collected. Functional studies were performed on the different missense changes and related to the phenotype. RESULTS: A 2714G>A (R905Q) mutation was identified in Family A. The TSC phenotype in this family was unusually mild and characterized by hypomelanotic macules or focal seizures that remitted spontaneously or were easily controlled with medication. Diagnostic criteria were met in only a minority of mutation carriers. Other families with the R905Q mutation were found to have a similar mild phenotype. In contrast, patients with a 2713C>T (R905W) or a 2713C>G (R905G) mutation had more severe phenotypes. Although all three amino acid substitutions were pathogenic, the R905W and R905G substitutions affected tuberin function more severely than R905Q. INTERPRETATION: Codon 905 missense mutations in TSC2 are relatively common. The TSC2 R905Q mutation is associated with unusually mild disease, consistent with functional studies. Combined with previous reports, it is apparent that certain TSC2 missense mutations are associated with a mild form of tuberous sclerosis, which in many patients does not meet standard diagnostic criteria. These findings have implications for the large number of patients with limited clinical features of TSC and for genetic counseling in these families.

Adolescent↗
PubMed2006

A real-time PCR approach to evaluate adipogenic potential of amniotic fluid-derived human mesenchymal stem cells.

Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.

Adipogenesis↗
PubMed2006

Friedreich ataxia: Detection of GAA repeat expansions and frataxin point mutations.

Friedreich ataxia (FA) is an autosomal-recessive disease primarily characterized by progressive neurological disability. A significant proportion of patients also present with a hypertrophic cardiomyopathy, which may, in some cases, cause premature death. FA is caused by insufficient levels of the protein, frataxin, which is involved in mitochondrial iron metabolism. All patients carry at least one copy of an intronic GAA triplet-repeat expansion that interferes with frataxin transcription. Normal chromosomes contain up to 35 to 40 GAA triplets in an Alu sequence localized in the first intron of the frataxin gene; FA chromosomes carry from approx 70 to more than 1000 GAA triplets. The molecular diagnosis of FA is, therefore, based on the detection of this expansion, which is present in homozygosity in more than 95% of the cases. The remaining patients are heterozygous for the GAA expansion and carry a frataxin point mutation as the other pathogenic allele. The expanded GAA triplet repeat may be detected by polymerase chain reaction (PCR) amplification followed by agarose gel electrophoresis analysis. In our hands, carefully performed PCR testing, in particular, if fragment detection is enhanced by hybridization with a GAA oligonucleotide probe, is as effective in identifying patients and carriers as is Southern blot analysis of genomic DNA, and allows a more accurate sizing of the repeat. Furthermore, in the case of smaller expansions, the amplified fragment may be directly sequenced to identify very rare nonpathogenic variant repeats, such as GAAGGA. Sequence analysis of the five coding exons of the frataxin gene should be performed in clinically affected individuals who are heterozygous for an expanded GAA repeat to identify point mutations.

Blotting, Southern↗
PubMed2005

The S18Y polymorphism in the UCHL1 gene is a genetic modifier in Huntington's disease.

An expanded polyglutamine stretch in the huntingtin protein has been identified as the pathogenetic cause of Huntington's disease (HD). Although the length of the expanded polyglutamine repeat is inversely correlated with the age-at-onset, additional genetic factors are thought to modify the variance in the disease onset. As linkage analysis suggested a modifier locus on chromosome 4p, we investigated the functional relevance of S18Y polymorphism of the ubiquitin carboxy-terminal hydrolase L1 in 946 Caucasian HD patients. In this group, the allelic variation on locus S18Y is responsible for 1.1% of the variance in the HD age-at-onset, and the rare Y allele is associated with younger-aged cases.

Age of Onset↗
PubMed2005

Hemicerebellectomy blocks the enhancement of cortical motor output associated with repetitive somatosensory stimulation in the rat.

Repetitive peripheral stimulation is associated with an enhancement of the intensity of corticomotor responses. We analysed the effects of hemicerebellectomy on the modulation of cortical motor output associated with repetitive electrical stimulation of the sciatic nerve in the rat. Hemicerebellectomy blocked the enhancement of the corticomotor response. The cerebellum is a key player in this form of short-term plasticity.

