Phosphate Transporters Expression in Patients with Primary Familial Brain Calcifications.

Primary familial brain calcification (PFBC), formerly known as Fahr disease, is a rare neurological disorder characterized by extensive calcification deposits in the brain. So far, four genes have been reported with variations associated with PFBC, SLC20A2, PDGFß, PDGFRß, and XPR1. Using real-time qPCR, we analyzed the expression of three inorganic phosphate (Pi) transporters (SLC20A1, SLC20A2, and XPR1) in patients with PFBC. Our results showed a significant reduction (~40%) of SLC20A2 expression in the patients carrying mutation whereas no significant change was observed within the patients without known mutations. No difference was detected in SLC20A1 and XPR1 expression between the groups compared to control. The results suggest that mutations in SLC20A2 gene by itself play an import role by reducing its expression in blood of PFBC patients. At the same time, we could not demonstrate a direct co-regulation between the three Pi transporters at mRNA level, once their expression did not change among the groups.

Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications ("Fahr's disease"). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr's disease and normal aging.

First report of a de novo mutation at SLC20A2 in a patient with brain calcification.

Primary familial brain calcification (PFBC) is identified by mineralization of the basal ganglia and other brain regions in the absence of known causes. The condition is often inherited in an autosomal dominant pattern and can manifest itself clinically with neuropsychiatric symptoms such as Parkinsonism, headaches, psychosis, and mood swings. Mutations in the SLC20A2 gene account for ~40% of inherited cases, and this gene encodes an inorganic phosphate transporter (PiT-2), a transmembrane protein associated with Pi homeostasis. The p.Y386X mutation in SLC20A2 was identified in a patient who presented migraines, brain calcification, and mild but chronic hypovitaminosis D. SLC20A2 c.1158C > G single-nucleotide heterozygous mutation results in a premature stop codon and a putative truncated protein of 385 amino acids. Proband parents do not present the mutation, which is also not present in major public SNP databases, suggesting a de novo sporadic trait. This study describes for the first time a de novo SLC20A2 mutation in a PFBC patient with migraine and mild hypovitaminosis D. This data further reinforces the pathogenic role of SLC20A2 mutations as causal factors in PFBC physiopathology.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension.

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

Revising the M235T polymorphism position for the AGT gene and reporting a modifying variant in the Brazilian population with potential cardiac and neural impact.

There is a growing need to curate the overwhelming amount of sequencing data which is available in many public databases. For instance, new information shows that the M235T polymorphism at the angiotensinogen gene (AGT) is actually positioned at the position corresponding to the amino acid 268 and not 235. This polymorphism is filled as rs699 in the NCBI SNP database and results in the synthesis of a threonine (T) instead of a methionine (M). It has been widely studied and associated as an important risk factor for several vascular and neuropsychiatric conditions. We faced this new situation during the targeted sequencing of 360 chromosomes from Brazilian subjects studied for the M235T polymorphism, leading to the identification of a novel variation (rs141900991). This report explores the potential impact of such a dinucleotide variation, which promotes the change of alanine (A) to serine (S) at the AGT protein structure (A237S). Considering the previous M268T variation at the four possible haplotypes combined (MA, MS, TA and TS), we performed a comparative hydrophobicity simulation, using the Kyte-Doolittle algorithm, available at the CLB Bio workbench, in the four possible haplotypes. Additional simulations were performed using the programs PolyPhen, I-Mutant and SIFT, in order to evaluate the pathogenicity of both mutations. The predicted hydrophobicity decreases of a similar magnitude, with both MS and TA haplotypes, but the presence of both variations induces a major decrease in hydrophobicity, suggesting a cumulative effect, with possible modifying effect since that this variation per se would limit the hydrophobicity range and the latter chances in finding significant phenotype differences. A better characterization of this kind of variant is particularly important because the current genome wide scan analyses in complex disorders with cardiac or neural etiology are not generating reliable findings, especially if we consider the huge investment with such approach. Additional and unknown variations like this one, with potential modifying effect, might be more common than previously expected.

Exploring the implications of INDELs in neuropsychiatric genetics: challenges and perspectives.

