Dysfunction in the coagulation system and schizophrenia.

Although different hypotheses have been formulated to explain schizophrenia pathogenesis, the links between them are weak. The observation that five psychotic patients on chronic warfarin therapy for deep-vein thrombosis showed long-term remission of psychotic symptoms made us suspect that abnormalities in the coagulation pathway, specifically low tissue plasminogen activator (tPA) activity, could be one of the missing links. Our hypothesis is supported by a high prevalence of conditions affecting tPA activity in drug-naive schizophrenia, such as antiphospholipid antibodies, elevated cytokine levels, hyperinsulinemia and hyperhomocysteinemia. We recently screened a group of schizophrenia patients and controls for conditions affecting tPA activity. Free-protein S deficiency was highly prevalent among patients, but not found in controls. Free-protein S and functional protein C are natural anticoagulants that form complexes that inhibit tPA inhibitors. All participants had normal protein C levels, suggesting that protein S could have a role in schizophrenia, independent of protein C. Chronic patients and those studied during acute episodes had between three and six conditions affecting tPA and/or protein S activity, while patients in remission had up to two, which led us to postulate that multiple conditions affecting tPA and/or protein S activity could contribute to the full expression of schizophrenia phenotype. This paper describes the physiological roles of tPA and protein S, reviewing how their activity influences pathogenesis and comorbidity of schizophrenia. Next, it analyzes how activity of tPA and protein S is influenced by biochemical abnormalities found in schizophrenia. Last, it suggests future directions for research, such as studies on animal models and on therapeutic approaches for schizophrenia aiming at increasing tPA and protein S activity.

[Molecular diagnosis of Gaucher disease in Tunisia]. / Diagnostic moléculaire de la maladie de Gaucher en Tunisie.

Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme acid ß-glucosidase. In order to determine the mutation spectrum in Tunisia, we performed recurrent mutation screening in 30 Tunisian patients with Gaucher disease. Screening of recurrent mutation by PCR/RFLP and direct sequencing had shown that N370S was the most frequent mutation (22/50 mutant alleles, 44%), followed by L444P mutation, which is found in 16% (8/50 mutant alleles). The recombinant allele (RecNciI) represented 14%. Our findings revealed that the genotype N370S/RecNciI was mosst frequent in patients with childhood onset and it was associated with severe visceral involvement. The screening of these three mutations provided a simple tool for molecular diagnosis of Gaucher disease in Tunisian patients and allowed also genetic counselling for their family members.

Molecular analysis of mucopolysaccharidosis type IIIB in Portugal: evidence of a single origin for a common mutation (R234C) in the Iberian Peninsula.

Mucopolysaccharidosis type IIIB (Sanfilippo B disease) is a rare autosomal recessive disorder caused by defective alpha-N-acetylglucosaminidase (NAGLU). We examined the NAGLU gene in 11 MPS IIIB Portuguese patients, having identified five novel (M1K, W147X, G304V, S522P, and R533X) and four previously reported mutations (W168X, R234C, R565W and R643C). R234C attained the high prevalence of 32% of the mutated alleles. Because R234C had already been reported to be common in Spanish patients, a haplotypic analysis was conducted to address the question of its origin in the Iberian Peninsula. Three neutral markers were studied that allowed for the identification of the probable founder haplotype (174-234-G) on which R234C arose. The sharing of the ancestral haplotype by Portuguese and Spanish patients clearly implied a common origin of the mutation in Iberia, through an event that was inferred to have been rather recent. Therefore, the reconstructed history of R234C explains the high incidence of the mutation in Iberian patients with Sanfilippo B disease.

[Mutation spectrum of Gaucher disease in Tunisia: high frequency of N370S/Rec NciI compound heterozygous]. / Spectre mutationnel de la maladie de Gaucher en Tunisie: forte fréquence des hétérozygotes composites N370S/Rec NciI.

