Circulating microRNAs as potential biomarkers for genetic generalized epilepsies: a three microRNA panel.

BACKGROUND AND PURPOSE: Genetic generalized epilepsies (GGEs) encompass a group of syndromes of mainly genetic causes, characterized by the involvement of both hemispheres. MicroRNAs (miRNAs) are small non-coding RNAs with a critical role in the regulation of neuronal biological processes through gene expression modulation. Dysregulated miRNA expression has been shown in epilepsy. Due to their stability in biological fluids like serum, miRNAs have assumed a prominent role in biomarker research. Our aim was to evaluate circulating levels of three miRNAs in GGE patients and assess their putative diagnostic value. METHODS: MiR-146a, miR-155 and miR-132 were quantified by real-time polymerase chain reaction in the serum of 79 GGE patients (47 women, 32 men, 35.1 ± 12.4 years) and 67 healthy individuals (41 women, 26 men, 42.4 ± 10.1 years). Relative expression values were calculated using the 2-ΔΔCt method. Receiver operating characteristic curve analysis was performed to assess diagnostic value. MiRNA expression was correlated with clinicopathological features. RESULTS: Serum levels of miR-146a and miR-155 were significantly upregulated in GGE patients relative to controls (3.13 and 6.05, respectively). Combined miR-146a, miR-155 and miR-132 serum levels performed well as a diagnostic biomarker, discriminating GGE patients from controls with an area under the curve of 0.85, 80% specificity and 73% sensitivity. CONCLUSIONS: Our results indicate that miR-146a, miR-155 and miR-132 may partake in GGE epileptogenesis. A panel of three circulating miRNAs with potential value as a GGE biomarker is reported for the first time. Novel biomarkers may help to identify new treatment targets and contribute to improved patients' quality of life through earlier diagnosis and a more precise prognosis.

Association between vitamin D receptor (VDR) gene polymorphisms and systemic lupus erythematosus in Portuguese patients.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown origin, in which both genetic and environmental factors are involved. One such environmental factor is vitamin D, a vital hormone that plays a specific function in the immune system homeostasis, acting through a nuclear receptor (VDR) expressed in all immune cells. Several polymorphisms of the gene that encodes this receptor have been described. Though inconsistently, these polymorphisms have been associated with clinical manifestations and SLE development.The aim of this study was to determine the possible association between VDR gene polymorphisms (BsmI, ApaI, TaqI e FokI) and SLE susceptibility and severity, in a cohort of lupus patients from the north of Portugal.A total of 170 patients (F = 155, M = 15; age = 45 ± 13.4 years) with SLE (diagnosed according the American College of Rheumatology criteria) with at least five years of disease evolution and followed in the Autoimmune Disease Clinical Immunology Unit of Centro Hospitalar do Porto were studied. Patients and 192 ethnicity-matched controls were genotyped for BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236) and FokI (rs2228570) polymorphisms by TaqMan allelic discrimination assay. Disease severity was assessed by SLICC damage score, number of affected organs, number of severe flares and pharmacological history.SLE patients with the CT genotype of FokI polymorphism have a higher SLICC value (p = 0.031). The same result was observed for the group of patients with the TT genotype of TaqI polymorphism (p = 0.046). No differences were observed in VDR genotype between patients and controls. Also, we observed that the other clinical features analysed were not influenced by VDR polymorphisms.Our study confirms a possible role of VDR gene polymorphisms in SLE. A positive association was found between VDR polymorphisms and SLE severity (chronic damage). The presence of CT genotype of FokI and TT genotype of TaqI seems to confer a worse prognosis and may constitute a risk factor for higher long-term cumulative damage in SLE patients.

