New CACNA1A deletions are associated to migraine phenotypes.

BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is a form of migraine with aura caused by heterozygous mutations in 4 genes: CACNA1A, ATP1A2, SNC1A and PRRT2, but further heterogeneity is expected. Here have been described clinical and molecular features in patients suffering from migraine with Aura (MA), without (MO) and hemiplegic migraine attacks. Next Generation Sequencing by TruSeq Custom Amplicon for CACNA1A and ATP1A2 gene has been performed. All genetic variants have been confirmed by Sanger sequencing and all samples were also analyzed with MLPA assay for ATP1A2-CACNA1A genes to detect duplication or deletion. All MLPA data were verified by Real Time PCR. RESULTS: Sequencing analysis showed 3 point mutations, two novel variants and one already described in literature. Moreover, MLPA analysis showed 3 deletions in 9 sporadic hemiplegic migraine (18%), in 3 patients with non-hemiplegic migraine (4.1%) and in 3 patients affected by episodic ataxia (20%). Two sporadic patients showed a deletion in exons 41-43, while the rest of HM patients (5) showed a deletion in the terminal part of the CACNA1A gene. About episodic ataxia, we have identified deletions in exon 12-15 and in exon 47. Finally, in migraine patients, we have found different subjects affected by different phenotypes deleted in exon 47. CONCLUSION: This work highlights the importance to complement analysis as direct sequencing with quantitative analysis (MLPA). In fact, intragenic CACNA1A rearrangements have been detected. Our work demonstrated that deletions in CACNA1A gene may be associated also to different migraine phenotypes.

Circulating microRNAs and diabetes mellitus: a novel tool for disease prediction, diagnosis, and staging?

Diabetes is a complex, multifactorial group of metabolic diseases characterized by chronic hyperglycaemia due to pancreatic beta-cell dysfunction and/or loss. It is characterized by an asymptomatic and highly variable prodromic phase, which renders diabetes mellitus difficult to be predicted with sufficient accuracy. Despite several efforts in the identification and standardization of newly trustable. Biomarkers able to predict and follow-up diabetes and to specifically subtype its different forms, few of them have proven of clinical utility. Recently, a new class of endogenous non-coding small RNAs, namely microRNAs, have been indicated as putative biomarkers, being released by cells and tissues and found in a cell-free circulating form in many biological fluids, including serum and/or plasma. MicroRNAs have been initially identified as promising biomarkers in cancer, and nowadays their application has been extended to other diseases, including diabetes. Although an increasing number of studies focused on the evaluation of circulating microRNAs in diabetes, few reproducibly identified microRNAs as biomarkers for disease prediction or follow-up. Technological problems as well as the need to obtain highly standardized operating procedures and methods are still an issue in such research field. In this review, we comprehensively resume the main and most recent findings on circulating microRNAs, and their possible use as biomarkers to predict and follow-up diabetes and its complications, as well as the methodological challenges to standardize accurate operating procedures for their analysis.

The reaction of rhenium nitrosyl with a sterically hindered NHC-carbene.

In this article, we present the serendipitous synthesis of the unknown Re(I) complex [(OPPh3)Re(NO)2Cl3] (3) that we obtained reacting the Re(V) complex trans-[(PPh3)2ReOCl3] (1) with NO gas in presence of CH3COOH. We found that 3 reacts with 1,3-bis (2,4,6-trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene (IMes) to yield a stable oximate-Re(III) complex [(OPPh3)Re(NO)(ONIMes)Cl3] (4). We speculate that the IMes reacts with a bent NO, because the DFT calculations excluded the formation of both dimeric and η2-NO complexes in solution. The reactivity of the NO toward the carbene is probably due to an internal fluxional process in which the NO passes from linear to bent, triggered by the π-electrons given by the three chlorides to the Re through the mesomeric effect.

Myelinated retinal fibers in autosomal recessive spastic ataxia of Charlevoix-Saguenay.

BACKGROUND: Myelinated retinal nerve fibers are considered a hallmark of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) in French Canadian patients. The demonstration of a worldwide distribution of this disease, as well as the almost invariable presence of a normal retina on fundoscopy in cases outside Canada, suggests that more quantitative methodologies are needed to assess the retina in ARSACS. METHODS: To characterize better the retinal features of ARSACS, we studied five Italian patients by means of optical coherence tomography (OCT), a processing method that allows the creation of three-dimensional images with micrometer resolution. We compared OCT characteristics in ARSACS with those obtained from five subjects with persistent myelination of the retina, a rare congenital non-progressive anomaly. RESULTS: Four patients with ARSACS showed myelinated retinal nerve fibers on ophthalmoscopy, corresponding to an increased thickness of the retina on OCT, a characteristic not present in the subjects with persistent myelination of the retina. CONCLUSIONS: Myelinated retinal fibers are not rare in Italian patients with ARSACS. This finding may be the consequence of the thickening of the retina, as detected by OCT.