Gaucher disease is the most common lysosomal storage disorder, it results from the inherited deficiency of the enzyme glucocerebrosidase, the accumulation of its substrate causes many clinical manifestations. Since the discovery of GBA gene, more than 200 different mutations have been identified, but only handful mutations are recurrent (N370S, L444P and c.84insG). In order to determine the mutation spectrum in Tunisia, we performed recurrent mutation screening in ten unrelated Tunisian children with Gaucher disease. Screening of recurrent mutation by PCR/RFLP and direct sequencing, has shown that N370S is the most frequent mutation (6/20 mutant alleles, 30%), followed by recombinant allele (RecNciI) which is found in five patients (5/20 mutant alleles, 25%), the L444P mutation represent 20% (4/20 mutant alleles). Our findings revealed that five among ten studied patients, were compound heterozygous N370S/RecNciI (50%). The screening of these mutations provides a simple tool for molecular diagnosis of Gaucher disease in Tunisian patients and allows also genetic counselling for their family members.

Allelic frequency determination of the 24-bp chitotriosidase duplication in the Portuguese population by real-time PCR.

Chitotriosidase is a human chitinase produced by macrophages. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal storage disorders, as well as other diseases in which macrophages are activated. Therefore, it is a useful tool as a secondary marker in the diagnosis of several disorders including Gaucher disease type 1 and Niemann-Pick disease. The determination of chitotriosidase levels as a diagnosis complement in some lysosomal storage disorders and in enzyme replacement therapy follow-up of Gaucher disease patients is of great importance. However, the fact that a mutation caused by a 24-bp duplication in the CHIT1 gene resulting in deficiency of plasma chitotriosidase activity is very frequent makes the establishment of the frequency of this mutation in different population groups necessary. Furthermore, in order to validate the use of chitotriosidase activity as a marker, it is indispensable to screen individuals for this particular mutation. In this work, we present the results of a study where the allelic frequency of the above mentioned CHIT1 gene mutation was determined in the Portuguese population by real-time PCR. The frequency of carriers encountered in this sample of Portuguese individuals was of 37%.

Adult-onset neuronopathic form of Gaucher's disease: a case report.

We report a patient with Gaucher's disease (GD) developing prominent neurological abnormalities in adult life confirming the existence of an adult neuronopathic form of GD. In this adult-onset form, an akinetic-rigid syndrome poorly responsive to dopatherapy, supranuclear gaze palsy, myoclonic jerks, seizures, cerebellar ataxia, cognitive and psychotic disturbances are frequent manifestations. The widely used clinical classification seems inadequate since it does not consider this rare form of GD. Until further understanding of the pathogenesis of the disease is achieved it is not possible to predict accurately which patients will or will not have late-onset nervous system involvement.

Use and misuse of benzodiazepines in Brazil: a review.

Benzodiazepines are among the most prescribed and consumed medication groups in the world. Although benzodiazepines are used in the treatment of several psychiatric and non-psychiatric disorders, and are generally safe and well-tolerated, the potential for misuse and abuse is considerable. This makes the study and regulation of benzodiazepine prescription and consumption an item of concern in public health around the world. Most developed countries have consistent data of benzodiazepine sales and consumption; however, data from developing countries is scarce, making health policies on the use of benzodiazepines a much tougher issue in these countries. This article aims to review the epidemiology of benzodiazepine use in Brazil, as well as to analyze how legislation, physician misinformation and economic factors might contribute to making benzodiazepine abuse a problem in the country.

Niemann-Pick type C disease: NPC1 mutations associated with severe and mild cellular cholesterol trafficking alterations.

Niemann-Pick type C disease (NPC) is a rare neurodegenerative disorder characterised by lysosomal/late endosomal accumulation of endocytosed unesterified cholesterol and delayed induction of cholesterol homeostatic reactions. The large majority of mutations in the NPC1 gene described thus far have been associated with severe cellular cholesterol trafficking impairment (classic biochemical phenotype, present in about 85% of NPC patients). In our population of 13 unrelated NP-C1 patients, among which 12 were of Portuguese extraction, we observed an unusually large proportion of families presenting mild alterations of intracellular cholesterol transport (variant biochemical phenotype), without strict correlation between the biochemical phenotype and the clinical expression of the disease. Mutational studies were carried out to compare molecular lesions associated with severe and mild cholesterol traffic impairment. Levels of NPC1 protein were studied by Western blot in cultured fibroblasts of four patients with homozygous mutant alleles. Ten novel mutations were identified (Q92R, C177Y, R518W, W942C, R978C, A1035V, 2129delA, 3662delT, IVS23+1 G>A and IVS16-82 G>A). The mutational profile appeared to be correlated with the biochemical phenotype. Splicing mutations, I1061T and A1035V, corresponded to "classic" alleles, while three missense mutations, C177Y, R978C and P1007A, could be defined as "variant" alleles. All "variant" mutations described so far appear to be clustered within the cysteine-rich luminal loop between TM 8 and 9, with the remarkable exception of C177Y. The latter mutant allele, at variance with P1007A, was correlated to a decreased level of NPC1 protein and a severe course of the disease, and disclosed a new location for "variant" mutations, the luminal loop located at the N-terminal end of the protein.

