Large deletion mutation of SPAST in a multi-generation family from Sardinia.

BACKGROUND AND PURPOSE: The hereditary spastic paraplegias (HSP) are characterized by progressive spasticity of the lower limbs, mostly inherited as an autosomal dominant trait. Analyses of large HSP pedigrees could help to better characterize the phenotype due to a single causative mutation. Patients in a seven-generation kindred carrying a large deletion in SPAST/SPG4 are described. METHODS: Individuals originating from Sardinia were clinically and genetically studied. RESULTS: Sixty-seven subjects carried a heterozygous deletion encompassing exons 2-17 of SPAST. Fifty patients (53.2 ± 15.4 years) presented a pure form of spastic paraparesis characterized by mild impairment and slow progression. Most patients showed spasticity, increased tendon reflexes in the lower limbs and Babinski sign, whilst weakness was rarely detected and urinary disturbances occasionally reported. Amongst the 17 asymptomatic carriers of the mutation, minimal neurological signs were detected in 11 cases. CONCLUSIONS: A focus on spasticity, increased tendon reflexes and Babinski sign, more than on weakness, could help clinicians to promote early diagnosis in asymptomatic carriers of SPAST deletions.

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.

Clinical and neuroimaging features of the m.10197G>A mtDNA mutation: New case reports and expansion of the phenotype variability.

Complex I (CI) is the largest component of the mitochondrial respiratory chain (MRC) and it is made up of 7 mitochondrial DNA (mtDNA)-encoded and at least 38 nuclear DNA-encoded subunits. Isolated CI deficiency is the most common single enzyme deficiency in the heterogeneous group of MRC disorders and it is a relatively common etiology of Leigh-like syndrome (LS). With a few exceptions, descriptions of the clinical spectrum of specific mutations in CI are scarce. We here present three unrelated Italian children who harbored the homoplasmic m.10197G>A mutation in MT-ND3 associated with reduced enzyme activity of CI in muscle. Compared with the spectrum of phenotypes seen in 13 previously described families with the same mutation, these children showed some novel clinical features. Two of the boys presented with subacute onset of dystonia, which showed a remitting-relapsing clinical course in one of them. The third boy presented acute symptoms consisting of speech impairment, progressive left-sided hemiparesis, and also vertebral and arterial malformations. In all the children, molecular studies identified a similar mutation load in tissues, and neuroimaging findings were consistent with the features seen in LS. Functional investigations in cultured skin fibroblasts suggested low ATP production in homoplasmic cells. Our results confirm that the m.10197G>A mutation is relevant to these patients' clinical and biochemical phenotypes, which thus expand the array of phenotypes associated with this variant.

Mitochondrial syndromic sensorineural hearing loss.

Mitochondrial diseases (MD) are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) is often associated to mitochondrial dysfunctions both in syndromic, nonsyndromic forms. SNHL has been described in association to different mitochondrial multisystemic syndromes, often characterized by an important neuromuscular involvement. Because of the clinical relevance of the associated neurological symptoms, the occurrence of SNHL is often underestimated and undiagnosed. In this study we evaluated the incidence of SNHL in a group of 17 patients with MD. We detected some degree of hearing impairment in 8/17 patients (47%), thus confirming the frequency of hearing impairment in MD. Furthermore, we want to highlight the role of the audiologist and otolaryngologist in the diagnosis and characterization of a MD, which should be suspected in all the cases in which the hearing loss is associated to signs and symptoms characteristic of mitochondrial dysfunction, especially if the family history is positive for hearing loss or MD in the maternal line.

Powerhouse failure and oxidative damage in autosomal recessive spastic ataxia of Charlevoix-Saguenay.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease due to mutations in SACS, which encodes sacsin, a protein localized on the mitochondrial surface and possibly involved in mitochondrial dynamics. In view of the possible mitochondrial involvement of sacsin, we investigated mitochondrial activity at functional and molecular level in skin fibroblasts obtained from ARSACS patients. We observed remarkable bioenergetic damage in ARSACS cells, as indicated by reduced basal, adenosine triphosphate (ATP)-linked and maximal mitochondrial respiration rate, and by reduced respiratory chain activities and mitochondrial ATP synthesis. These phenomena were associated with increased reactive oxygen species production and oxidative nuclear DNA damage. Our results suggest that loss of sacsin is associated with oxidative stress and mitochondrial dysfunction, and thus highlight a novel mechanism in the pathogenesis of ARSACS. The involvement of mitochondria and oxidative stress in disease pathogenesis has been described in a number of other neurodegenerative diseases. Therefore, on the basis of our findings, which suggest a potential therapeutic role for antioxidant agents, ARSACS seems to fall within a larger group of disorders.

Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia.

BACKGROUND: Mixed cryoglobulinaemia (MC), a systemic vasculitis associated with hepatitis C virus (HCV) infection in >90% of cases, is frequently complicated by multiple organ involvement. The prevalence of thyroid disorders in MC has not yet been studied. AIM: To investigate the prevalence and clinical features of thyroid involvement in patients with HCV-associated MC (HCV + MC). DESIGN: Case-control study. METHODS: HCV + MC patients (n = 93, 17 men and 76 women, mean +/- SD age 63 +/- 10 years, mean disease duration 14 +/- 7 years) consecutively referred to the Rheumatology Unit were matched by sex and age (+/- 2 years) to (i) 93 patients with chronic C hepatitis (CH) without MC and (ii) 93 healthy (HCV-negative) controls from the local population. Measurements included prevalence of hypo- or hyperthyroidism, thyroid autoantibodies, thyroid nodules and thyroid cancer. RESULTS: By McNemar's chi(2) test, the following thyroid abnormalities were significantly more frequent in HCV + MC patients than in HCV-negative controls: serum anti-thyroperoxidase autoantibody (AbTPO) (28% vs. 9%, p = 0.001); serum AbTPO and/or anti-thyroglobulin autoantibody (31% vs. 12%, p = 0.004); subclinical hypothyroidism (11% vs. 2%, p = 0.038); thyroid autoimmunity (35% vs. 16%, p = 0.006). Serum AbTPO were also significantly more frequent in HCV + MC patients than in CH controls (28% vs. 14%, p = 0.035). DISCUSSION: The prevalence of thyroid disorders is increased in patients with HCV-related mixed cryoglobulinaemia. We suggest careful monitoring of thyroid function in these patients.

Micronuclei assay and FISH analysis in human lymphocytes treated with six metal salts.

The capability of some metal compounds for inducing micronuclei (MN) in human lymphocytes was studied. In this investigation, Al (III), Cd (II), Hg (II), Sb (V), Te (VI), and Tl (I) salts were considered. The FISH (fluorescence in situ hybridization) technique with a centromeric probe was coupled with the MN assay in binucleated cells in order to detect both centromere-positive MN (C+ MN) due to malsegregation phenomena and centromere-negative MN (C- MN) due to chromosome breakage. The blood of two young nonsmoking male donors was employed for all experiments. In both donors, all the tested metal compounds, with the exception of Tl(2)SO(4), showed a statistically significant increase of MN compared to controls, at least at one dose. FISH analysis revealed an increase in the fraction of C+ MN for Al, Cd, and Hg compounds, and of C- MN for the Sb salt; however, this was not a statistically significant increase. A different efficiency was observed for the different metal compounds, in particular, KSbO(3) and CH(3)HgCl, which were highly genotoxic, whereas the others showed minimal effects.

Thyroid cancer in HCV-related mixed cryoglobulinemia patients.

OBJECTIVE: The prevalence of thyroid cancer in a series of unselected HCV-related mixed cryoglobulinemic patients was investigated in comparison with a control group. METHODS: Among 107 consecutive patients with mixed cryoglobulinemia (MC), 94 were eligible for the study. A control group was obtained from a sample of the general population (2,401 subjects), age > 50 years, who had undergone thyroid ultrasonography (582 subjects); 5 sex-matched controls were randomly assigned to each MC patients (470 individuals). The mean age was similar in the MC patients and controls (64.2 +/- 10.0 vs. 63.4 +/- 7.0). RESULTS: The prevalence of thyroid nodules was higher, although not significantly so, in control subjects than in MC patients (65.3 vs. 54.8%). Two patients with papillary thyroid cancer were found in the MC series, while no case was observed among controls (p = 0.001, chi-square P value; p = 0.02, Fisher's exact test). In both MC patients with papillary thyroid cancer lymphocytic infiltration was observed in the thyroid tissue. CONCLUSION: The possible association between HCV-related MC and thyroid cancer indicates that a careful monitoring of the thyroid would be opportune during the clinical follow-up of HCV-associated MC patients, especially in those with signs of thyroid autoimmune disorders.

