In vitro effects of fermented papaya (Carica papaya, L.) on platelets obtained from patients with type 2 diabetes.

BACKGROUND AND AIM: Oxidative stress is associated with insulin resistance pathogenesis, insulin secretion deficiency, and complication onset. Fermented papaya preparation (FPP), a dietary supplement obtained by fermentation of the papaya fruit, may be used as an antioxidant in the prevention of diabetic complications. METHODS AND RESULTS: Platelets from 30 patients with type 2 diabetes mellitus (DM 2) and 15 healthy subjects were analyzed to evaluate the in vitro effects of FPP incubation. Na(+)/K(+)-adenosine triphosphatase (ATPase) activity, membrane fluidity, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity, and conjugated diene levels were determined. In vitro FPP incubation improved platelet function, by enhancing Na(+)/K(+)-ATPase activity and membrane fluidity, and ameliorated the antioxidant system functionality, through an increase in TAC and SOD activity and a parallel decrease in conjugated diene levels in patients with DM 2. CONCLUSION: Our data suggest that the incubation with FPP may have a protective effect on platelets from patients with DM 2, by preventing the progression of oxidative damage associated with diabetes and its complications.

Effect of consumption of dark chocolate on oxidative stress in lipoproteins and platelets in women and in men.

The goal of this research was to investigate the effects of 3 weeks consumption of 50 g flavonoid-rich dark chocolate on lipoprotein oxidative stress in vitro and in vivo in 25 women compared to 25 men. Levels of thiobarbituric acid-reactive substances, conjugated dienes and hydroperoxide levels in HDL and LDL before and after consumption of dark chocolate were determined. Moreover in platelets of the same subjects NO and peroxynitrite levels were studied. TBARs concentration in women's HDL decreased by 26.7% while in men's HDL 23.4%; lipid hydroperoxides decreased in women's HDL by 62.8% while in men's HDL they decreased by 21.1%. Conjugate diene formation decreased in women's HDL by 55.9%, while in men's HDL it decreased by 49.2%. Moreover TBARs concentration decreased in women's LDL by 26.7% after supplementation and in men's LDL by 21.6%; lipid hydroperoxides decreased in women's LDL by 83.6% while in men's LDL they decreased by 64.7%. Moreover conjugate diene formation decreased in women's LDL by 48.2%, while in men's LDL it decreased by 21.6%. After supplementation peroxynitrite values decreased in women by 24% and in men by 18.6% while NO increased after supplementation by 15.7% compared to basal determination in women, and by 32.2% in men. This study showed that a short-term intake of dark chocolate might improve the lipoprotein profile in healthy humans, more so in women than in men, and this might exert a protective effect on the cardiovascular system.

Platelet amyloid precursor protein isoform expression in Alzheimer's disease: evidence for peripheral marker.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by a progressive cognitive and memory decline. Among peripheral markers of AD, great interest has been focused on the amyloid precursor protein (APP). In this regard, platelets represent an important peripheral source of APP since it has been demonstrated that the three major isoforms, that are constituted of 770, 751 and 695 aa residues, are inserted in the membrane of resting platelets. APP 751 and APP 770 contain a Kunitz-type serine protease inhibitor domain (APP KPI) and APP 695 lacks this domain. To address this issue, we first examined the platelet APP isoform mRNAs prospectively as biomarker for the diagnosis of AD by means of real-time quantitative PCR, and then evaluated the correlation between APP mRNA expression levels and cognitive impairment of enrolled subjects. Differential gene expression measurements in the AD patient group (n=18) revealed a significant up-regulation of APP TOT (1.52-fold), APP KPI (1.32-fold), APP 770 (1.33-fold) and APP 751 (1.26-fold) compared to controls (n=22). Moreover, a statistically significant positive correlation was found between APP mRNA levels (TOT, KPI, 770 and 751) and cognitive impairment. Since AD definitive diagnosis still relies on pathological evaluation at autopsy, the present results are consistent with the hypothesis that platelet APP could be considered a potential reliable peripheral marker for studying AD and could contribute to define a signature for the presence of AD pathology.