Evoked potentials for the evaluation of latent hepatic encephalopathy in pediatric liver transplant candidates.

BACKGROUND: Visual evoked potentials (VEPs) and brain stem auditory evoked potentials (BAEPs) have been proposed as tools in the diagnosis of subclinical hepatic encephalopathy (HE). However, little information exists to determine their usefulness in pediatric patients. This study was undertaken to evaluate both methods in the detection of subclinical HE in pediatric liver transplant candidates. METHODS: VEPs and BAEPs were recorded in 15 pediatric liver transplant candidates with no clinical signs of HE. The wave latencies found in these examinations were then compared with those in 16 healthy controls of similar age. Laboratory data on liver function and electroencephalographic data from the patients were also recorded to examine their correlation with the evoked potentials results. RESULTS: No differences were found in the BAEP results between patients and controls. However, in the VEPs, the liver transplant candidates had significantly prolonged N1 (N75) latencies when compared with controls; no significant delay was found in the other waves. In contrast, among the children with liver disease, higher BAEP peak latencies correlated positively with electroencephalographic abnormalities, but this correlation was not observed in VEPs. CONCLUSIONS: Evoked potentials might be of use in detecting alterations related to HE in children. However, further studies are necessary to determine their sensitivity and specificity in this situation.

Altered ATP hydrolysis induced by pentylenetetrazol kindling in rat brain synaptosomes.

The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Adenosine has potent anticonvulsant effects on various models of epilepsy. One of these models is pentylenetetrazol (PTZ) kindling, in which repeated administration of subconvulsive doses of this drug induces progressive intensification of seizure activity. In this study, we examine the effect of a single convulsive injection (60 mg/kg, i.p.) or 10 successive (35 mg/kg, i.p.) injections of PTZ on synaptosomal ectonucleotidases. Our results have shown that no changes in ectonucleotidase activities were seen at 0, 1, and 24 h or at 5 days after a single convulsive PTZ injection. However, after PTZ-kindling, rats which were more resistant to seizure development presented an increase in ATP hydrolysis in synaptosomes from hippocampus and cerebral cortex (44% and 28%, respectively). These results suggest that changes in nucleotide hydrolysis may represent an important mechanism in the modulation of chronic epileptic activity in this model.

Gaucher disease: the origins of the Ashkenazi Jewish N370S and 84GG acid beta-glucosidase mutations.

Type 1 Gaucher disease (GD), a non-neuronopathic lysosomal storage disorder, results from the deficient activity of acid beta-glucosidase (GBA). Type 1 disease is panethnic but is more prevalent in individuals of Ashkenazi Jewish (AJ) descent. Of the causative GBA mutations, N370S is particularly frequent in the AJ population, (q approximately .03), whereas the 84GG insertion (q approximately .003) occurs exclusively in the Ashkenazim. To investigate the genetic history of these mutations in the AJ population, short tandem repeat (STR) markers were used to map a 9.3-cM region containing the GBA locus and to genotype 261 AJ N370S chromosomes, 60 European non-Jewish N370S chromosomes, and 62 AJ 84GG chromosomes. A highly conserved haplotype at four markers flanking GBA (PKLR, D1S1595, D1S2721, and D1S2777) was observed on both the AJ chromosomes and the non-Jewish N370S chromosomes, suggesting the occurrence of a founder common to both populations. Of note, the presence of different divergent haplotypes suggested the occurrence of de novo, recurrent N370S mutations. In contrast, a different conserved haplotype at these markers was identified on the 84GG chromosomes, which was unique to the AJ population. On the basis of the linkage disequilibrium (LD) delta values, the non-Jewish European N370S chromosomes had greater haplotype diversity and less LD at the markers flanking the conserved haplotype than did the AJ N370S chromosomes. This finding is consistent with the presence of the N370S mutation in the non-Jewish European population prior to the founding of the AJ population. Coalescence analyses for the N370S and 84GG mutations estimated similar coalescence times, of 48 and 55.5 generations ago, respectively. The results of these studies are consistent with a significant bottleneck occurring in the AJ population during the first millennium, when the population became established in Europe.