Animals↗
PubMed2005

A new clinical and molecular form of Unverricht-Lundborg disease localized by homozygosity mapping.

Progressive myoclonus epilepsy (PME) has a number of causes, of which Unverricht-Lundborg disease (ULD) is the most common. ULD has previously been mapped to a locus on chromosome 21 (EPM1). Subsequently, mutations in the cystatin B gene have been found in most cases. In the present work we identified an inbred Arab family with a clinical pattern compatible with ULD, but mutations in the cystatin B gene were absent. We sought to characterize the clinical and molecular features of the disorder. The family was studied by multiple field trips to their town to clarify details of the complex consanguineous relationships and to personally examine the family. DNA was collected for subsequent molecular analyses from 21 individuals. A genome-wide screen was performed using 811 microsatellite markers. Homozygosity mapping was used to identify loci of interest. There were eight affected individuals. Clinical onset was at 7.3 +/- 1.5 years with myoclonic or tonic-clonic seizures. All had myoclonus that progressed in severity over time and seven had tonic-clonic seizures. Ataxia, in addition to myoclonus, occurred in all. Detailed cognitive assessment was not possible, but there was no significant progressive dementia. There was intrafamily variation in severity; three required wheelchairs in adult life; the others could walk unaided. MRI, muscle and skin biopsies on one individual were unremarkable. We mapped the family to a 15-megabase region at the pericentromeric region of chromosome 12 with a maximum lod score of 6.32. Although the phenotype of individual subjects was typical of ULD, the mean age of onset (7.3 years versus 11 years for ULD) was younger. The locus on chromosome 12 does not contain genes for any other form of PME, nor does it have genes known to be related to cystatin B. This represents a new form of PME and we have designated the locus as EPM1B.

Adolescent↗
PubMed2005

Depression of extra-cellular GABA and increase of NMDA-induced nitric oxide following acute intra-nuclear administration of alcohol in the cerebellar nuclei of the rat.

Gamma-aminobutyric acid (GABA) and nitric oxide are two key-transmitters in cerebellar nuclei, the major output of cerebellar circuitry. The aims of this study were to investigate the effects of acute intra-cerebellar administration of ethanol (20 mM) on extra-cellular levels of GABA and on the NMDA-induced nitric oxide (NO) production using microdialysis in the rat. We also studied: (i) the effects of a pre-administration of DNQX, a specific antagonist of AMPA receptors, on NO production, (ii) the effects of a pre-administration of 7-NI (7-nitroindazole, an inhibitor of neuronal nitric oxide synthase NOS) and APV (D-2-amino-5-phosphonovaleric acid, a specific blocker of the NMDA type glutamate receptors) on the actions of alcohol/NMDA on glutamate receptors, and (iii) the in vivo interaction between DNQX, ethanol and NMDA receptor activation. We found that ethanol decreased the amount of extra-cellular GABA, and that this effect was counterbalanced by administration of tiagabine 1 mg/kg, a potent inhibitor of GAT-1 GABA transporter, given by the i.p. route. In loco administration of NMDA increased the levels of NO, as previously reported. A pre-administration of DNQX (500 microM) increased significantly the production of NO up to toxic levels, as well as ethanol administration. A pre-administration of 7-NI or APV reduced significantly the amounts of NO when NMDA and alcohol were infused simultaneously. The combination of ethanol with DNQX was associated with a marked enhancement of the concentrations of NO. The activity of GAT-1 in cerebellar nuclei and around this target, including in glial cells expressing GAT-1 activated by ambient GABA, seems to be spared by ethanol. Tiagabine could be considered as a candidate for future investigational treatments of acute ethanol-induced dysfunction of cerebellar nuclei. We found a potentiation of the production of NO when AMPA antagonists are given simultaneously to ethanol. The hypothesis of AMPA neurotoxicity, which has convincing arguments during chronic exposure, is challenged in this model of acute cerebellar nuclear toxicity of alcohol.

Animals↗
PubMed2004

Multifaceted role of galectin-3 on human glioblastoma cell motility.