Predictive genetic tests in neurodegenerative disorders: a methodological approach integrating psychological counseling for at-risk individuals and referring clinicians.

The identification of the molecular basis of numerous hereditary neurological disorders allowed the feasibility of predictive genetic tests for at-risk family members. In agreement with international guidelines, we tested a protocol for a predictive test to optimize cooperation among specialists, well-being of participants, and organization of clinical activities. The psychiatrist/psychologist did not meet the at-risk subjects, but cooperated with the team, integrating psychological support for participants and clinicians. We enrolled 60 subjects at risk for Huntington disease, and 32 at risk for spinocerebellar ataxias. Seventy-two subjects (78%) continued the visit program; 55 (60%) received the genetic result, and 38 subjects (41%) completed the program. Participation and outcome were similar in both groups. Mean psychological scores were all below significant levels; however, the need for psychological support was recognized for 5 mutation carriers and a non-carrier. Our data provide a methodological example of a simple and safe procedure for a predictive test, and indicate that the clinical conference represents a good setting to handle psychosocial impact associated with disclosure of genetic results in hereditary late-onset disorders.

Different patterns of movement-related cortical oscillations in patients with myoclonus and in patients with spinocerebellar ataxia.

OBJECTIVE: To assess whether different patterns of EEG rhythms during a Go/No-go motor task characterize patients with cortical myoclonus (EPM1) or with spinocerebellar ataxia (SCA). METHODS: We analyzed event-related desynchronization (ERD) and synchronization (ERS) in the alpha and beta-bands during visually cued Go/No-go task in 22 patients (11 with EPM1, 11 with SCA) and 11 controls. RESULTS: In the Go condition, the only significant difference was a reduced contralateral beta-ERS in the EPM1 patients compared with controls; in the No-go condition, the EPM1 patients showed prolonged alpha-ERD in comparison with both controls and SCA patients, and reduced or delayed alpha- and beta-ERS in comparison with controls. In both conditions, the SCA patients, unlike EPM1 patients and controls, showed minimal or absent lateralization of alpha- and beta-ERD. CONCLUSIONS: EPM1 patients showed abnormal ERD/ERS dynamics, whereas SCA patients mainly showed defective ERD lateralization. SIGNIFICANCE: A different behavior of ERS/ERD distinguished the two patient groups: the pattern observed in EPM1 suggests a prominent defect of inhibition occurring in motor cortex contralateral to activated segment, whereas the pattern observed in SCA suggested a defective lateralization attributable to the damage of cerebello-cortical network, which is instead marginal in patients with cortical myoclonus.

Sialic acid and sialidase activity in acute stroke.

Stroke is a heterogeneous syndrome caused by multiple disease mechanisms, resulting in a disruption of cerebral blood flow with subsequent tissue damage. It is well known that erythrocytes have a large amount of sialic acid and could represent a model to investigate changes occurring in a pathology like stroke. The aim of this study was to investigate a possible relationship among erythrocyte membrane, plasma and sialic acid content. The possible impact of the sialic acid content and the activity of sialidase on stroke severity was also evaluated. The study population consisted of 54 patients with a first stroke and of 53 age-and sex matched healthy volunteers. The total bound sialic acid was substantially decreased in patients. There was a significant correlation between the sialidase activity values and the severity of the neurological deficit defined by the National Institute of Health Stroke Scale. This study shows that low sialic acid erythrocyte concentrations with contemporary high sialic acid plasma levels and elevated sialidase activity can be considered as markers of ischemic stroke. Further investigations are needed to clarify the possible role of these biochemical changes in producing and sustaining cerebral ischemic damage.

Role of raloxifene on platelet metabolism and plasma lipids.