Gaucher disease: expression and characterization of mild and severe acid beta-glucosidase mutations in Portuguese type 1 patients.

Type 1 Gaucher disease (GD), the most prevalent lysosomal storage disease, results from the deficient activity of acid beta-glucosidase. Molecular analysis of 12 unrelated Portuguese patients with type 1 GD identified three novel acid beta-glucosidase mutations (F109V, W184R and R395P), as well as three previously reported, but uncharacterized, lesions (R359Q, G377S and N396T). The type 1 probands were either heteroallelic for the well-characterized common lesion, N370S, and the F109V, W184R, R359Q or N396T lesions or homoallelic for the G377S or N396T mutations. Expression of the W184R, R359Q and R395P mutations revealed very low specific activities based on cross-reacting immunologic material (CRIM SAs of 0.0004, 0.016 and 0.045, respectively), consistent with their being found only in type 1 patients who had a neuroprotective N370S allele. In contrast, the F109V, G377S and N396T alleles had significant acid beta-glucosidase activity (CRIM specific activities of 0.15, 0.17, 0.14, respectively), in agreement with their being mild type 1 alleles. Thus, these studies identified additional acid beta-glucosidase mutations in the Portuguese population and demonstrated that the G377S and N396T mutations were neuroprotective, consistent with the mild clinical phenotypes of the type 1 patients who were homoallelic for the G377S and N396T lesions.

Increased sensitivity to seizures in mice lacking cellular prion protein.

PURPOSE: The physiologic role of the cellular prion protein (PrPc) is unknown. Mice devoid of PrPc develop normally and show only minor deficits. However, electrophysiologic and histologic alterations found in these mice suggest a possible role for PrPc in seizure threshold and/or epilepsy. METHODS: We tested the sensitivity of PrPc knockout mice to seizures induced by single convulsant or repeated subconvulsant (kindling) doses of pentylenetetrazol (PTZ), and to status epilepticus (SE) induced by kainic acid or pilocarpine. RESULTS: In PTZ kindling, seizure severity progressed faster in the PrPc knockout group, in which 92.8% reached stage 5 or death after 4 days of stimulation, as opposed to 38.4% in wild-type animals. After 10 injections, mortality was 85.7% among knockouts and 15.3% among controls. After a single PTZ injection (60 mg/kg), overall mortality due to seizures was 91% in knockout mice, but only 33% among wild-type animals. Pilocarpine-induced SE (320 mg/kg) caused an 86.7% mortality in knockouts, as opposed to 40% in wild-type animals. Finally, after kainic acid injections (10 mg/kg), 70% of the knockouts developed at least one severe seizure, and 50% showed repetitive seizures, whereas no wild-type animal exhibited observable seizures. CONCLUSIONS: Animals lacking cellular prion protein expression are more susceptible to seizures induced by various convulsant agents. This is perhaps the most striking alteration yet found in PrPc-null mice, who at first analysis appeared to be completely normal. A possible role for PrPc in chronic and idiopathic (familial), secondary, or cryptogenic epilepsies in humans remains to be investigated.

Dose-dependent impairment of inhibitory avoidance retention in rats by immediate post-training infusion of a mitogen-activated protein kinase kinase inhibitor into cortical structures.

