Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification.

Golgi damage caused by dysfunction of PiT-2 in primary familial brain calcification.

The Golgi apparatus is vital for protein modification and molecular trafficking. It is essential for nerve development and activity, and damage thereof is implicated in many neurological diseases. Primary familial brain calcification (PFBC) is a rare inherited neurodegenerative disease characterized by multiple brain calcifications. SLC20A2, which encodes the inorganic phosphate transporter 2 (PiT-2) protein, is the main pathogenic gene in PFBC. The PiT-2 protein is a sodium-dependent phosphate type III transporter, and dysfunction leads to a deficit in the cellular intake of inorganic phosphate (Pi) and calcium deposits. Whether the impaired Golgi apparatus is involved in the PFBC procession requires elucidation. In this study, we constructed induced pluripotent stem cells (iPSCs) derived from two PFBC patients with different SLC20A2 gene mutations (c.613G > A or del exon10) and two healthy volunteers as dependable cell models for research on pathogenic mechanism. To study the mechanism, we differentiated iPSCs into neurons and astrocytes in vitro. Our study found disruptive Golgi structure and damaged autophagy in PFBC neurons with increased activity of mTOR. We also found damaged mitochondria and increased apoptosis in the PFBC dopaminergic neurons and astrocytes. In this study, we prove that dysfunctional PiT-2 leads to an imbalance of cellular Pi, which may disrupt the Golgi apparatus with impaired autophagy, mitochondria and apoptosis in PFBC. Our study provides a new avenue for understanding nerve damage and pathogenic mechanism in brain calcifications.

Role of phosphate transporter PiT-2 in the pathogenesis of primary brain calcification.

Primary brain calcification (PBC), also known as idiopathic basal ganglia calcification (IBGC), primary familial brain calcification (PFBC) and so on, is a rare intractable disease characterized by abnormal mineral deposits, including mostly calcium in the basal ganglia, thalamus, and cerebellum. The causative gene of familial PBC is SLC20A2, which encodes the phosphate transporter PiT-2. Despite this knowledge, the molecular mechanism underlying SLC20A2-associated PBC remains unclear. In the present study, we investigated whether haploinsufficiency or a dominant-negative mechanism reduced Pi uptake in two PiT-2 variants (T115 M and R467X). We demonstrated that the presence of T115 M or R467X had no dominant-negative effect on Pi transport activity of wild-type (WT). In addition, the subcellular localization of R467X completely differed from that of WT, indicating that there is no interaction between R467X and WT. Conversely, T115 M and WT showed almost the same localization. Therefore, we examined the interaction between T115 M and WT using the bioluminescence resonance energy transfer (BRET) method. Although WT and T115 M interact with each other, T115 M does not inhibit WT's Pi transport activity. These results suggest that the role of SLC20A2 in the pathogenesis of PBC may involve decreased intracellular Pi uptake by a haploinsufficiency mechanism rather than a dominant-negative mechanism; agents promoting PiT-2 dimerization may be promising potential therapeutic agents for PBC.

Brain Calcifications: Genetic, Molecular, and Clinical Aspects.

Many conditions can present with accumulation of calcium in the brain and manifest with a variety of neurological symptoms. Brain calcifications can be primary (idiopathic or genetic) or secondary to various pathological conditions (e.g., calcium-phosphate metabolism derangement, autoimmune disorders and infections, among others). A set of causative genes associated with primary familial brain calcification (PFBC) has now been identified, and include genes such as SLC20A2, PDGFB, PDGFRB, XPR1, MYORG, and JAM2. However, many more genes are known to be linked with complex syndromes characterized by brain calcifications and additional neurologic and systemic manifestations. Of note, many of these genes encode for proteins involved in cerebrovascular and blood-brain barrier functions, which both represent key anatomical structures related to these pathological phenomena. As a growing number of genes associated with brain calcifications is identified, pathways involved in these conditions are beginning to be understood. Our comprehensive review of the genetic, molecular, and clinical aspects of brain calcifications offers a framework for clinicians and researchers in the field.

SLC20A2-Associated Idiopathic basal ganglia calcification (Fahr disease): a case family report.