BACKGROUND: This study was performed to understand the metabolic effects of raloxifene, a selective oestrogen receptor modulator, on platelets in healthy non-obese postmenopausal women. The data were compared to untreated subjects. MATERIALS AND METHODS: Platelet nitric oxide activity (NO) and peroxynitrite level, platelet inducible and endothelial nitric oxide synthase expression and plasma lipids were evaluated at baseline and after 12 months of raloxifene or placebo treatment. RESULTS: A significant increase of platelet NO and reduction of platelet peroxynitrite levels, as well as a decrease of inducible nitric oxide synthase expression, was observed 12 months after raloxifene therapy as compared to baseline or placebo treatment. Moreover, raloxifene treatment caused a significant increase in high-density lipoprotein cholesterol and a decrease of total cholesterol and low-density lipoprotein cholesterol were observed versus baseline values (P < 0.05). A significant positive correlation was observed between high-density lipoprotein cholesterol and platelet NO (r = 0.76, P < 0.005) in the raloxifene group. CONCLUSION: Our results showed that raloxifene improves platelet metabolism in healthy postmenopausal women through an increase of the bioavailability of platelet NO by a reduction of iNOS and the beneficial effects on lipid metabolism. This mechanism of action of raloxifene on platelet activity may explain some cardiovascular protective effects of this selective oestrogen receptor modulator.

Plasma levels of nitric oxide and stroke outcome.

Production of reactive oxygen species after cerebral blood flow disruption may enhance tissue damage through multiple molecular pathways. Changes in nitric oxide (NO) metabolism and oxidative stress status were investigated in 47 patients with ischemic stroke by measuring plasma nitric oxide (NO) and peroxynitrite (ONOO(-)) levels.A correlation was sought between these two parameters and i) baseline stroke severity based on the National Institute of Health stroke scale (NIHSS) and ii) neurological outcome in terms of NIHSS changes from entry (T(0)) to 30 days after symptom onset (T(1)). The control group consisted of 30 age- and sex-matched healthy subjects. Mean plasma levels of ONOO(-) (arbitrary fluorescence number +/- SD) were significantly higher in patients (7.70 +/- 1.71 vs 5.35 +/- 0.69, p < 0.001), whereas mean NO levels (nmol/mg protein) were significantly higher in controls (115.40 +/- 12.40 vs. 51.10 +/- 12.50, p < 0.001). Plasma ONOO(-) was significantly higher among patients with non-lacunar stroke (8.48 +/- 1.50 vs. 6.95 +/- 1.58 in those with lacunar stroke; p = 0.001), whereas NO levels were significantly higher among lacunar stroke patients (60.00 +/- 7.86, vs. 41.77 +/- 9.29 in patients with nonlacunar stroke; p < 0.001). Nitric oxide plasma levels were also associated with an unfavorable evolution in non-lacunar stroke, since a 10 unit increase in NO predicted a 1 point reduction in the NIHSS score at T1. Findings show that changes in NO metabolism may be considered as markers of brain injury in patients with ischemic stroke. Further work is needed to establish whether the amount of biochemical changes related to oxidative stress may influence outcome in these patients.

Nitric oxide and peroxynitrite platelet levels in women with small-for-gestational-age fetuses.

OBJECTIVE: The placenta produces reactive oxygen species (ROS) including nitric oxide (NO) and peroxynitrite (ONOO(-)) that have pronounced effects on placental function. Excessive ROS production may occur in pathological pregnancies, such as those complicated by small-for-gestational-age (SGA) fetuses. DESIGN: The aim of the present work was to study NO and ONOO(-) levels in platelets of pregnant women with SGA fetuses compared with a control group. SETTING AND POPULATION: The study was performed on 30 pregnant women with SGA fetuses (SGA group) and on 30 healthy pregnant women (appropriate-for-gestational-age [AGA] group) matched for maternal and gestational age. All women included in this study were in the third trimester of pregnancy. METHODS: Platelets were isolated by differential centrifugation. NO metabolites, after enzymatic conversion followed by the Griess reaction, were measured as nitrite by spectrophotometric detection. Peroxynitrite (ONOO(-)) levels were evaluated using the fluorescence probe 2,7-dichlorofluorescein diacetate (DCFDA). MAIN OUTCOME MEASURES: The following determinations were made: platelet nitric oxide and peroxynitrite levels in the SGA group and controls; inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and nitrotyrosine (N-Tyr) expression in the same groups. RESULTS: Our results show that both platelet NO and ONOO(-) levels were significantly higher in the SGA group than in the controls. CONCLUSION: Increased platelets levels of nitric oxide and peroxynitrite might play a role in the pathophysiology of intrauterine growth restriction. Further investigations are in progress to clarify if these molecules are pathogenetic factors, an epiphenomenon or a pathophysiological marker.

