R. R. Lemos

Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice

Calcifications in the basal ganglia are a common incidental finding and are sometimes inherited as an autosomal dominant trait (idiopathic basal ganglia calcification (IBGC)). Recently, mutations in the PDGFRB gene coding for the platelet-derived growth factor receptor β (PDGF-Rβ) were linked to IBGC. Here we identify six families of different ancestry with nonsense and missense mutations in the gene encoding PDGF-B, the main ligand for PDGF-Rβ. We also show that mice carrying hypomorphic Pdgfb alleles develop brain calcifications that show age-related expansion. The occurrence of these calcium depositions depends on the loss of endothelial PDGF-B and correlates with the degree of pericyte and blood-brain barrier deficiency. Thus, our data present a clear link between Pdgfb mutations and brain calcifications in mice, as well as between PDGFB mutations and IBGC in humans.

Update and Mutational Analysis of SLC20A2: A Major Cause of Primary Familial Brain Calcification

Primary familial brain calcification (PFBC) is a heterogeneous neuropsychiatric disorder, with affected individuals presenting a wide variety of motor and cognitive impairments, such as migraine, parkinsonism, psychosis, dementia, and mood swings. Calcifications are usually symmetrical, bilateral, and found predominantly in the basal ganglia, thalamus, and cerebellum. So far, variants in three genes have been linked to PFBC: SLC20A2, PDGFRB, and PDGFB. Variants in SLC20A2 are responsible for most cases identified so far and, therefore, the present review is a comprehensive worldwide summary of all reported variants to date. SLC20A2 encodes an inorganic phosphate transporter, PiT‐2, widely expressed in various tissues, including brain, and is part of a major family of solute carrier membrane transporters. Fifty variants reported in 55 unrelated patients so far have been identified in families of diverse ethnicities and only few are recurrent. Various types of variants were detected (missense, nonsense, frameshift) including full or partial SLC20A2 deletions. The recently reported SLC20A2 knockout mouse will enhance our understanding of disease mechanism and allow for screening of therapeutic compounds. In the present review, we also discuss the implications of these recent exciting findings and consider the possibility of treatments based on manipulation of inorganic phosphate homeostasis.

Brain calcification process and phenotypes according to age and sex: Lessons from SLC20A2, PDGFB, and PDGFRB mutation carriers

Primary Familial Brain Calcification (PFBC) is a dominantly inherited cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Three causative genes have been identified: SLC20A2, PDGFRB and, recently, PDGFB, whose associated phenotype has not yet been extensively studied. We included in the largest published case series of genetically confirmed PFBC, 19 PDGFB (including three new mutations), 24 SLC20A2 (including 4 new mutations), and 14 PDGFRB mutation carriers, from two countries (France and Brazil). We studied clinical features and applied our visual rating scale on all 49 available CT scans. Among the symptomatic mutation carriers (33/57, 58%), the three most frequently observed categories of clinical features were psychiatric signs (72.7%, 76.5%, and 80% for PDGFB, SLC20A2, and PDGFRB, respectively), movement disorders (45.5%, 76.5%, and 40%), and cognitive impairment (54.6%, 64.7%, and 40%). The median age of clinical onset was 31 years, 25% had an early onset (before 18) and 25% a later onset (after 53). Patients with an early clinical onset exhibited mostly isolated psychiatric or cognitive signs, while patients with a later onset exhibited mostly movement disorders, especially in association with other clinical features. CT scans rating allowed identifying four patterns of calcification. The total calcification score was best predicted by the combined effects of gene (SLC20A2 > PDGFB > PDGFRB mutations), sex (male), and (increasing) age, defining three risk classes, which correlated with the four patterns of calcification. These calcification patterns could reflect the natural history of the calcifying process, with distinct risk classes characterized by different age at onset or rate of progression.

Reporting a new mutation at the SLC20A2 gene in familial idiopathic basal ganglia calcification.

Mutations at the SLC20A2 gene, at chromosome 8, were recently linked to familial idiopathic basal ganglia calcification (IBGC) in families from China, Spain and Brazil [1]. Due to the increasing use of neuroimaging procedures, brain calcifications are visualized more often than ever before. Patients burdening these calcifications, mainly in basal ganglia but occasionally also in the cerebellum and white matter, present a wide variety of symptoms, such as parkinsonism, headaches, psychosis, dementia and mood symptoms [2,3]. We have collected clinical history and neuroimages from two available subjects from a Brazilian family with idiopathic brain calcinosis and Caucasian background. The rest of the family was not available for additional analysis. These results are part of a study approved by the ethics committee from the Federal University of Pernambuco (CAEE-0296.0172.000-08). Figure 1 shows the pedigree chart, an eletropherogram displaying the missense mutation and 3D models of brain calcifications in each affected, using 3D-Doctor software (Able Software, Lexington, MA, USA). The clinical manifestation of I-2, an 84-year-old woman, is mild depression and parkinsonism of late manifestation, despite the presence of large calcifications (10.85 cm) in basal ganglia and cerebellum. The II-1 subject presents calcifications in basal ganglia, pineal and plexus choroid (20.73 cm). The rest of the family was not available for a thorough analysis. II-1 is a 43-year-old man, one of seven children from I-2, with rapid progression of parkinsonism features in the late third decade of life. Until then, he was a fully functional person with regular employment. In the last 5 years, a progressive presentation of general bradykinesia, rigidity and paresis in the right arm has developed. Recently, cognitive and speech disorders were added, characterized by dysarthria, mild cognitive impairment and moderate depressive state. No other cause was disclosed, such as stroke, demyelinating conditions, autoimmune processes or metabolic disorder. During the screening of mutations at the SLC20A2 gene, using automated sequencing in a MegaBACE 1000 (Sunnyvale, CA, USA), we identified a new missense mutation in NM_006749.4 sequence at the exon 8, position c. 2140G > A (A495T), predicted to be pathogenic by SIFT, I-Mutant2.0 and Polyphen analysis. This is a novel mutation with no previous report at the 1000 Genomes Project databases, at the ESP6500 data set from the National Heart, Lung and Blood Institute (consisting of 4300 exomes from European–Americans and 2203 from African–American individuals) or in other four Brazilian families excluded during the same screening. This finding reinforces the relevance of the SLC20A2 gene to the etiopathogeny of IBGC, and points to exon 8 as a region, so far, with more mutations reported. Despite the limited number of subjects studied, more severe symptoms and larger calcifications in subject II-1 favor the hypothesis of genetic anticipation, which was previously reported in IBGC and other neuropsychiatric conditions with autosomal dominat pattern of inheritance [4]. Ongoing analysis of genome-wide scans in additional families showed new candidate regions at chromosomes 2, 7, 9 and 14, indicating genetic heterogeneity. The study of new families with this condition will be crucial to identify other genes and to reinforce the relevance of the SLC20A2 gene to the etiopathogeny of IBGC [5–7]. URLs1000 Genomes Project, http:// www.1000genomes.org/; NHLBI Exome