Mitogen-activated protein kinase (MAPK) is a serine/threonine protein kinase abundantly expressed in postmitotic neurons of the developed nervous system. MAPK is activated in and required for both the induction of long-term potentiation (LTP) in hippocampal slices and the acquisition of fear conditioning training in rats. The present work was performed in order to test the effect of the specific inhibitor of MAPK kinase (MAPKK), PD 098059, on retention of a step-down inhibitory avoidance (IA). Adult male Wistar rats were bilaterally injected (0.5 microl/side) with PD 098059 (at 0.5, 5, or 50 microM) or vehicle into the entorhinal cortex or into the parietal cortex immediately after IA training using a 0.4 mA footshock. Retention testing was carried out 24 h after training. PD 098059 impaired retention when injected into the entorhinal cortex at the dose of 50 microM, but not at the doses of 5 or 0.5 microM. When infused into the parietal cortex, PD 098059 was amnestic at the doses of 5 and 50 microM. The drug had no effect when infused at the highest dose in either structure 6 h after training. Our results suggest that the MAPKK inhibitor impairs IA retention memory in a dose-dependent manner when injected immediately after training into entorhinal cortex or parietal cortex. The effective dose is variable according to the neocortical structure studied.

T cell numbers relate to bone involvement in Gaucher disease.

The major elements of bone pathology in Gaucher disease are a failure of osteoclast and osteoblast function, resulting in osteopenia and also osteonecrosis. T lymphocytes have recently been found to be involved in the regulation of osteoblast/osteoclast activity in vitro. In the present report the peripheral blood T major lymphocyte subsets were investigated in a group of genotyped type 1 Gaucher disease patients. A total of 31 patients were studied: 21 non-splenectomized (5 N370S homozygotes) and 10 splenectomized (of whom 1 was a N370S homozygote). The results show that non-splenectomized patients present a decrease in absolute numbers of peripheral blood T lymphocytes, specially the CD4+ T subset. However, when patients were analyzed with respect to the presence of bone disease, the number of CD8+ T lymphocytes was found to be statistically significantly lower in patients presenting bone involvement. Furthermore, lower numbers of CD8+ T lymphocytes were significantly correlated with higher levels of plasma tartrate resistant acid phosphatase (TRAP) activity, a putative marker of osteoclast cell activity. These in vivo findings are in agreement with the results reached in vitro by others. They provide an additional marker of disease severity in Gaucher disease. In the group of genotyped Gaucher disease patients, the majority of the N370S homozygous patients presented a clinically milder phenotype, including the absence of bone involvement, confirming earlier reports predicting that a number of these patients may remain undiagnosed. Collectively the homozygosity for the N370S mutation and normal T cell numbers may provide additional markers for the clinical heterogeneity of Gaucher disease.

Metachromatic leucodystrophy in Portugal-finding of four new molecular lesions: C300F, P425T, g.1190-1191insC, and g.2408delC. Mutations in brief no. 232. Online.

The mutation identification rate achieved in the study of Portuguese MLD patients was found to be extremely high (100%), thus revealing the power of the association of vertical and horizontal PCR-SSCA. The identification of new mutations adds to the large number of mutations already described to be associated to MLD. Nevertheless, mutation g.1238G>A has been found in most of the Portuguese patients, either in homozygosity or heterozygosity, suggesting this mutation to be more common in Portuguese patients than in patients with other ethnic backgrounds. Two new missense mutations (C300F and P425T) were found to be associated to late infantile and juvenile forms, respectively. Two novel microlesions (g.1190-1191insC, g.2408delC) were identified in two late infantile patients. It should be noted that both C300F and g.2408delC were detected in homozygosity. The approach used and the results here presented may provide useful information for the study of other MLD patients, as well as new insights about the effect of mutations, such as C300F, in the structure/function of ARSA.

Distinct haplotype in non-Ashkenazi Gaucher patients with N370S mutation.

A new polymorphism, in intron 7 of glucocerebrosidase gene, has been identified in Gaucher Disease patients. It seems to appear only in Pv1.1- alleles bearing the N370S mutation. This new sub-haplotype was only identified in Portuguese patients, of origins spanning all of the Portuguese continental territory. This finding indicates that, in the Portuguese, mutation N370S has existed in the context of two slightly different haplotypes and thus must be relatively ancient.