Correlation between age and DNA damage detected by FADU in human peripheral blood lymphocytes.

Fluorometric analysis of DNA unwinding (FADU) is a fast and reliable method for detecting single strand DNA breaks as an index of DNA damage induced by clastogenic agents. A study of damage detected by FADU was conducted on DNA extracted from peripheral blood lymphocytes of 128 healthy nonsmoking regular donors (ranging in age from 19 to 67 years) and from 5 umbilical cord blood samples. DNA damage was measured as percentage of unwound DNA after alkalinization. Statistical analyses, both parametric (Pearson r correlation coefficient, b regression coefficient, ANOVA) and nonparametric (Kruskal-Wallis H test, Spearman rs rank correlation coefficient), support a significant correlation between age of donors and amount of DNA damage. The same results are found when adult donors are divided in four age classes and the ANOVA test performed among the mean percentages of unwound DNA of each class. Furthermore, donors of the same age belonging to different blood groups (A, B, AB and O) do not show any difference in DNA damage detected by FADU.

Clinical validity of the elbow flexion test for the diagnosis of ulnar nerve compression at the cubital tunnel.

We evaluated the elbow flexion test in 216 elbows without compression of the ulnar nerve at the cubital tunnel and without other neuropathies. We used Rayan's four positions as our test. The percentage of positive tests was only 3.6% at one minute, whereas evaluating the responses at three minutes we saw positive results in 16.2%. Therefore we find that if the test is performed for one minute it may be useful to help in diagnosing ulnar nerve compression at the cubital tunnel.

Our experience in the treatment of fractures of the astragalus.

The authors evaluate the results of a series of cases treated by surgery for fractures of the astragalus at the IInd Orthopaedic Division of the University of Pisa between 1978 and 1998. Treatment consisted in reduction and stabilization of the fractures by percutaneous route or by anterolateral approach. Prognosis for fractures of the neck worsens as the Hawkins radiographic classification of degrees increases. Based on the Hawkins clinical evaluation form, excellent and good results were obtained in 56% of cases. Radiographic evaluation confirmed the effectiveness of the Hawkins sign for the prediction of necrosis of the body; this necrosis occurred in 37.5% of cases; arthrosis of the astragalus developed in about half of the patients, which was, however, not always associated with stiffness of this joint. A stiff astragalus was the most fastidious complication, after necrosis of the astragalar body.

Our results in the treatment of fractures dislocations of Lisfranc's joint.

The authors report their experience in the treatment of traumatic injuries of Lisfranc's joint based on 30 cases treated by surgery between 1984 and 1999. All of the patients were re-evaluated clinically and radiographically. What emerges from the study is the need for surgical stabilization with percutaneous Kirschner wires or by open procedure in cases where there are doubts or where reduction is impossible. The prognosis is worse in injuries of the medial column and in exposed fractures or when mortification of the soft tissues is present.

Investigation of interactions between poly-L-lysine-coated boron nitride nanotubes and C2C12 cells: up-take, cytocompatibility, and differentiation.

Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-l-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription - polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.

Diagnostic approach to mitochondrial disorders: the need for a reliable biomarker.

Mitochondrial diseases (MD) are disorders caused by impairment of the mitochondrial electron transport chain (ETC). Phenotypes are polymorphous and may range from pure myopathy to multisystemic disorders. The genetic defect can be located on mitochondrial or nuclear DNA. The ETC is needed for oxidative phosphorylation (which provides the cell with the most efficient energetic outcome in terms of ATP production), and consists of five multimeric protein complexes located in the inner mitochondrial membrane. The ETC also requires cytochrome c and a small electron carrier, coenzyme Q10. One of the pathogenic mechanisms of ETC disorders is excessive accumulation of reactive oxygen species (ROS). Mitochondrial dysfunction and oxidative stress appear to have a strong impact also on the pathogenesis of neurodegenerative diseases. At present, diagnosis of MD requires a complex approach: measurement of serum lactate, exercise testing, electromyography, magnetic resonance spectroscopy, muscle histology and enzymology, and genetic analysis. Biomarkers are molecules associated with biological processes or regulatory mechanisms. A reliable biomarker for the screening or diagnosis of MD is still needed. In this paper we review the diagnostic approach to MD, from serum lactate to other blood and urinary markers, from muscular biopsy to imaging studies, and we highlight some potentially interesting perspectives in this field.

