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.

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.

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.

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.