Characterization and evaluation of magnesite ore mining by-products of Gerakini mines (Chalkidiki, N. Greece).

The qualitative and quantitative characterization of several mining by-product samples, were collected from the magnesite mine of "Grecian Magnesite SA" company (Gerakini, Chalkidiki, North Greece), was aiming to evaluate the possibility of upgrading their refractory properties by applying thermal treatment. The concentration range of main components for the selected best qualified samples was 38.7-43 wt% for MgO, 37.5-44.1 wt% for SiO2 and 6.5-7.3 wt% for FeO. The mineralogical characterization revealed the presence of olivine, pyroxenes and serpentine, with the concentration of the latter positively correlated to LOI. Microprobe analyses clarified the presence of olivine [(Mg1.79Fe0.19Ni0.01)SiO4], consisting mainly of 90 wt% of forsterite (Mg2SiO4) and 10 wt% of fayalite (Fe2SiO4), as well as of pyroxene group minerals [(Mg0.87Fe0.08Ca0.01Cr0.01)(Si0.98Al0.04)O3], consisting mainly of 91 wt% enstatite (MgSiO3) and 9 wt% of ferrosilite (FeSiO3), respectively. The thermal treatment of the qualified samples demonstrated that at the temperature of 650-680 °C serpentine is almost totally decomposed and at the temperature of 850 °C it has been totally recrystallized to olivine and pyroxenes. At higher temperature treatment (1300 °C), it seems that there is a recrystallization process that favors the deformation of olivine and the further formation of pyroxenes, due to the excess of Si available from the initial decomposition of serpentine, while the presence of magnesite resulted to the restriction of olivine deformation through the partial capture of available Si. For increasing the olivine percentage and, subsequently, the improvement of refractory properties of this material, at temperature > 1300°C, the ideal theoretical addition dose of wt% MgO for optimizing the formation of olivine was calculated, ranging from 7.4 to 17.5 wt%. The latter calculations are reported for the first time in the literature regarding this kind of materials.

The wolframin His611Arg polymorphism influences medication overuse headache.

Homozygosis for wolframin (WFS1) mutations determines Wolfram syndrome (WS), and common polymorphisms of WFS1 are associated with psychiatric illnesses and dependence behaviour. To test the influence of WFS1 polymorphisms on medication overuse headache (MOH), a chronic headache condition related to symptomatic drugs overuse, we analyzed 82 MOH patients for the WFS1 His611Arg polymorphism, and performed a comparison between clinical features of Arg/Arg (R/R) and non-R/R individuals. Individuals harbouring the R/R genotype showed significantly higher monthly drug consumption (t=-3.504; p=0.00075) and more severe depressive symptoms on the BDI questionnaire (t=-3.048; p=0.003) than non-R/R. WFS1 polymorphism emerged as the only significant predictor of drug consumption, at the multivariate regression analysis (F=12.277; d.f.=1,80; p=0.00075, adjusted R2=0.122). These results implicate WFS1 in the clinical picture of MOH, may be through an influence on need for drugs as in other conditions of abuse behaviour.

Early-onset progressive ataxia associated with the first CACNA1A mutation identified within the I-II loop.

Familial hemiplegic migraine type 1, spinocerebellar ataxia type 6 (SCA6) and episodic ataxia type 2 (EA2) are allelic disorders associated with mutations in the CACNA1A gene, which encodes the alpha1 subunit of the P/Q-type calcium channel (Ca(V)2.1). SCA6 and EA2 share a number of clinical features, such as prominent cerebellar involvement and good response to acetazolamide therapy. However, while SCA6 develops as a late-onset, progressive ataxia, EA2 has an earlier, and episodic, onset. We report on two sisters with a heterogeneous clinical phenotype. The first developed progressive cerebellar ataxia after age 30, without noticeable episodes of vertigo or headache. A 1 year trial with acetazolamide did not produce significant results. The other reported episodes of vertigo, headache and gait imbalance since late childhood, with good response to acetazolamide, before developing moderate chronic cerebellar ataxia. Brain MRI showed cerebellar atrophy, especially in the vermis, in both patients. Direct sequencing of CACNA1A identified a heterozygous 1360G>A mutation in exon 11 resulting in the substitution of alanine for threonine at residue 454 (p.Ala454Thr). This is the first description of a change residing in the cytoplasmic I-II loop associated with a clinical phenotype.

Skeletal muscle wasting in tumor-bearing rats is associated with MyoD down-regulation.