Lack of association between mtDNA haplogroups and Alzheimer's disease in Tuscany.

Mitochondrial DNA (mtDNA) haplogroup-specific polymorphisms were previously related to several neurodegenerative diseases, including Alzheimer's disease (AD). However, the precise role of mtDNA haplogroups in the neurodegenerative cascade leading to AD is still unclear. In this work we have genotyped predefined European mtDNA haplogroups in 209 patients with AD and 191 matched controls. In order to minimise the risk of "genetic contamination", which could lead to false associations between gene markers and disease, we were careful to enrol in the study only patients and controls of clear Tuscan origin (with at least three generations of Tuscanborn relatives). The frequency of the haplogroups did not differ between the two groups, and no correlation with gender, ApoE genotype, age of onset or disease status was observed. Further studies will be required to define the contribution of mtDNA haplogroups, if any, to the pathogenesis of AD. A correct population selection, in order to minimise the risk of genetic contamination, is essential in these studies.

Possible identity of transcription and translation signals in early vital systems.

The distribution of codons was analysed in three classes of eukaryote proteins having widely different evolutionary rates: 78 histones, 40 tubulins, and seven fibrinogens. In this set of genes, (i) it was confirmed that codons which are components of known transcription signals, like ATA, are used infrequently when a synonym is available, particularly in the more constrained proteins, and (ii) it was observed that the three codons which have an iso-accepting transfer with anticodon UAA, UAG or UGA are also suppressed. Then, the distribution of UAA, UAG and UGA trimers was studied in 498 tDNAs and 198 rDNAs. It was found that these trimers are weakly but significantly suppressed in tDNAs and to a lesser extent in rDNAs. It was advanced that the present suppression of ATA, which codes for Methionine in several mitochondria, and of the TAA, TAG and TGA trimers in tDNAs, might be an indication that at the very early stages of the evolution of translation and transcription the signals for initiation and termination were shared by the two processes.

Phylogeny inferred from codon usage pattern in 31 organisms.

We employed the Codonusage database to analyze the codon usage pattern in 31 organisms from all the main biological taxa. We tested the similarity in codon usage pattern between each organism and all the others by the Pearson linear correlation coefficient. The 465 values obtained were located in a 31 x 31 triangular matrix from which a correlation distance matrix was calculated. An evolutionary dendrogram was then constructed from these distances. The results showed a fair correlation between codon usage patterns and phylogenetic relationships, at least for organisms which diverged in rather recent times (end of Jurassic--beginning of Cretaceous).

A set of Alu-free frequent decamers from mammalian genomes enriched in transcription factor signals.

We have recently reported that the statistical analysis of the frequency distribution of short oligonucleotides within mammalian and viral genomes allows the production of sets of DNA sequences enriched in signals for transcription factors. Such statistical approaches could facilitate the identification of new promoter regions playing a role in the transcriptional regulation of gene expression. In the case of mammalian oligonucleotides, we found that the published set of frequent decamers enriched in transcriptional motifs is not suitable for studies on genes of Homo sapiens and evolutionarily related genomes, because it contains decameric sequences belonging to genomic repeats. We report here that most of the decameric sequences of DNA repeats belong to Alu repeats. Accordingly, we produced a subset of Alu-free frequent decamers. In addition, we eliminated from the subset of Alu-free frequent decamers those that are frequently present within other common human repeats, including (GT)n, (AT)n, (CA)n, (ATT)n, (CAA)n and (GTT)n. The Alu-free (repeats-free) subset of frequent mammalian decamers is enriched in signals for transcription factors and allows the identification of putative signals in genes, such as those coding for plasminogen activator, adenosine deaminase and p53, that contain a large number of Alu-like repeats interspersed within our genomic sequences. The newly generated compilation of frequent decamers described here might be used to locate genomic regions playing functional roles in the expression of genes of Homo sapiens and related primates.