Astrocytic tumors' aggressiveness results from an imbalance between cell proliferation and cell death favoring growth, but also from the propensity of tumor cells to detach from the primary tumor site, migrate, and invade the surrounding parenchyma. Astrocytic tumor progression is known to be associated with an increased expression of galectin-3. We investigated in cell culture how galectin-3 expression affects astrocytoma cell motility. Galectin-3 deficient cells were obtained by stable transfection of the U373 glioblastoma cell line with a specific expression antisense plasmid. Cultured galectin-3 deficient glioblastoma cells showed increased motility potential on laminin and modifications in the cytoskeleton reorganization. In addition, c-DNA microarrays and quantitative immunofluorescence analysis showed that galectin-3 deficient U373 cells have an increased expression of integrins-alpha6 and -beta1, proteins known to be implicated in the regulation of cell adhesion.

Actins↗
PubMed2004

The gene for paroxysmal non-kinesigenic dyskinesia encodes an enzyme in a stress response pathway.

Paroxysmal non-kinesigenic dyskinesia (PNKD) is characterized by spontaneous hyperkinetic attacks that are precipitated by alcohol, coffee, stress and fatigue. We report mutations in the myofibrillogenesis regulator 1 (MR-1) gene causing PNKD in 50 individuals from eight families. The mutations cause changes (Ala to Val) in the N-terminal region of two MR-1 isoforms. The MR-1L isoform is specifically expressed in brain and is localized to the cell membrane while the MR-1S isoform is ubiquitously expressed and shows diffuse cytoplasmic and nuclear localization. Bioinformatic analysis reveals that the MR-1 gene is homologous to the hydroxyacylglutathione hydrolase (HAGH) gene. HAGH functions in a pathway to detoxify methylglyoxal, a compound present in coffee and alcoholic beverages and produced as a by-product of oxidative stress. Our results suggest a mechanism whereby alcohol, coffee and stress may act as precipitants of attacks in PNKD. Stress response pathways will be important areas for elucidation of episodic disease genetics where stress is a common precipitant of many common disorders like epilepsy, migraine and cardiac arrhythmias.

Animals↗
PubMed2004

Frataxin overexpressing mice.

Friedreich ataxia, the most common autosomal recessive ataxia, is caused by frataxin deficiency. Reduction of frataxin has been associated with iron accumulation and sensitivity to iron induced oxidative stress. To better understand the function of frataxin, transgenic mice (tgFxn) overexpressing human frataxin were generated. Iron metabolism parameters in tgFxn were normal and no signs of ataxia or other obvious abnormalities were observed, indicating that overexpression of frataxin in mouse is innocuous. Several hypotheses for frataxin function were evaluated in tgFxn mice. In particular, we observed that TgFxn mice show an altered response during hematopoietic differentiation, suggesting that frataxin may directly affect heme synthesis.

Animals↗
PubMed2004

Spinocerebellar ataxia type 8: molecular genetic comparisons and haplotype analysis of 37 families with ataxia.

We reported elsewhere that an untranslated CTG expansion causes the dominantly inherited neurodegenerative disorder spinocerebellar ataxia type 8 (SCA8). SCA8 shows a complex inheritance pattern with extremes of incomplete penetrance, in which often only one or two affected individuals are found in a given family. SCA8 expansions have also been found in control chromosomes, indicating that separate genetic or environmental factors increase disease penetrance among SCA8-expansion-carrying patients with ataxia. We describe the molecular genetic features and disease penetrance of 37 different families with SCA8 ataxia from the United States, Canada, Japan, and Mexico. Haplotype analysis using 17 STR markers spanning an approximately 1-Mb region was performed on the families with ataxia, on a group of expansion carriers in the general population, and on psychiatric patients, to clarify the genetic basis of the reduced penetrance and to investigate whether CTG expansions among different populations share a common ancestral background. Two major ancestrally related haplotypes (A and A') were found among white families with ataxia, normal controls, and patients with major psychosis, indicating a common ancestral origin of both pathogenic and nonpathogenic SCA8 expansions among whites. Two additional and distinct haplotypes were found among a group of Japanese families with ataxia (haplotype B) and a Mexican family with ataxia (haplotype C). Our finding that SCA8 expansions on three independently arising haplotypes are found among patients with ataxia and cosegregate with ataxia when multiple family members are affected further supports the direct role of the CTG expansion in disease pathogenesis.

Case-Control Studies↗