Mutations in the motor and stalk domains of KIF5A in spastic paraplegia type 10 and in axonal Charcot-Marie-Tooth type 2

Spastic paraplegia type 10 (SPG10) is an autosomal dominant form of hereditary spastic paraplegia (HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy chain implicated in anterograde axonal transport. KIF5A mutations were found in both pure and complicated forms of the disease; a single KIF5A mutation was also detected in a CMT2 patient belonging to an SPG10 mutant family. To confirm the involvement of the KIF5A gene in both CMT2 and SPG10 phenotypes and to define the frequency of KIF5A mutations in an Italian HSP patient population, we performed a genetic screening of this gene in a series of 139 HSP and 36 CMT2 affected subjects. We identified five missense changes, four in five HSP patients and one in a CMT2 subject. All mutations, including the one segregating in the CMT2 patient, are localized in the kinesin motor domain except for one, falling within the stalk domain and predicted to generate protein structure destabilization. The results obtained indicate a KIF5A mutation frequency of 8.8% in the Italian HSP population and identify a region of the kinesin protein, the stalk domain, as a novel target for mutation. In addition, the mutation found in the CMT2 patient strengthens the hypothesis that CMT2 and SPG10 are the extreme phenotypes resulting from mutations in the same gene.     

P-selectin glycoprotein ligand-1 variable number of tandem repeats (VNTR) polymorphism in patients with multiple sclerosis

P-selectin glycoprotein ligand-1 (PSGL-1) is an important adhesion molecule involved in lymphocyte recruitment into the brain, which represents a crucial step in the pathogenesis of multiple sclerosis (MS). Three hundred twenty-one MS patients and 342 controls were genotyped for the presence of a polymorphism in the PSGL-1 gene, consisting of a variable number of tandem repeats (VNTR) originating three possible alleles: A, B and C, in order to test whether they influence the susceptibility and the course of the disease. No significant differences among allelic frequencies of A, B and C alleles in MS as compared with controls were observed. Stratifying patients according to the course of the disease, a significantly increased frequency of the shortest C allele in PP-MS was found (7.1%), either in comparison with controls (P=0.011) or with all other MS patients, who had acute inflammatory attacks at onset and an initial RR form (P=0.036). Besides, none of SP-MS patients was a carrier of the C allele and B carriers converted later from RR to SP course as compared with A/A subjects (after 15.8 rather than 8.8 years, P=0.01). In conclusion, the C allele of the VNTR polymorphism in PSGL-1 is likely to be associated with PP-MS. As this allele has been demonstrated to have a very low efficiency in mediating lymphocyte binding to brain endothelium during attacks, its high frequency in PP-MS could be related to the absence of exacerbations in such patients.     

The T-786C NOS3 polymorphism in Alzheimer's disease: association and influence on gene expression

A common single nucleotide polymorphism (SNP), consisting in a T-->C transition (T-786C) in endothelial nitric oxide synthase (NOS3), has been reported to be associated with vascular pathologies, but no information are available on a possible association with AD. T-786C genotype was determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in an Italian population of 432 AD patients compared with 360 healthy controls, matched for ethnic background, age and gender. Peripheral blood mononuclear cells (PBMC) from 22 subjects (11 AD and 11 controls) carrying different genotypes were isolated. Total RNA was extracted and analyzed by real-time PCR. No significant differences either in allelic or genotypic frequencies of the T-786C polymorphism between AD and normal population were observed, even stratifying AD patients by age at onset, gender, or ApoE status. However, expression of NOS3 in PBMC seems to be influenced by the presence of the C mutated allele, as demonstrated by a tendency towards a decrease in mRNA levels in C carriers, assessed by real-time PCR assay. This effect was observed both in patients and controls, independently from the cognitive impairment, and is likely to be dose-dependent, being mostly evident in CC homozygous. In conclusion, the T-786C SNP does not seem to be a risk factor for sporadic AD, but its presence correlates with a trend toward lower NOS3 expression rate, possibly exerting a beneficial effect in AD by contributing to lower oxidative damage.     

Clinical and genetic variability of glycogen storage disease type IIIa: seven novel AGL gene mutations in the Mediterranean area

Deficiency of amylo-1,6-glucosidase, 4-alpha-glucanotransferase enzyme (AGL or glycogen debrancher enzyme) is responsible for glycogen storage disease type III, a rare autosomal recessive disorder of glycogen metabolism. The AGL gene is located on chromosome 1p21, and contains 35 exons translated in a monomeric protein product. The disease has recognized clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different subjects. The clinical manifestations of GSD III are represented by hepatomegaly, hypoglycemia, hyperlipidemia, short stature and, in a number of subjects, cardiomyopathy and myopathy. In this article, we discuss the genotypic-phenotypic heterogeneity of GSD III by the molecular characterization of mutations responsible for the disease on a collection of 18 independent alleles from the Mediterranean area. We identified by heteroduplex band shift, DNA direct sequencing, and restriction analysis, seven novel mutations (four nonsense point-mutations: R34X, S530X, R1218X, W1398X; two microinsertions: 1072insT and 4724insAA; and one bp deletion: 676DeltaG), together with two new cases carrying a IVS21 + 1 G --> A splicing site mutation previously described in Italian patients. Altogether, 15 alleles were characterized. The correlation between type of mutation and clinical severity was studied in six patients in whom both mutated alleles were detected. Our data confirm the extreme genetic heterogeneity of this disease, thus precluding a strategy of mutation finding based on screening of recurrent common mutations.     

Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin

Ataxia with oculomotor apraxia (AOA) type 2 (AOA2 MIM 606002) is a recessive subtype of AOA characterized by cerebellar atrophy, oculomotor apraxia, early loss of reflexes, and peripheral neuropathy. Various mutations either in homozygous or compound heterozygous condition were so far identified in the associated gene SETX (MIM 608465). SETX encodes a large protein called senataxin with a DNA-RNA helicase domain and a putative N-terminus protein interaction domain. Here, we report the identification of two novel homozygous mutations in SETX gene, c.340_342delCTT (p.L114Del) and c.1669C?>?T (p.R557X), in two AOA2 families. The characterization of the mutant lymphoblastoid cell lines for sensitivity to oxidative DNA-damaging agents indicates that the p.L114Del deletion confers an increased sensitivity to H₂O₂, camptothecin, and mitomycin C, previously found to induce death in lymphoblasts harbouring other SETX mutations; the cells carrying the nonsense mutation display instead values within the normal range. Further analysis of a neuronal cell model SKNBE, transfected with the mutant senataxin proteins, reveals increased sensitivity also to staurosporine and excitotoxicity associated with the p.L114Del mutant only. We also demonstrate that the sensitizing effect of p.L114Del on apoptosis can be reversed by senataxin silencing. The ability of a single amino acid deletion to sensitize cells to death by different agents, compared to the lack of effect of a whole protein deletion, seems to exclude a protective role played by the native protein while suggesting that a specific mutation confers to the protein the ability to enhance the toxic effect of various cell damaging agents.     

Antinociceptive, anti-inflammatory and wound healing features in animal models treated with a semisolid herbal medicine based on Aleurites moluccana L. Willd. Euforbiaceae standardized leaf extract: Semisolid Herbal

Ethnopharmacological relevance

Aleurites moluccana L. (Willd) Euforbiaceae is a native tree of Indonesia and India that has become acclimatized and well-adapted to the South and Southwest of Brazil. It is commonly used in traditional medicine to treat pain, fever, inflammation, asthma, hepatitis, headache, gastric ulcer, cuts, skin sores and other ailments. The oral antinociceptive effects of standardized 70:30 (v/v) ethanol:water spray dried extract of A. moluccana leaf, as well as its flavonoids 2?-O-rhamnosylswertisin (I) and swertisin (II), have previously been reported.

Aim

The aim of this study was to develop a stable and effective semisolid herbal medicine for topical use in the treatment of pain, inflammation and wound healing, containing 0.5 and 1.0% of standardized dried extract of A. moluccana.

Materials and methods

The chemical markers I and II were assayed by HPLC-UV analysis after extraction by matrix solid dispersion phase (MSDP) followed analytical validation as ICH Guidelines. The semisolid preparations of Hostacerin CG^® vehicle containing 0.5 and 1.0% of dried extract of A. moluccana were submitted to stability studies (180 day of accelerated and long-term studies). The phytomedicine semisolid was analysed in croton oil-induced ear oedema model in mice, in the healing process, using the excisional wound model in rats, and to prevent mechanical sensitization following plantar incision in rats in the postoperative model of pain.

Results

The MSDP method showed average recovery of 101.6 and 105.7% for I and II, respectively, with good precision (RSD<2.0%) and selectivity, without interference of the excipients. The formulations were approved in the stability studies, maintaining conformity after 180 day of accelerated and long-term studies, with variation<10% in the analytical parameters. The phytomedicine reduced the ear oedema in 37.6±5.7% and 64.8±6.2%, for 0.5 and 1.0% of dried extract, respectively. The formulation also accelerated the healing process by up to 50.8±4.1% and 46.0±4.0% at 0.5 and 1.0% of extract, respectively, and both amounts were capable of preventing the development of mechanical sensitization following plantar incision in rats.

Conclusions

The MSDP followed by HPLC-UV analytical method was appropriate for the quality control of the topical phytomedicine based on A. moluccana. The formulation developed at 0.5 and 1.0% of A. moluccana dried extract proved to be effective as an analgesic, anti-inflammatory and wound healing in the pre-clinical studies, which is in agreement with the ethnopharmacological data.