The decade passed after publishing the Human Genome first draft faced an enormous growth at the understanding of the genomic variation among different subjects, populations, and groups of patients. Single nucleotide polymorphisms (SNPs) and insertion or deletions (INDELs) have been increasingly recognized as a major type of genetic variations, with potential impact in protein activities and gene expression changes observed in complex genetic traits, like neuropsychiatric diseases. INDELs represent the second most common class of variations after SNPs, but there is still an important gap between the number of INDELs reported and the actual knowledge about the functional implications of such variations. There are approximately 10 million SNPs already reported, and the human populations are expected to collectively harbor at least 1.6-2.5 million INDELs. One of the major challenges is to find better platforms to screen for INDELs in a high throughput manner. The discordance in between the data from different studies might be explained by the diverse approaches employed to sequence the genomes with variable platforms. Short INDEL variations increased the scope of genetic markers in human genetic diseases, and various studies showed that common microdeletions and smaller INDELs might be highly associated with neuropsychiatric diseases such as schizophrenia, autism, mental retardation, and Alzheimer disease. The rapidly increasing amount of resequencing, genotyping, and personal genome data generated by large-scale genetic human projects require the development of integrated bioinformatics tools able to efficiently manage and analyze these genetic data. Our group is currently dealing with different approaches that might optimize sequencing and bioinformatics analyses of short INDELs to broaden our research capabilities of identifying those intriguing genetic variations. Hopefully, INDELs might become a new trend in association studies in neuropsychiatric genetics since so far the level of significant and positive associations with the standard SNPs reported presents limited predictive application.

In silico identification of new genetic variations as potential risk factors for Alzheimer's disease in a microarray-oriented simulation.

Genomic and proteomic studies of neurodegenerative disorders require complementary approaches to integrate the massive amount of data generated in high throughput experimental procedures. We propose a Bioinformatics pipeline in which expression studies guide the selection of candidate genes that should be screened for potential new genetic variations from a public expressed site tags (ESTs) database. Motivated by the former interest of our group in genetic polymorphisms involved with the immune system, we selected five genes from a previous expression microarrays study of hippocampal cornu ammonis (CA1) area of Alzheimer's Disease subjects (AD). The CLCbio Workbench Combined version 3.6.2. was initially used to build ESTs and mRNA files retrieved respectively from the Goldenpath of University of California Santa Cruz (UCSC) and National Center for Biotechnology Information (NCBI) databases and latter to perform multiple batches of Smith-Waterman alignments. A total of 116 ESTs sequences were selected after proper stringent parameters were applied to the first set of mismatches. The annotation revealed various classes of variations, most of them deletions (176). Amongst this specific group, some were frameshift deletions (35) and the virtual translation of a few others (5) were predicted to induce no change other than a single aminoacid removal, with no subsequent repercussions at the protein sequence. In addition, the analysis identified transitions (three), transversions (52), synonymous (41), non-synonymous (12), and deletions in 36 ESTs located in Untranslated Regions -UTRs (Supplementary data). Deletions are often associated to major genetics syndromes with dysmorphic features. However, various recent studies show that common microdeletions might be highly associated with common neuropsychiatric disorders such as schizophrenia, autism, mental retardation, or even in various ethnicities, detected in whole genome sequencing experiments. A virtual validation confirmed that some of the variations identified were previously reported and confirmed in DNA samples, showing that this method is a feasible way to detect genetic variations that merit further exploration in AD genetic risk factor association studies.

Linkage studies in familial idiopathic basal ganglia calcification: separating the wheat from the chaff.

Idiopathic Basal Ganglia Calcification (IBGC) is a neuropsychiatric condition characterized by brain calcinosis, heterogeneous motor impairment and behavioral symptoms. The IBGC1 locus was the first region linked to this phenotype in an American family, but another kindred from Spain was also reported as possibly associated with this locus. Our group excluded this locus in additional families together with an independent study of an Australian pedigree with IBGC, but without clinical symptoms. Recently, a large Italian family from a population isolate was excluded from IBGC1. However, there are unusual aspects concerning this Tyrolean family, especially if we consider that almost all the clinically affected subjects manifested symptoms and signs suggestive of a dysmorphic syndrome, associated with neuropsychiatric symptoms. Curiously, some of the clinical features in this kindred match with the autosomal dominant chromosomal instability syndrome reported in Japan. Previous studies show that the definition of an autosomal dominant pattern of inheritance is an assumption that might be considered cautiously in familial IBGC, due to the limited clinical penetrance for the brain calcifications and especially when there is no access to all the parents neuroimaging data. Families from an Italian isolate, such as Tyrol, with high inbreeding rates, are more likely to manifest recessive syndromes. Nevertheless, the current debate regarding the nosology of this heterogeneous phenotype demands the establishment of standard diagnostic criteria. The current identification of loci or mutations responsible for FIBGC might help to elucidate this intriguing neuropsychiatric condition.