CCR5-Delta32: implications in SLE development.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease with strong genetic and environmental components. Previous studies have shown increased levels of several chemokines in active SLE. C-C chemokine receptor type 5 (CCR5) is involved in the recruitment of inflammatory cells into tissues, and mechanisms modulating CCR5 expression and function may interfere in SLE development, influencing the clinical course of the disease. The aim of this study was to evaluate the possible association between the CCR5∆32 base-pair deletion polymorphism and SLE disease in a group of Portuguese patients. A total of 219 patients with SLE and 205 healthy individuals were studied. The frequency of CCR5/∆32 heterozygotes was lower in patients with SLE than in controls (8% vs. 15% OR = 0.5162; P = 0.0319), suggesting a protective association between CCR5∆32 allele and SLE. These results highlight the protective role of Th1 cells that express CCR5 in SLE pathogenesis.

HFE gene polymorphisms and severity in Portuguese patients with multiple sclerosis.

BACKGROUND: High iron concentrations have been reported in oligodendrocytes, myelin and macrophages in multiple sclerosis (MS) lesions. It has been proposed that HFE gene polymorphisms could have a role in MS. METHODS: The C282Y and H63D HFE variants frequencies were determined in 373 patients with MS and compared with a normal population. RESULTS: No significant association was found between HFE polymorphisms and disease susceptibility. An analysis of the association of genotypes with disease severity was performed, and the C282Y allele was more frequent in the aggressive group. CONCLUSIONS: Patients carrying the C282Y variant seem to have a worse prognosis.

High degree of genetic diversity among genotypes of the forage grass Brachiaria ruziziensis (Poaceae) detected with ISSR markers.

The grasses of the genus Brachiaria account for 80% of the cultivated pastures in Brazil. Despite its importance for livestock production, little information is available for breeding purposes. Embrapa has a population of B. ruziziensis from different regions of Brazil, representing most of existing variability. This population was used to initiate an improvement program based on recurrent selection. In order to assist the genetic improvement program, we estimated the molecular variability among 93 genotypes of Embrapa's collection using ISSR (inter-simple sequence repeat) markers. DNA was extracted from the leaves. Twelve ISSR primers generated 89 polymorphic bands in the 93 genotypes. The number of bands identified by each primer ranged from two to 13, with a mean of 7.41. Cluster analysis revealed a clearly distinct group, containing most of the B. ruziziensis genotypes apart from the outgroup genotypes. Genetic similarity coefficients ranged from 0.0 to 0.95, with a mean of 0.50 and analysis of molecular variance indicated higher variation within (73.43%) than among species (26.57%). We conclude that there is a high genetic diversity among these B. ruziziensis genotypes, which could be explored by breeding programs.

Radiographical evaluation of bone quality after extraction of mandibular impacted and semi-impacted third molars.

AIM: The association between bone quality and fracture risk in the mandibular angle with the presence of impacted/semi-impacted third molars and after their extraction is controversial. This study aimed to assess mandibular bone quality in digital radiographies of patients after extraction of impacted/semi-impacted lower third molars. METHODS: A total of 130 sets of digital panoramic radiographies were selected and divided into the following three groups: Group 1 with 50 panoramic radiographies of patients with impacted/semi-impacted lower third molars, Group 2 with 30 panoramic radiographies of patients with lower third molar agenesis, and Group 3 with 50 panoramic radiographies of patients after extraction of impacted/semi-impacted lower third molars. The mandibular angular cortex was the anatomical structure used as parameter for bone quality assessment. ANOVA and Student's t test were applied for comparison between groups. RESULTS: Mandibular angular cortical width was significantly lower when the third molar was present in both genders (P<0.05). Agenesis of the third molar in women was associated with lower thickness of the mandibular cortex when compared with patients who had their third molar extracted (P<0.05). CONCLUSION: It can be concluded that the absence of impacted/semi-impacted lower third molars, was associated with a significant increase in cortical width.

New insights of HLA class I association to Behçet's disease in Portuguese patients.