BACKGROUND: Idiopathic basal ganglia calcification (IBGC) is a genetic disorder of the nervous system commonly known as Fahr disease. IBGC patients with a genetic background are considered to have primary familial brain calcification (PFBC), also known as familial basal ganglia calcification (FBGC), or familial Fahr disease. It is a rare degenerative neurological disorder characterized by extensive bilateral basal ganglia calcification that can lead to a range of extrapyramidal symptoms and neuropsychiatric manifestations. Studies have suggested that more than 50 variants of SLC20A2 gene mutations account for approximately 50% of IBGC cases. There is a wide spectrum of mutation types, including frameshift, nonsense, and splice site mutations in addition to deletion and missense mutations. Here we report a case of familial basal ganglia calcification caused by a frameshift mutation in the SLC20A2 gene. We identified a heterozygous mutation in the SLC20A2 gene, c.1097delG (p.G366fs*89). To our knowledge, this mutation site has not been reported before. CASE PRESENTATION: A 57-year-old male patient was admitted to the hospital with "unstable walking and involuntary movements between the eyes and eyebrows for 6 months". Based on the patient's family history, symmetrical calcification foci in the bilateral caudate nucleus head, thalamus, cerebellum and parietal lobe indicated by head CT, and gene test results, the diagnosis of familial Fahr disease caused by mutations in the SLC20A2 gene, c.1097delG p.G366fs*89) was confirmed. CONCLUSION: For the first time, we identified c.1097delG (p.G366fs*89) as a frameshift mutation in the IBGC family. This frameshift mutation caused the condition in this family of patients. This mutation not only broadens the range of known SLC20A2 mutations but also aids in the genetic diagnosis of IBGC.

SLC20A2-related primary familial brain calcification with purely acute psychiatric symptoms: a case report.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare inherited neurological disorder characterized by bilateral basal ganglia calcification with a series of motor and nonmotor symptoms. Mutations in the SLC20A2 gene, encoding the PiT2 protein, are the major cause of the disease. Here, we report a Chinese PFBC family carrying a SLC20A2 gene mutation, and the proband presented with purely acute psychiatric symptoms, which has been rarely reported in this disease. CASE PRESENTATION: A 38-year-old woman was hospitalized due to disorganized speech; disordered thought contents; disorganized behaviour; emotional instability and lability; and grandiose words, actions and facial expressions. Brain computerized tomography (CT) revealed calcification in the basal ganglia; cerebellar dentate nuclei; and subcortical, periventricular, and deep white matter regions in she and her family members. Through mutation analysis, a heterozygous truncating mutation, c.1723G > T, p.(Glu575*), was identified in the SLC20A2 gene in this family. Thus, this patient was diagnosed with genetically confirmed PFBC, and she responded well to a low dose of antipsychotic drugs. The penetrance of the disease in this family was only 33%, which was significantly lower than that in most families carrying SLC20A2 gene mutations. CONCLUSIONS: Patients with SLC20A2-related PFBC might present with psychiatric symptoms alone, and the penetrance of the disease may be quite low, which adds to the clinical heterogeneity of the disease.

Familial idiopathic basal ganglia calcification with a heterozygous missense variant (c.902C>T/p.P307L) in SLC20A2 showing widespread cerebrovascular lesions.

We describe a postmortem case of familial idiopathic basal ganglia calcification (FIBGC) in a 72-year-old Japanese man. The patient showed progressive cognitive impairment with a seven-year clinical course and calcification of the basal ganglia, thalami, and cerebellar dentate nuclei. A novel heterozygous missense variant in SLC20A2 (c.920C>T/p.P307L), a type III sodium-dependent phosphate transporter (PiT-2), was subsequently identified, in addition to typical neuropathological findings of FIBGC, such as capillary calcification of the occipital gray matter, confluent calcification of the basal ganglia and cerebellar white matter, widespread occurrence of vasculopathic changes, cerebrovascular lesions, and vascular smooth muscle cell depletion. Immunohistochemistry for PiT-2 protein revealed no apparent staining in endothelial cells in the basal ganglia and insular cortex; however, the immunoreactivity in endothelial cells of the cerebellum was preserved. Moreover, Western blot analysis identified preserved PiT-2 immunoreactivity signals in the frontal cortex and cerebellum. The variant identified in the present patient could be associated with development of FIBGC and is known to be located at the large intracytoplasmic part of the PiT-2 protein, which has potential phosphorylation sites with importance in the regulation of inorganic phosphate transport activity. The present case is an important example to prove that FIGBC could stem from a missense variant in the large intracytoplasmic loop of the PiT-2 protein. Abnormal clearance of inorganic phosphate in the brain could be related to the development of vascular smooth muscle damage, the formation of cerebrovascular lesions, and subsequent brain calcification in patients with FIBGC with SLC20A2 variants.

Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis.

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

Expanding the genetic spectrum of primary familial brain calcification due to SLC2OA2 mutations: a case series.

Primary familial brain calcification (PFBC) is a neurological condition characterized by the presence of intracranial calcifications, mainly involving basal ganglia, thalamus, and dentate nuclei. So far, six genes have been linked to this condition: SLC20A2, PDGFRB, PDGFB, and XPR1 inherited as autosomal-dominant trait, while MYORG and JAM2 present a recessive pattern of inheritance. Patients mainly present with movement disorders, psychiatric disturbances, and cognitive decline or are completely asymptomatic and calcifications may represent an occasional finding. Here we present three variants in SLC20A2, two exonic and one intronic, which we found in patients with PFBC associated to three different clinical phenotypes. One variant is novel and two were already described as variants of uncertain significance. We confirm the pathogenicity of these three variants and suggest a broadening of the phenotypic spectrum associated with mutations in SLC20A2.