The genetics of Alzheimer's disease in Brazil: 10 years of analysis in a unique population.

Alzheimer's Disease (AD) is the most common type of dementia among the elderly, with devastating consequences for the patient, their relatives, and caregivers. More than 300 genetic polymorphisms have been involved with AD, demonstrating that this condition is polygenic and with a complex pattern of inheritance. This paper aims to report and compare the results of AD genetics studies in case-control and familial analysis performed in Brazil since our first publication, 10 years ago. They include the following genes/markers: Apolipoprotein E (APOE), 5-hidroxytryptamine transporter length polymorphic region (5-HTTLPR), brain-derived neurotrophin factor (BDNF), monoamine oxidase A (MAO-A), and two simple-sequence tandem repeat polymorphisms (DXS1047 and D10S1423). Previously unpublished data of the interleukin-1alpha (IL-1alpha) and interleukin-1 beta (IL-1beta) genes are reported here briefly. Results from others Brazilian studies with AD patients are also reported at this short review. Four local families studied with various markers at the chromosome 21, 19, 14, and 1 are briefly reported for the first time. The importance of studying DNA samples from Brazil is highlighted because of the uniqueness of its population, which presents both intense ethnical miscegenation, mainly at the east coast, but also clusters with high inbreeding rates in rural areas at the countryside. We discuss the current stage of extending these studies using high-throughput methods of large-scale genotyping, such as single nucleotide polymorphism microarrays, associated with bioinformatics tools that allow the analysis of such extensive number of genetics variables, with different levels of penetrance. There is still a long way between the huge amount of data gathered so far and the actual application toward the full understanding of AD, but the final goal is to develop precise tools for diagnosis and prognosis, creating new strategies for better treatments based on genetic profile.

Modeling of skeletal muscle: the influence of tendon and aponeuroses compliance on the force-length relationship.

The aim of this study was to investigate the influence of changing elastic properties of tendon and aponeuroses on force production and muscle geometry. A three-dimensional, structural, continuum mechanics model of the cat medial gastrocnemius was used for this purpose. Increasing compliance in tendon and aponeuroses caused a decrease in the peak isometric force and a shift of the force-length relationship to the right of the length axis (i.e. toward greater muscle lengths). This result can be explained with the stability condition of the force-length relationship which produced a history dependence of force production that is conceptually in agreement with experimental observations.

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin.

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.

A framework for structured modeling of skeletal muscle.

The aim of this study is to present a detailed continuum mechanics formulation, and the corresponding algorithms, to predict the deformation of skeletal muscle at different structural levels, starting from the muscle fiber level. The model is used to investigate force production and structural changes during isometric and dynamic contractions of the cat medial gastrocnemius. From a comparison with experimental data obtained in our own laboratories, we conclude that the model faithfully predicts all of the observations pertaining to force production, fascicle length and angle of pennation under various test conditions.

Plasmacytoma of the urethra treated by excisional biopsy.

Plasmacytoma is an unusual disease of plasma cells, its presentation in the urethra is rare. We report the sixth known case of primary urethral plasmacytoma, which was treated by surgical excision, without recurrence for 3 years of follow-up.

[Self-medication profile in a city of south Brazil]. / Perfil da automedicação em município do Sul do Brasil.

OBJECTIVE: The characterization of drug users, especially as regards self-medication and the determination of its prevalence in the population studied. MATERIAL AND METHOD: About 413 people that had used drugs in the previous month were interviewed. RESULTS: From the interviewed, 69.9% used medicines and of those 76.1% were self medicated. Headache (28.8%) was the main complaint among the self-medicated group. Acetilsalicilic acid was the most frequently used medicine (25.4%). As regards the drugs utilized, 51.2% of the users had received a recommendation from a third party and 51.7% used old prescriptions, given in previous consultations. CONCLUSION: Age, schooling and absence of periodic medical consultation were significant statistical factors in self-medication.