Human leukocyte antigen (HLA)-B*51 is a well-known genetic factor associated with Behçet's disease (BD). To analyse the influence of HLA-B*51 and other HLA class I alleles in BD susceptibility in a Portuguese population and its association with disease severity, we studied 78 BD patients and 208 healthy controls. The patients were classified into two severity groups as described by Gul et al. As expected, a higher frequency of HLA-B*51 was found. The frequency of HLA-Cw*16 alleles was significantly higher in patients. Regarding severity, HLA-B*27 frequency was higher in the severe group compared with controls and with the mild group. Thus, HLA-B*51 and HLA-Cw*16 seem to confer susceptibility to BD in this patients. HLA-B*27 may be important as a prognostic factor.

Biochemical marker in familial amyloidotic polyneuropathy, Portuguese type. Family studies on the transthyretin (prealbumin)-methionine-30 variant.

A transthyretin variant with a methionine for valine substitution at position 30 [TTR(Met30)] is found in Portuguese patients with familial amyloidotic polyneuropathy (FAP). Effective, rapid, small- and semimicro-scale (immunoblotting) procedures were developed to determine whether or not TTR(Met30) is present in the plasma of an individual subject. The immunoblotting procedure employs only 0.10 ml of serum and can serve as a reliable procedure for the screening of large numbers of persons for the presence of TTR(Met30). In family studies of seven FAP kindreds, TTR(Met30) was found in 21 out of 41 asymptomatic FAP offspring, and its presence was not related to either age or sex. Thus, the mutant TTR segregated in accordance with the known autosomal dominant mode of inheritance of FAP. Total plasma TTR levels were not reduced in asymptomatic FAP offspring who were carriers of TTR(Met30), and no difference was observed between carriers and noncarriers of the mutant TTR. The ratios of the variant to normal TTR in plasma were estimated in asymptomatic FAP offspring and were similar to those found in FAP patients. In contrast, TTR(Met30) was relatively enriched in cerebrospinal fluid samples from two FAP patients. The significance of this finding is not known, but might relate to the preferential deposition of amyloid in the nervous system in FAP. A limited study was conducted involving simultaneous analysis of both stored (collected in 1975) and fresh serum from 20 FAP offspring, all of whom had been asymptomatic in 1975. In every subject, the results obtained with the stored and the fresh serum samples were in agreement. Six of these subjects developed clinical FAP since 1975; TTR(Met30) was present in each of these subjects. These several studies strongly suggest that the presence of TTR(Met30) in plasma constitutes a predictive biochemical marker of FAP in the preclinical phase of the disease.

Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin).

Amyloid fibril protein in patients with familial amyloidotic polyneuropathy is known to be chemically related to transthyretin (TTR), the plasma protein that is usually referred to as prealbumin. A genetically abnormal TTR may be involved in this disease. Studies were conducted on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with familial amyloidosis, and on TTR isolated from sera of patients with this disease. AFp, purified by affinity chromatography on retinol-binding protein linked to Sepharose, resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and retinol-binding protein. The structural studies included: (a) comparative peptide mappings by reverse-phase high performance liquid chromatography (HPLC) after trypsin digestion; (b) cyanogen bromide cleavage studies; and (c) amino acid microsequence analysis of selected tryptic and CNBr peptides. On the basis of the known amino acid sequence of TTR, comparative tryptic peptide maps showed the presence of a single aberrant tryptic peptide (peptide 4, residues 22-34) in AFp as compared with TTR. This aberrant peptide contained a methionine residue, not present in normal tryptic peptide 4. CNBr cleavage of AFp produced two extra peptide fragments, which were demonstrated, respectively, by HPLC analysis and by sodium dodecyl sulfate-gel electrophoresis. Sequence analyses indicated the presence of a methionine-for-valine substitution at position 30 in AFp as compared with TTR. Thus, the purified amyloid fibril protein comprised a TTR variant with a methionine-forvaline substitution at position 30. A single nucleotide change in a possible codon for valine 30 could explain the substitution. The variant TTR was also present in the TTR isolated from the pooled sera of amyloidoses patients, together with larger (four- to six-fold) amounts of the normal TTR. Thus, in these patients, the variant TTR was circulating in plasma, along with larger amounts of normal TTR. We suggest that the variant TTR represents the specific biochemical cause of the disease, and that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Characterization of a basic transthyretin variant--TTR Arg 102--in the German population.

