Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

Expansion of the Primrose syndrome phenotype through the comparative analysis of two new case reports with ZBTB20 variants.

Primrose syndrome (PRIMS), a rare genetic disorder with several clinical findings including intellectual disability, macrocephaly, typical facial features, and muscle wasting, is caused by heterozygous variants in the ZBTB20 gene. We report the cases of two males diagnosed with PRIMS at different ages, emphasizing the likely progressive nature of the disorder, as well as the differences and similarities of presentation during infancy and adulthood. Patient 1 is a 2-year-old American male with a medical history marked by impaired hearing, developmental delays, and fainting spells. Patient 2 is a 28-year-old Brazilian male, who presents with a phenotype similar to that seen in Patient 1 with additional features of ectopic calcifications and prominent muscular and skeletal abnormalities. Additionally, Patient 2 has a history of fainting spells and diminished body height and weight, with the latter features having only been reported in one PRIMS patient so far. Both Patients 1 and 2 were found to carry heterozygous likely pathogenic missense variants, detected in the last coding exon of ZBTB20 (c.1822T>C, p.Cys608Arg, de novo, and c.1873A>G, p.Met625Val, respectively), consistent with PRIMS. Overall, these case reports highlight PRIMS's likely progressive nature and contribute to the understanding of the natural history of this condition.

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.

Mutations in SLC20A2 are a major cause of familial idiopathic basal ganglia calcification.

Familial idiopathic basal ganglia calcification (IBGC) or Fahr's disease is a rare neurodegenerative disorder characterized by calcium deposits in the basal ganglia and other brain regions, which is associated with neuropsychiatric and motor symptoms. Familial IBGC is genetically heterogeneous and typically transmitted in an autosomal dominant fashion. We performed a mutational analysis of SLC20A2, the first gene found to cause IBGC, to assess its genetic contribution to familial IBGC. We recruited 218 subjects from 29 IBGC-affected families of varied ancestry and collected medical history, neurological exam, and head CT scans to characterize each patient's disease status. We screened our patient cohort for mutations in SLC20A2. Twelve novel (nonsense, deletions, missense, and splice site) potentially pathogenic variants, one synonymous variant, and one previously reported mutation were identified in 13 families. Variants predicted to be deleterious cosegregated with disease in five families. Three families showed nonsegregation with clinical disease of such variants, but retrospective review of clinical and neuroimaging data strongly suggested previous misclassification. Overall, mutations in SLC20A2 account for as many as 41% of our familial IBGC cases. Our screen in a large series expands the catalog of SLC20A2 mutations identified to date and demonstrates that mutations in SLC20A2 are a major cause of familial IBGC. Non-perfect segregation patterns of predicted deleterious variants highlight the challenges of phenotypic assessment in this condition with highly variable clinical presentation.

COVID-19 Unveiling Brain Calcifications.

Neurological symptoms in COVID-19 patients have attracted the interest of the scientific community, yet their mechanisms remain unknown. In some circumstances, the presence of neurological manifestations may result in an incidental diagnosis after a detailed investigation. In the present letter, we discuss a case published by Demir et al., in which the diagnosis of COVID-19 enabled the diagnosis of a rare neurological disorder, characterized by bilateral brain calcifications, commonly known by the eponym Fahr's syndrome. In addition, we report a case of primary brain calcifications unveiled by a suspected coronavirus infection.

New Data from Pdfgb ret/ret Mutant Mice Might Lead to a Paradoxical Association Between Brain Calcification, Pericytes Recruitment and BBB Integrity.

Data of mice with PDGF-B-truncating mutation (Pdgfb ret/ret) from different research groups indicate that the malfunction of this protein leads to reduced pericyte recruitment, loss of Blood-Brain Barrier (BBB) integrity and bilateral brain calcification. This makes these mice important models for Primary Brain Calcification and pericyte-BBB correlation studies. The global brain pericyte count is reduced in Pdgfb ret/ret mice, with higher BBB permeability. We have overlapped the data from other research groups into a figure to further analyze the findings. Calcifications form within midbrain, interbrain, basal forebrain, and pons. Interestingly, these calcification-prone regions have a comparably higher pericyte count and lower BBB leakage in relation to other non-calcifying regions of the Pdgfb ret/ret mouse (such as the cortex and striatum). A comparatively higher BBB integrity in regions prone to calcification seems paradoxical and indicates that other region-specific changes are the cause of the calcifications.

Brain calcifications and PCDH12 variants.

OBJECTIVE: To assess the potential connection between PCDH12 and brain calcifications in a patient carrying a homozygous nonsense variant in PCDH12 and in adult patients with brain calcifications. METHODS: We performed a CT scan in 1 child with a homozygous PCDH12 nonsense variant. We screened DNA samples from 53 patients with primary familial brain calcification (PFBC) and 26 patients with brain calcification of unknown cause (BCUC). RESULTS: We identified brain calcifications in subcortical and perithalamic regions in the patient with a homozygous PCDH12 nonsense variant. The calcification pattern was different from what has been observed in PFBC and more similar to what is described in in utero infections. In patients with PFBC or BCUC, we found no protein-truncating variant and 3 rare (minor allele frequency <0.001) PCDH12 predicted damaging missense heterozygous variants in 3 unrelated patients, albeit with no segregation data available. CONCLUSIONS: Brain calcifications should be added to the phenotypic spectrum associated with PCDH12 biallelic loss of function, in the context of severe cerebral developmental abnormalities. A putative role for PCDH12 variants remains to be determined in PFBC.