Alzheimer's Disease Risk and Progression: The Role of Nutritional Supplements and their Effect on Drug Therapy Outcome.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly population. Despite significant advancements in understanding the genetic and molecular basis of AD, the pathology still lacks treatments that can slow down or reverse the progression of cognitive deterioration. Recently, the relationship between nutrient deficiency and dementia onset has been highlighted. AD is in fact a multifactorial pathology, so that a multi-target approach using combinations of micronutrients and drugs could have beneficial effects on cognitive function in neurodegenerative brain disorders leading to synaptic degeneration. Primarily, this review examines the most recent literature regarding the effects of nutrition on the risk/progression of the disease, focusing attention mostly on antioxidants agents, polyunsaturated fatty acids and metals. Secondly, it aims to figure out if nutritional supplements might have beneficial effects on drug therapy outcome. Even if nutritional supplements showed contrasting evidence of a likely effect of decreasing the risk of AD onset that could be studied more deeply in other clinical trials, no convincing data are present about their usefulness in combination with drug therapies and their effectiveness in slowing down the disease progression.

Peroxynitrite production and NOS expression in astrocytes U373MG incubated with lipoproteins from Alzheimer patients.

Apolipoprotein E (apo E), a plasma protein involved both in the metabolism of cholesterol and triglycerides, particularly in nervous tissue, has been associated with a higher risk of Alzheimer's disease. It has been shown that apo E increased the production of nitric oxide (NO) from human monocyte-derived macrophages (MDM); this effect could represent an important link between tissue redox balance and inflammation, since inflammation and oxidative stress are involved in chronic neurodegenerative disorders. Moreover, it has been evidenced that an overproduction of NO in the central nervous system (CNS) may play a key role in aging and that the glial cells (microglials cells and probably astrocytes) are able to form consistent amounts of NO through the induction of a nitric oxide synthase (iNOS) isoform so-called inducible or inflammatory. This report was performed in order to elucidate the effects produced by lipoproteins from control subjects, AD patients and first degree relatives (offspring) on human astrocyte cells after a short incubation. Peroxynitrite and NO production and NOS expression in cultured astrocytes were measured. We observed a decreased NO production after incubation with both LDL and HDL and an increased peroxynitrite production. As it concerns NOS expression, densitometric analysis of bands indicated that iNOS protein levels were significantly higher in the cells incubated with both AD lipoproteins and offspring lipoproteins compared to cells incubated with control lipoproteins. These findings suggest the possibility to identify in NO pathway a precocious marker of AD.

Modification induced by homocysteine and low-density lipoprotein on human aortic endothelial cells: an in vitro study.

The aim of the present study was to investigate the effect exerted by low-density lipoprotein (LDL) modified by homocysteine (Hcy)-thiolactone (Hcy-LDL) on functional properties on human endothelial cells. Hcy-thiolactone, a reactive product formed in human cells from enzymatic conversion of Hcy, was hypothesized to play an important role in Hcy-induced vascular damages. Using endothelial cultured cells [human aortic endothelial cells (HAEC)] as cellular model, we evaluated nitric oxide (NO) production, cytoplasmic Ca(2+) levels, Na(+)/K(+)-ATPase activity, and peroxynitrite production in cells incubated in the presence of control LDL or Hcy-LDL. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy subjects, with 100 microm Hcy-thiolactone. A significant increase in cytoplasmic Ca(2+) levels and peroxynitrite production and a decrease in Na(+)/K(+)-ATPase and NO production in HAEC incubated with Hcy-LDL compared with HAEC incubated with control LDL were observed. Moreover, a positive correlation was found between Na(+)/K(+)-ATPase activity and cytoplasmic Ca(2+) content and between peroxynitrite activity and cytoplasmic Ca(2+) content. In conclusion, our results demonstrated that LDL homocysteinylated in vitro induced alterations of functional properties and NO metabolism of human endothelial cells.