A basic transthyretin (TTR) variant, apparently non-pathogenic, has been reported in a German family. Protein analysis of this TTR variant revealed the substitution of arginine for proline at position 102 of the TTR polypeptide chain. This result was confirmed by DNA analysis of PCR amplified DNA.

Thyroxine binding in a TTR Met 119 kindred.

Recently, a transthyretin variant, TTR Met 119, in which methionine substitutes for threonine 119, a component of the protein's iodothyronine binding site, was identified in individuals with transient euthyroid hyperthyroxinemia. Healthy carriers of Met 119 have normal serum thyroid hormone concentrations, but two studies of Met 119 carriers have differed as to whether T4 binding to TTR is increased. An additional kindred has been identified by hybrid isoelectric focusing in an ongoing screening program for TTR variants in the Portuguese population with TTR Met 30 associated familial amyloidotic polyneuropathy. Cyanogen bromide peptide mapping and DNA restriction length polymorphism analyses showed that the propositus was a compound heterozygote for two TTR variants: Asn 90 and Met 119. Family analysis revealed that he inherited the TTR Met 119 variant from the mother and the TTR Asn 90 variant from the father. Neither the compound heterozygote nor his parents had symptoms of familial amyloidotic polyneuropathy. Serum dialysis with stepwise saturation of iodothyronine binding sites confirmed that TTR binding of T4 is increased in TTR Met 119. The increased binding is due to a higher TTR concentration rather than an increased association constant for T4. Because of the small proportion of serum T4 bound by TTR, increased T4 binding by TTR did not affect the ratio of free to bound T4 or T4 concentrations. In contrast, plasma retinol binding protein, almost all of which is bound by TTR, was elevated. The Asn 90 mutation does not affect either the concentration or the hormone binding characteristics of the protein. Possible long-term effects of these mutations and the combined heterozygotic state remain to be determined.

Genetic expression of a transthyretin mutation in typical and late-onset Portuguese families with familial amyloidotic polyneuropathy.

Two studies were conducted to explore questions concerning the expression of a mutant transthyretin (TTR) gene, found in Portuguese patients with familial amyloidotic polyneuropathy (FAP). In a kindred with typical onset of the disease, complete agreement between genotype and phenotype was seen for all carriers of the variant TTR with a methionine-for-valine substitution at position 30 (TTR[Met30]). In another study involving a FAP kindred with a late onset of clinical disease, TTR(Met30) was found in plasma in asymptomatic persons with ages above the usual age of onset of the disease. No evidence was obtained for the existence of a different mutation in TTR or for repression of the expression of the mutant TTR gene in this kindred. The factors responsible for the delay in the development of clinical manifestations in late-onset patients are not known and warrant further study.

A Danish kindred with familial amyloid cardiomyopathy revisited: identification of a mutant transthyretin-methionine111 variant in serum from patients and carriers.