Monoamine oxidase a polymorphism in Brazilian patients: risk factor for late-onset Alzheimer's disease?

Different studies have attempted to find polymorphisms involved in the serotonergic pathway that could be involved in mood disorders and late-onset Alzheimer's disease (LOAD) symptoms. Here, we compared the frequency of two polymorphisms: monoamine oxidase A (MAOA) and serotonin transporter in LOAD patients versus controls. No evidence of association was observed when these polymorphisms were compared separately; however, the combination of the MAOA allele 1+the short allele of 5-HTTLPR+ApoE-epsilon4 was significantly more frequent in patients than in controls. It reinforces the hypothesis that different genes acting together might play a role in AD susceptibility. Based on these data, we suggest replicating these studies in larger samples of LOAD patients belonging to different ethnic groups.

Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export.

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.

Analysis of IL-1alpha, IL-1beta, and IL-1RA [correction of IL-RA] polymorphisms in dysthymia.

Investigators of independent studies reported alterations in cytokine serum levels in patients with different mood disorders. Several polymorphisms associated with neuropsychiatric disorders such as schizophrenia and Alzheimer's disease have been reported at the interleukin-1 (IL-1) panel. Here we report the results of three specific polymorphisms at the IL-1alpha, IL-1beta, and IL-1RA genes, which were analyzed in 128 Brazilian subjects: 59 dysthymic patients and 69 normal controls. We found a statistically significant difference (p = 0.002) in the frequency of haplotypes with alleles 2+ (IL-1RA), T+ (IL-1alpha), and C+ (IL-1beta) in patients as compared to controls. We also observed that haplotype IL-1RA1.2/IL-1alpha CT/IL-1beta CC, present in 6 dysthymic patients (10%) was absent in the normal control group (p = 0.012). These results suggest that these polymorphisms might confer a greater susceptibility to develop dysthymia in Brazilian patients. However, to validate these data it will be of great interest to repeat this study in larger samples and other ethnic groups.

Lack of association between the brain-derived neurotrophin factor (C-270T) polymorphism and late-onset Alzheimer's disease (LOAD) in Brazilian patients.

After the identification of the apolipoprotein E gene isoform (APOE-epsilon4) as a risk factor for late-onset Alzheimer's disease (LOAD), the search for other polymorphisms associated with AD has been undertaken by many groups of investigators around the world. These studies have shown controversial results in many populations. More recently, a single nucleotide polymorphism in the promoter region of the brain-derived neurotrophin factor (BDNF) was found to be a risk factor for AD in two independent population studies. Here we report the analysis of this polymorphism in a group of 188 LOAD Brazilian patients compared to matched normal controls. A strong association between the APOE-epsilon4 polymorphism and LOAD was observed, but there was no significant association between this BNDF polymorphism and affected patients. The possibility that other polymorphisms or mutations in this gene play a role in the development of AD cannot be ruled out. However, the results of the present study suggest that in opposition to the two reported studies, this polymorphism does not seem to be implicated in LOAD Brazilian patients. It also shows the importance of replication studies in different populations, as susceptibility loci might differ in different ethnic groups; this will have important implications in future treatments with pharmacological agents.

Psychiatric symptoms are present in most of the patients with idiopathic normal pressure hydrocephalus.

Normal pressure hydrocephalus (NPH) is characterized by gait disturbance, dementia and/or urinary incontinence associated with dilation of ventricular system with normal opening cerebrospinal fluid pressure. Wide scientifical evidence confirms association between NPH and psychiatric symptoms. We selected 35 patients with idiopathic normal pressure hydrocephalus from January 2010 to January 2012 in a Brazilian tertiary hospital and performed a formal psychiatric evaluation to identify psychiatric disorders. Psychiatric disorders were present in 71% of these patients, especially anxiety, depression and psychotic syndromes. NPH patients may develop symptoms with frontal dominance, such as personality changes, anxiety, depression, psychotic syndromes, obsessive compulsive disorder, Othello syndrome; shoplifting and mania. Unusual appearances of NPH symptoms may hinder early diagnosis and consequently proper treatment.

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.

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 ß (PDGF-Rß) 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-Rß. 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.

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.

Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis.

Familial idiopathic basal ganglia calcification (IBGC) is a genetic condition with a wide spectrum of neuropsychiatric symptoms, including parkinsonism and dementia. Here, we identified mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), in IBGC-affected families of varied ancestry, and we observed significantly impaired phosphate transport activity for all assayed PiT2 mutants in Xenopus laevis oocytes. Our results implicate altered phosphate homeostasis in the etiology of IBGC.

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.