Cachexia is a syndrome characterized by profound skeletal muscle wasting that frequently complicates malignancies. A number of studies indicate that protein hypercatabolism, largely mediated by classical hormones and cytokines, is the major component of muscle depletion. Impaired regeneration has been suggested to contribute to the reduction of muscle size. In particular, it has been shown that the expression of MyoD, a muscle-specific transcription factor, is down-regulated by cytokines such as TNFalpha and IFNgamma in a NF-kappaB-dependent posttranscriptional manner. The present study investigated whether modulations of the transcription factor MyoD are associated with the onset of muscle wasting in a well established model of cancer cachexia. Rats bearing the Yoshida AH-130 hepatoma develop a condition of muscle protein hypercatabolism, largely dependent on TNFalpha bioactivity. In the gastrocnemius of these animals the expression of MyoD was markedly reduced, paralleling the decrease of muscle weight. This pattern is associated with increased nuclear translocation of AP-1, while DNA-binding assays did not detect any change in NF-kappaB activity. This is the first observation demonstrating that muscle depletion in tumor-bearing rats is associated with a down-regulation of MyoD levels. Although the underlying mechanisms remain to be clarified, this change is compatible with the hypothesis that a reduced expression of molecules involved in the regulation of the regenerative response may concur to muscle wasting in cancer cachexia.

Autosomal dominant external ophthalmoplegia and bipolar affective disorder associated with a mutation in the ANT1 gene.

The authors report on a family with dominantly inherited progressive external ophthalmoplegia and a diagnostic and statistical manual (fourth revised edition) diagnosis of bipolar psychiatric disorder in several members. Skeletal muscle biopsy from the proposita showed decreased cytochrome c oxidase staining, several ragged-red fibers, and multiple mtDNA deletions. The authors identified a missense mutation (leucine 98-->proline) in the adenine nucleotide translocator 1 gene. The presence of bipolar affective disorder expands the phenotype of adenine nucleotide translocator 1 allelic variants.

Hypertrophic obstructive cardiomyopathy. Late follow-up after ventricular myotomy-myectomy.

A series of 25 patients with hypertrophic obstructive cardiomyopathy, isolated or associated with minor anomalies, were operated upon using a modified Morrow's procedure. The indication for operation was based on either the presence of severe symptoms despite treatment with beta-blocking agents, or a significant peak gradient of over 50 mmHg even in asymptomatic patients. All patients survived and none was lost to follow-up. Long-term results were evaluated with 2-dimensional echocardiography. Surgical versus pharmacological treatment is discussed, with particular consideration given to paediatric patients.

Recruitment and early treatment in a multicenter study of acute spinal cord injury.

STUDY DESIGN: Post hoc secondary analysis of data from 1992 to 1998 in the trial of Sygen in Acute Spinal Cord Injury. OBJECTIVES: Quasi-epidemiologic understanding of injury and treatment patterns and of recruitment in an SCI trial. No drug efficacy results. SUMMARY OF BACKGROUND DATA: The most recent large epidemiologic study was the National SCI Database by Stover and colleagues around 1980. METHODS: Emphasis on descriptive, rather than inferential, statistics: consistent with secondary analysis. RESULTS: The study involved 760 patients at 28 centers in North America. Cervical injuries were more common than thoracic, and complete injuries were more common than incomplete injuries. Recruitment in the complete cervical stratum was 332, but the incomplete thoracic strata had only 31 patients combined. Vital signs at arrival and on randomization show fair stability. Clock times show more injuries on weekends and nights but suggest immediate attention was given. Elapsed times to treatment (especially EMT and Medevac arrival) are short. The rate of direct admission to tertiary centers, traction weight, and time to surgery vary among centers. Inpatient rehabilitation appeared driven by insurance in addition to severity. CONCLUSIONS: The imbalances in favor of cervical and of complete injuries would make it hard for studies to attain results for SCI in general. The vital signs and time patterns suggest local protocol-driven stabilization to prevent secondary physiologic injury early after SCI. Some features of care vary among centers, but the sparseness of prospective data in specific injury and treatment categories suggests that treatment guidelines have limited empirical support and should be made cautiously.

Measurements and recovery patterns in a multicenter study of acute spinal cord injury.

STUDY DESIGN: Post hoc, secondary analysis of data from 1992 to 1998 in the trial of Sygen in acute spinal cord injury. OBJECTIVES: Quasi-epidemiologic understanding of measurement tools and of recovery patterns. No drug efficacy results. SUMMARY OF BACKGROUND DATA: Many authors have studied individual scales for measuring the severity of spinal cord injury. METHODS: Emphasis on descriptive, rather than inferential, statistics: consistent with secondary analysis. RESULTS: Of the 760 patients, 43 died within 365 days. The rate was higher for complete injuries (7.1% vs. 3.2%, P = 0.017). Marked recovery at 26 weeks was more frequent in those with better baseline American Spinal Injury Association (ASIA) Impairment Scale (AIS) scores, but was not different for methylprednisolone within versus after 3 hours. Light touch scores improved at each visit, more so in those with higher scores at baseline. Bladder control similarly improved. Motor and sensory scores exhibited departures from assumptions underlying normal-theory statistical techniques: t test and analysis of variance. Furthermore, they were mixtures of differing distributions from different study strata, so that overall conclusions depend on the mixture of patients seen. CONCLUSIONS: The prognosis of these patients with spinal cord injury seen at 28 centers in North America during the mid-1990s appears better than was often assumed earlier. The general patterns are similar across different measurement scales, although there are intriguing differences. The patterns in different strata are different in specifics, and complete injuries do less well. Pooling data from different strata may result in probability distributions that depart from normal-theory assumptions and give misleading results depending on recruitment patterns.