PURPOSE: In familial amyloid cardiomyopathy of Danish origin, the amyloid microfibrils contain a mutant transthyretin (TTR) with a methionine-for-leucine substitution at the molecular position 111. We studied the possible occurrence of this variant TTR-Met111 in serum from afflicted as well as nonafflicted family members and their offspring, in order to define its possible role as predictor of the disease and to describe its mode of inheritance. PATIENTS AND METHODS: Stored, frozen serum samples obtained from 1959 through 1960 from 36 of 40 living members of the kindred were analyzed. The method employed to detect the abnormal TTR was based on the electrophoretic separation of fragments produced by cyanogen bromide cleavage at the two methionine sites. RESULTS: All sera from family members with amyloid cardiomyopathy contained the variant transthyretin TTR-Met111 as did sera from half of their offspring. In contrast, nonafflicted family members and their offspring were seronegative for TTR-Met111. Three cousins from the second generation died between 1980 and 1986 of amyloid cardiomyopathy. The presence of variant TTR-Met111 preceded their deaths by 20 to 26 years. CONCLUSIONS: The occurrence in serum of the mutant transthyretin TTR-Met111 is linked to the occurrence of amyloid cardiomyopathy in patients and their offspring, while unafflicted branches of the family are negative for the variant protein. That the occurrence in serum of TTR-Met111 precedes the onset of clinical amyloid cardiomyopathy by several decades makes the variant TTR a marker for the disease. The distribution of afflicted family members and seropositivity for the variant TTR shows an autosomal dominant mode of inheritance. CLINICAL SIGNIFICANCE: The results make possible early detection of potential patients and provide tools for genetic counseling. Cardiac transplantation may provide a new therapeutic option.

Unmasking antigen determinants in amyloid.

Rehydrated paraffin sections of formalin-fixed, amyloid-containing tissues were treated with denaturing agents (guanidine and urea) and reducing agents (DDT and mercaptoethanol) before immunostaining, in an attempt to expose antigenic determinants hidden in the rigid structure of amyloid fibrils. Pre-treatment overnight with 6 M guanidine or urea was found beneficial, especially in specimens from familial amyloid polyneuropathy of the Portuguese type. Addition of reducing agents had no major effect. Modifications of this method may be useful in unmasking other antigens that are polymerized or considered destroyed by fixation and paraffin embedding.

Family studies of the genetic abnormality in transthyretin (prealbumin) in Portuguese patients with familial amyloidotic polyneuropathy.

Amyloid deposits in several heredofamilial forms of amyloidosis are chemically related to transthyretin (TTR, the protein usually referred to as prealbumin). A genetically abnormal TTR may be involved. Studies were conducted on TTR isolated from sera of patients with familial amyloidotic polyneuropathy (FAP), and on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with FAP. AFp, purified by affinity chromatography on retinol-binding protein (RBP), resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and RBP. Purified AFp was found to comprise a TTR variant with a methionine for valine substitution at position 30. This conclusion was based upon studies that included: (i) comparative peptide mapping by reverse-phase high-performance liquid chromatography after trypsin digestion; (ii) cyanogen bromide (CNBr) cleavage studies; and (iii) amino acid microsequence analysis of selected tryptic and CNBr peptides. The variant TTR was also found to be present in serum samples from FAP patients, along with larger amounts of normal TTR. An effective, small-scale procedure was developed to determine whether or not the variant TTR was present in the plasma of an individual subject. This procedure involved isolation of TTR by affinity chromatography on RBP, followed by CNBr cleavage, and analysis for the presence of specific aberrant CNBr peptides. Studies with six kindreds, including 21 asymptomatic children of 6 patients with FAP, showed that the "abnormal" TTR can be detected and used as a preclinical marker of the disease in affected children of patients with FAP. It is likely that the variant TTR represents a point mutation within the TTR structural gene, and that the normal and mutant genes act as co-dominant alleles at a single locus in FAP. The distribution of the mutant TTR within the six families was consistent with the autosomal dominant mode of inheritance of FAP. The mutant TTR apparently selectively deposits in tissues as the amyloid characteristic of the disease.

Specific removal of transthyretin from plasma of patients with familial amyloidotic polyneuropathy: optimization of an immunoadsorption procedure.