An update on primary familial brain calcification.

Patients with primary familial brain calcifications (PFBC) present bilateral calcifications, often affecting basal ganglia, thalamus, and cerebellum, inherited in an autosomal dominant pattern of segregation. Affected individuals display a wide variety of motor and cognitive impairments such as parkinsonism, dystonia, migraine, dementia, psychosis, and mood symptoms. Worldwide growth in the availability of neuroimaging procedures, combined with careful screening of patients and their relatives, has increased detection of PFBC. Recently, mutations in the SLC20A2 gene coding for the inorganic phosphate transporter PiT2 were linked to PFBC, thereby implicating impaired phosphate transport as an underlying disease mechanism. To date, around 20 families of various ethnicities carry different mutations in SLC20A2 correlate with ~40% of PFBC cases. More recently, two French families were recently reported with mutations in PDGFRB: c.1973T>C, p.L658P and c.2959C>T, p.R987W, a class III tyrosine kinase receptor. Six other families were found with mutations in PDGFB, and, in general, mutations at the PDGF pathway add a new dimension to the physiopathology of PFBC so far explained by a disturbance in phosphate homeostasis with SLC20A2. The identification of SLC20A2, PDGFRB, and PDGFB provides a new avenue for potential treatments based on compounds such as bisphosphonates and those modulating the PDGFB pathway.

Population and Computational Analysis of the MGEA6 P521A Variation as a Risk Factor for Familial Idiopathic Basal Ganglia Calcification (Fahr’s Disease)

Familial idiopathic basal ganglia calcification, also known as “Fahr’s disease” (FD), is a neuropsychiatric disorder with autosomal dominant pattern of inheritance and characterized by symmetric basal ganglia calcifications and, occasionally, other brain regions. Currently, there are three loci linked to this devastating disease. The first one (IBGC1) is located in 14q11.2-21.3 and the other two have been identified in 2q37 (IBGC2) and 8p21.1-q11.13 (IBGC3). Further studies identified a heterozygous variation (rs36060072) which consists in the change of the cytosine to guanine located at MGEA6/CTAGE5 gene, present in all of the affected large American family linked to IBGC1. This missense substitution, which induces changes of a proline to alanine at the 521 position (P521A), in a proline-rich and highly conserved protein domain was considered a rare variation, with a minor allele frequency (MAF) of 0.0058 at the US population. Considering that the population frequency of a given variation is an indirect indicative of potential pathogenicity, we screened 200 chromosomes in a random control set of Brazilian samples and in two nuclear families, comparing with our previous analysis in a US population. In addition, we accomplished analyses through bioinformatics programs to predict the pathogenicity of such variation. Our genetic screen found no P521A carriers. Polling these data together with the previous study in the USA, we have now a MAF of 0.0036, showing that this mutation is very rare. On the other hand, the bioinformatics analysis provided conflicting findings. There are currently various candidate genes and loci that could be involved with the underlying molecular basis of FD etiology, and other groups suggested the possible role played by genes in 2q37, related to calcium metabolism, and at chromosome 8 (NRG1 and SNTG1). Additional mutagenesis and in vivo studies are necessary to confirm the pathogenicity for variation in the P521A MGEA6.

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.

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.

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.

Exaggerated blood pressure response during exercise treadmill testing: functional and hemodynamic features, and risk factors

The factors which contribute to an exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) are not wholly understood. The association between the insertion/deletion polymorphisms of the angiotensin-converting enzyme (ACE) and M235T of the angiotensinogen with EBPR during ETT still remains unstudied. To identify and compare the risk factors for hypertension between normotensive subjects with EBPR and those who exhibit a normal curve of blood pressure (BP) during ETT. In a series of EBPR cases from a historical cohort of normotensive individuals, a univariate analysis was performed to estimate the association of the studied factors with BP behavior during ETT. Additionally, logistic multivariate regression was conducted to analyze the joint effects of the variables. P-values above 0.05 were considered statistically significant. From a total of 10 027 analyzed examinations, only 219 met the criteria employed to define EBPR, which resulted in a prevalence of 12.6%. For the systolic component of the BP, hyperreactive subjects displayed a mean age and body mass index (BMI) significantly higher than the others (P=0.002 and <0.001, respectively). No association was observed between the polymorphisms cited above and EBPR. An analysis of the joint effect of variables has indicated that only age (P< 0.001) and BMI (P=0.001) were specifically associated with systolic BP during exercise. Age and BMI were the only factors that independently influenced EBPR during ETT.