Genotype-Phenotype Relations in Primary Familial Brain Calcification: Systematic MDSGene Review.

This systematic MDSGene review covers individuals with confirmed genetic forms of primary familial brain calcification (PFBC) available in the literature. Data on 516 (47% men) individuals, carrying heterozygous variants in SLC20A2 (solute carrier family 20 member 2, 61%), PDGFB (platelet-derived growth factor subunit B, 12%), XPR1 (xenotropic and polytropic retrovirus receptor, 16%), or PDGFRB (platelet-derived growth factor receptor beta, 5%) or biallelic variants in MYORG (myogenesis-regulating glycosidase, 13%) or JAM2 (junctional adhesion molecule 2, 2%), were extracted from 93 articles. Nearly one-third of the mutation carriers were clinically unaffected. Carriers of PDGFRB variants were more likely to be clinically unaffected (~54%), and the penetrance of SLC20A2 and XPR1 variants (<70%) was lower in comparison to the remaining three genes (>85%). Among the 349 clinically affected patients, 27% showed only motor and 31% only nonmotor symptoms/signs, whereas the remaining 42% had a combination thereof. While parkinsonism and speech disturbance were the most frequently reported motor manifestations, cognitive deficits, headache, and depression were the major nonmotor symptoms/signs. The basal ganglia were always calcified, and the cerebellum, thalamus, and white matter contained calcifications in 58%, 53%, and 43%, respectively, of individuals. In autosomal-dominant PFBC, mutation severity influenced the number of calcified brain areas, which in turn correlated with the clinical status, whereby the risk of developing symptoms/signs more than doubled for each additional region with calcifications. Our systematic analysis provides the most comprehensive insight into genetic, clinical, and neuroimaging features of known PFBC forms, to date. In addition, it puts forth the penetrance estimates and newly discovered genotype-phenotype relations that will improve counseling of individuals with mutations in PFBC genes. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element.

OBJECTIVE: Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes. METHODS: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic phosphate uptake assay, and introduced the deletion of a putative SLC20A2 enhancer mapping to this region in human embryonic kidney 293 (HEK293) cells by clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9). RESULTS: The 8p11.21 deletion, segregating with PFBC in a family, mapped 35 kb upstream of SLC20A2. The deletion carriers/normal controls ratio of relative SLC20A2 mRNA levels was 60.2% (P < 0.001). This was comparable with that of patients carrying an SLC20A2 premature stop codon (63.4%; P < 0.001). The proband exhibited a 39.3% decrease of inorganic phosphate uptake in blood (P = 0.015). In HEK293 cells, we observed a 39.8% decrease in relative SLC20A2 mRNA levels after normalization on DNA copy number (P < 0.001). DISCUSSION: We identified a deletion of an enhancer of SLC20A2 expression, with carriers showing haploinsufficiency in similar ranges to loss-of-function alleles, and we observed reduced mRNA levels after deleting this element in a cellular model. We propose a 3-step strategy to identify and easily assess the effect of such events. © 2020 International Parkinson and Movement Disorder Society.

[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

Spectrum of SLC20A2, PDGFRB, PDGFB, and XPR1 mutations in a large cohort of patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.

SLC20A2 variants cause dysfunctional phosphate transport activity in endothelial cells induced from Idiopathic Basal Ganglia Calcification patients-derived iPSCs.

Idiopathic Basal Ganglia Calcification (IBGC) is a rare neuropsychiatric illness also known as Fahr's disease or Primary Familial Brain Calcification (PFBC). IBGC is caused by SLC20A2 variants, which encodes the inorganic phosphate (Pi) transporter PiT-2, a transmembrane protein associated with Pi homeostasis. We have reported novel SLC20A2 variants in the Japanese population and established an induced pluripotent stem cells (iPSCs) from an IBGC patient carrying a SLC20A2 variant. To investigate the effect of these SLC20A2 variants identified in our previous study, we used Chinese hamster ovary (CHO) cells expressing these variant proteins using the Flp-In system (Flp-In CHO cells), and showed that variant SLC20A2 proteins significantly disrupted the Pi transport activity in Flp-In CHO cells. Endothelial cells (ECs) represent important target cells for elucidating the pathology of IBGC. Using patient-derived iPSCs in this study, we differentiated these cells into ECs and found no significant difference in their differentiation capacity into ECs compared with control iPSCs. However, the Pi transport activity of IBGC patient-derived iPS-ECs was significantly decreased compared with that of control iPS-ECs without changing the gene expression of the other SLC 20 family members. We confirmed that SLC20A2 variants caused the loss of function of the Pi transport activity in both Flp-In CHO cells and disease-specific iPSCs. This is the first report to show an in vitro model of iPSCs in IBGC with patient-identified SLC20A2 variants. These useful tools will help in elucidating IBGC pathogenesis and can be used for screening drug candidates.