The Sygen multicenter acute spinal cord injury study.

STUDY DESIGN: Randomized, double-blind, sequential, multicenter clinical trial of two doses of Sygen versus placebo. OBJECTIVES: To determine efficacy and safety of Sygen in acute spinal cord injury. SUMMARY OF BACKGROUND DATA: An earlier, single-center trial in 28 patients showed an improvement (50.0% vs. 7.1%, P = 0.034) in marked recovery with Sygen. METHODS: Standard clinical trial techniques. RESULTS: The prospectively planned analysis at the prespecified endpoint time for all patients was negative. There was a significant effect in all patients in the primary outcome variable (the percentage of marked recovery) at week 8, the end of the dosing period. There was a significant effect in all patients in the time at which marked recovery is first achieved. Restricted to severity Group B, which has small sample size, the primary efficacy analysis showed a trend but did not reach significance. There is a large, consistent and, at some time points, significant effect in the primary outcome variable in the nonoperated patients through week 26. The American Spinal Injury Association motor, light touch, and pinprick scores showed a consistent trend in favor of Sygen, as also did bowel function, bladder function, sacral sensation, and anal contraction. The less severely injured patients appeared to have a greater beneficial drug effect. Evidence against an effect of Sygen was minimal and scattered. CONCLUSIONS: Although not proven in the primary efficacy analysis of this trial, Sygen appears to be beneficial in patients with severe spinal cord injury.

The role of a modified intussusception jejunocolic valve in short-bowel syndrome.

Short-bowel syndrome results in malabsorption and malnutrition producing profound metabolic disorders. Attempts to correct this problem with various surgical procedures have so far proved unsuccessful. Studies made in our laboratory using experimental animals, showed that a modified jejunocolic intussusception valve appears to be effective in prolonging the small-bowel transit time and the absorptive capacity of the intestines in short-bowel syndrome. Furthermore, construction of the valve is uncomplicated and unaccompanied by any mortality or morbidity.

Association in the same patient of autosomal dominant progressive external ophthalmoplegia with multiple mtDNA deletions and X-linked ichthyosis: clinical, biochemical, histological, submicroscopic and molecular genetic study.

Autosomal dominant chronic progressive external ophthalmoplegia (AdPEO) is a muscle mitochondrial disorder due to multiple large scale rearrangements of the mitochondrial DNA. This disorder is probably due to a nuclear defect which causes genetic instability or an impairment in the replication of mitochondrial DNA. X-linked ichthyosis (XLI) is a skin disorder caused by a deletion in the steroid-sulphatase gene. Here we report the clinical, biochemical, morphologic and molecular genetic findings in a patient affected by both AdPEO, inherited by the father, and steroid-sulphatase-deficiency, inherited by the mother. The association in the same patient of the two inherited diseases is merely casual and does not seem to influence the phenotypic expression of the two diseases.

Effect of the 50 bp deletion polymorphism in the SOD1 promoter on SOD1 mRNA levels in Italian ALS patients.

The genetic association between homozygosity for a 50 bp deletion polymorphism in the SOD1 promoter, 1684 bp upstream of the ATG, and an increased age of symptom onset was observed in various populations of ALS patients. Moreover, it has been demonstrated that this deletion reduces SOD1 expression in vitro. The objective of the present study was to test whether the observed association is replicated in patients from an Italian population and to check whether the deletion correlates with reduced SOD1 mRNA expression in vivo. Genomic DNA from 235 Italian SALS cases and 245 age- and sex-matched donors from the same ethnic background was screened for the 50 bp SOD1 promoter deletion by real time PCR assays. No differences were observed between ALS patients and controls for the frequency of both the alleles (D=deleted, N=non-deleted; p=0.95) and genotypes (p=0.90). Furthermore, stratification of the ALS samples showed that this variation was not associated with increased age of onset in ND and DD patients in comparison to NN patients (p=0.48). Finally, we performed real-time RT-PCR to quantify SOD1 mRNA levels in 48 patients and we did not find a relevant difference among the three sub-groups of genotypes (p=0.30). Our data suggest that the studied polymorphism does not modulate SOD1 mRNA level and disease phenotype in an Italian population.