Familial amyloidotic polyneuropathy is characterized by the presence in patients plasma of a genetic variant of transthyretin. No specific treatment has been found and extracorporeal immunoadsorption on immobilized anti-transthyretin antibodies appears as a potentially attractive procedure. Parameters involved in specific immunoadsorption of transthyretin were studied and optimized. Several monoclonal anti-TTR antibodies were compared as affinity ligands and one of them was found to be suitable for such purposes. Optimum quantities of antibodies to be immobilized on the gel were determined. Three desorption agents were tested for regenerating immunoadsorbents and best results were obtained with basic variation of pH, allowing total desorption of TTR and possibility of multiple use without loss of adsorption capacity. Simulation of an immunoadsorption procedure in well-defined conditions showed efficiency and specificity of adsorption to remove TTR and the system thus should be subjected to clinical trials.

[Liver transplantation for the treatment of type I familial amyloidotic polyneuropathy]. / Trasplante hepático para el tratamiento de la polineuropatía amiloidótica familiar tipo I.

The first liver transplantation carried out in Spain for the treatment of type I familial amyloidotic polyneuropathy (FAP I) is presented. The reason for the operation was based on the liver being responsible for the synthesis of abnormal transtirretin (TTR) constituting the peculiar amyloid of the disease. Following transplantation a rapid and noticeable decrease in abnormal TTR was observed and the evolution of the clinical picture after 18 months of surgery is favorable with progressive improvement of the neurologic symptoms and normal function of the graft. These encouraging results coincide with those of the Swedish group of Umea, the pioneer of this procedure.

Síndrome de Eisenmenger em um gato / Eisenmenger syndrome in cat

A 6-month-old female, 1.0kg, uncastrated female Persian cat was brought to the Veterinary Hospital at State University of Ceara, with a history of dyspnea, prostration, hyporexia and progressive weight loss for a month. On physical examination, systolic cardiac murmur, cyanosis and dyspnea were detected. Unfortunately, the cat died during oxygen therapy. Necropsy examination revealed an increase in cardiac silhouette and ventricular septal defect of 2cm in diameter. Macroscopically the lungs were collapsed, with absent and diffusely reddish blackish crepitus, and the liver with blackish red coalescent multifocal areas, interspersed with lighter areas and lobular pattern with irregular brownish multifocal areas intercepted by brownish areas. Thus, the necropsy results together with the history and physical examination of the animal confirmed the diagnosis of Eisenmenger Syndrome, becoming the report of the first case, in a cat, in Brazil.(AU)

Towards a better understanding of Ipomoea asarifolia toxicity: evidence of the involvement of a leaf lectin.

Natural intoxication of livestock by ingestion of Ipomoea asarifolia leaves has been reported to occur widely in Brazil. Previous studies carried out by our research group provided strong evidence that a lectin could be involved with the toxic properties of I. asarifolia. To reinforce this hypothesis, a lectin-enriched fraction (LEF) was isolated from I. asarifolia leaves and its toxic effects were assessed. Leaves of I. asarifolia were excised from plants growing widely in the field, mechanically wounded and maintained in a chamber at 25 ± 3 °C for 72h in the dark, under near 100% relative humidity. The leaf proteins were extracted, ammonium sulfate precipitated, chromatographed on DEAE-cellulose and Phenyl-Sepharose to produce LEF that under SDS-PAGE showed a molecular mass of 44.0 kDa and after N-terminal amino acid analysis a primary sequence composed of AGYTPVLDIGAEVLAAGEPY. The in vivo toxicity of LEF assessed by intraorbital injection in mice showed induced severe uncoordinated movements without death. LEF reduced the muscular contraction in a dose depend way and at 29.8 µg/mL (CE(50)) it produces 50% inhibition of contraction, suggesting that LEF blunts autonomic neurotransmission. Isolated rat kidneys were perfused with LEF and no effects on the perfusion pressure or renal vascular resistance were observed, but urinary flow and glomerular filtration rate increased. Moreover, the percentage of tubular transport of Na(+), K(+) and Cl(-) decreased. Histological examination of the kidneys perfused with LEF exhibited little alterations. These toxic effects observed above were concomitant with the increase of LEF hemagglutination activity, which strongly suggest that one of the toxic principles of I. asarifolia is a lectin present in its leaves.