Reduced susceptibility to peroxidation of erythrocyte plasma membranes from centenarians.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

Sialic acid content in erythrocyte membranes from pregnant women affected by gestational diabetes.

Sialic acid (SA) content, membrane fluidity, and Na(+)/K(+)-adenosine triphosphatase (ATPase) activity were determined in erythrocyte membrane from 10 nonpregnant women (HNPW), 16 pregnant women affected by gestational diabetes mellitus (GDM), and 25 healthy pregnant women (HPW). In GDM patients the membrane erythrocyte SA content was significantly increased compared with HNPW and membrane fluidity was significantly increased in comparison with HPW. Erythrocyte membrane Na(+)/K(+)-ATPase activity was significantly reduced in GDM patients compared both to HNPW and to HPW subjects. A significant inverse correlation was found between 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) anisotropy and erythrocyte membrane SA content in HNPW and in HPW, while this significant correlation was not observed in GDM. The present results indicate that in comparison with normal pregnancy GDM is characterized by deep alterations of the erythrocyte plasma membrane physicochemical properties (increased fluidity) and functional activities (reduced Na(+)/K(+)-ATPase activity). These modifications might be at the basis of the altered blood viscosity and placental perfusion observed under such conditions. Moreover, these results show that in physiological pregnancy and in the nonpregnant state, the erythrocyte surface membrane fluidity is inversely correlated with SA content, while in GDM there is an unbalance of this relation, which might be associated with the microcirculatory abnormality present in this disease.

LDL and HDL affect nitric oxide metabolism in human astrocytoma cells.

Astrocytes provide structural, trophic and metabolic support to neurons and modulate synaptic activity. Under physiological conditions, neuronal-derived nitric oxide (NO) plays an important role in the modulation of a variety of central nervous system (CNS) functions. NO, although short lived, can travel sufficient distances to be able to act as an intercellular messenger in the brain. Its targets include adjacent neurons and astrocytes. The aim of the present study was performed in order to investigate the effects produced by incubation of lipoproteins, at different times, with human astrocytoma cells and thus measuring NO and its metabolite production. NO and peroxynitrite production, iNOS and nNOS expression by Western immunoblot were evaluated. The LDL and HDL-treated cells showed an increased production of NO, more evident after 12 h, compared to basal levels; concerning peroxynitrite production, LDL and HDL-treated cells showed a higher fluorescence, more evident at 3 h. nNOS and iNOS protein levels were significantly higher in the cells incubated with control LDL and HDL. The present work supports the hypothesis that lipoproteins can induce the formation of reactive astrocytes, inducing iNOS as reported by other authors, giving experimental support to a role played by LDL and HDL inducing a reactive response.

In vitro effects of resveratrol on oxidative stress in diabetic platelets.

To evaluate the in vitro effects of resveratrol (RSV) incubation on platelets from compensated and decompensated diabetic patients in order to use it as an adjuvant therapy. The study was performed on 77 diabetic patients and divided into two phases: 29 compensated and 48 decompensated diabetic platelets were analyzed at recruitment (T0) and after in vitro RSV incubation (20 µg/ml) for 3 h at 37 °C (T1). Lipoperoxide and nitric oxide (NO) levels, superoxide dismutase (SOD) and Na(+)/K(+) ATPase activities, total antioxidant capacity (TAC), and membrane fluidity tested by anisotropy of fluorescent probes TMA-DPH and DPH were determined. In vitro RSV incubation counteracts oxidative damage associated with diabetes and its complications; it is able to improve platelet function through augmented membrane fluidity and Na(+)/K(+) ATPase activity; it enhances antioxidant systems' functionality by increasing NO levels, SOD activity, and TAC and by decreasing lipoperoxide levels in both compensated and decompensated patients. Such platelet functionality enhancement suggests a new method of secondary prevention of complications associated with platelet dysfunction. Being free from one of the major risks associated with many antidiabetic agents, it can be assumed that RSV utilization in the diabetic diet may have a preventive and protective role in the progression of diabetic oxidative damage.

PP073. Gestational hypertension: A study on placental expression of endothelial and inducible nitric oxide synthase and no metabolism.

INTRODUCTION: Hypertension is one of the most common medical disorders in pregnancy and a major cause of maternal and perinatal morbidity and death. In primates adequate development of the embryo, and later of the fetus, depends on a successful hemomonochorial placentation. Nitric oxide (NO) a low molecular weight mediator, induces vasodilatation, inhibits platelet aggregation, and prevents the adhesion of platelets to endothelial cells. Till date, no data are available regarding gestational hypertension (GH) placenta and no metabolism and related enzyme expression and activity. OBJECTIVES: The present study aimed to evaluate eNOS and iNOS expression in the placentas of both normal and GH patients, by means of Real-Time quantitative PCR, measure placental nitric oxide and peroxynitrite levels in the same group of subjects, and correlate such findings with HELLP group already published. METHODS: Fifteen patients with gestational hypertension and thirty healthy pregnant controls comparable for maternal and gestational age were enrolled in the study. Placental tissue was taken immediately after delivery. eNOS and iNOS mRNA levels were evaluated Real-Time quantitative PCR, whereas nitric oxide and peroxynitrite production was measured by a commercially available kit. RESULTS: Placental eNOS and iNOS mRNA levels were significantly reduced in GH (2,02-fold reduction and 2,33-fold reduction, respectively) when compared to controls. Conversely, NO and ONOO(-) production were significantly higher in GH group compared to control group (31.56±4.15nmol NO/mg prot vs. 23.98±5.14nmol NO/mg prot and 68.49±8.57 arbitrary fluorescence units vs 17.31±2.25 arbitrary fluorescence units; p<0, 05). Such results were compared to HELLP group obtained in an already published study. CONCLUSION: As from results herein reported, we can hypothesize that complex mechanisms involving NO pathways cause a placental vasculature damage. However, it is not easy to understand if these changes could be interpreted as causes or consequences of this pathologic state.

Novel and recurrent spastin mutations in a large series of SPG4 Italian families.

BACKGROUND: Hereditary spastic paraplegias (HSP) are heterogeneous neurodegenerative disorders, genetically classified according to the identified disease gene or locus. Clinically, HSP are distinguished in pure and complicated forms. Mutations in the spastin gene (SPAST) are responsible for SPG4 and account approximately for 50% of the dominantly inherited paraplegias with a pure HSP phenotype. METHODS: Molecular screening of the SPAST gene allowed the identification of 31 Italian mutation carriers, from 19 unrelated families. Genetic testing was performed by direct sequencing and multiplex ligation-dependent probe amplification. Subjects carrying SPAST mutations were retrospectively evaluated for clinical phenotype and disability score assessment. RESULTS: We found 12 recurrent mutations, and 7 novel SPAST mutations. Twenty-eight patients exhibited a pure spastic paraplegia phenotype, while 3 subjects were asymptomatic mutation carriers. Four patients were sporadic cases. Age at onset ranged from 10 to 61 years. Disability score increased with age at examination and disease duration. Patients with onset >38 years presented a faster disease progression, and a higher disability functional index, than the patients with earlier onset (p<0.04). CONCLUSIONS: Our study enlarges the number of pathogenic SPAST mutations, and confirms the association with a pure spastic paraplegia phenotype. Age at onset was highly variable and correlates with the rate of disease progression. Future longitudinal clinical studies are needed to confirm these observations.