Identification of SLC20A2 deletions in patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurological disorder. Mutations in five genes (SLC20A2, PDGFRB, PDGFB, XPR1, and MYORG) have been linked to PFBC. Here, we used SYBR green-based real-time quantitative polymerase chain reaction (PCR) assay and denaturing high-performance liquid chromatography analysis to detect copy number variants (CNVs) in 20 unrelated patients with PFBC, negatively sequenced for the five known genes. We identified three deletions in SLC20A2, including a large de novo full gene deletion and two exonic deletions confined to exon 2 and exon 6, respectively. Subsequent linked-read whole-genome sequencing of the patient with the large deletion showed a 1.7 Mb heterozygous deletion which removed the entire coding regions of SLC20A2 as well as 21 other genes. In the family with a deletion of exon 6, a missense variant of uncertain significance (SLC20A2: p.E267Q) also co-segregated with the disease. Functional assay showed the deletion could result in significantly impaired phosphate transport, whereas the p.E267Q variant did not. Our results confirm that deletion in SLC20A2 is a causal mechanism for PFBC and highlight the importance of functional study for classifying a rare missense variant as (likely) pathogenic.

Pericytes in Primary Familial Brain Calcification.

Pericytes are perivascular cells along capillaries that are critical for the development of a functional vascular bed in the central nervous system and other organs. Pericyte functions in the adult brain are less well understood. Pericytes have been suggested to mediate functional hyperemia at the capillary level, regulate the blood-brain barrier and to give rise to scar tissue after spinal cord injury. Furthermore, pericyte loss has been suggested to precede cognitive decline in mouse models of Alzheimer's disease. Despite this observation, there is no convincing causality between pericyte loss and the pathogenesis of Alzheimer's disease. However, recent loss-of-function mutations in PDGFB and PDGFRB genes have implicated pericytes as the principle cell type affected in primary familiar brain calcification (PFBC), a neuropsychiatric disorder with dominant inheritance. Here we review the role of the PDGFB/PDGFRB signaling pathway in pericyte development and briefly discuss homeostatic functions of pericytes in the brain. We provide an overview of recent studies with mouse models of PFBC and discuss suggested pathogenic mechanisms for PFBC with special reference to pericytes.

Primary familial brain calcification with a novel SLC20A2 mutation: Analysis of PiT-2 expression and localization.

Primary familial brain calcification (PFBC) is an autosomal dominant rare disorder characterized by bilateral and symmetric brain calcifications, and neuropsychiatric manifestations. Four genes have been linked to PFBC: SLC20A2, PDGFRB, PDGFB, and XPR1. In this study, we report molecular and clinical data of a PFBC patient carrying a novel SLC20A2 mutation and we investigate the impact of the mutation on PiT-2 expression and function. Sanger sequencing of SLC20A2, PDGFRB, PDGFB, XPR1 led to the identification of a novel duplication of twelve nucleotides (c.1876_1887dup/ p.Trp626_Thr629dup) in SLC20A2 gene. SLC20A2 encodes for a cell membrane transporter (PiT-2) involved in maintenance of inorganic phosphate homeostasis. We performed an analysis of expression and functionality of PiT-2 protein in patient primary cultured fibroblasts. In patient fibroblasts, the mutation does not affect PiT-2 expression but alter sub-cellular localization. The Pi-uptake assay revealed a less Pi depletion in patient than in control fibroblasts, suggesting that SLC20A2 duplication may impair Pi internalization. This is the first study reporting sub-cellular expression analysis of mutant PiT-2 in primary cultured fibroblasts from a PFBC patient, showing that p.Trp626_Thr629dup in SLC20A2 alters PiT-2 sub-cellular localization and reduces Pi-uptake, leading to onset of PFBC in our patient.

SCL20A2 mutation presenting with acute ischemic stroke: a case report.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare disorder characterized by distinctive bilateral brain calcification and variable clinical presentations. However, cerebrovascular attack was rarely reported in PFBC patients. We here reported a SLC20A2 mutation patient presenting with acute ischemic stroke. CASE PRESENTATION: A 56 years old man was transferred to our hospital because of 6 days of melena and 3 days of somnolence, agitation and mood changes. Computed tomography (CT) scan showed symmetrical calcifications in bilateral basal ganglia, caudate nucleus, thalami, subcortical white matter and cerebellum, which is consistent with PFBC. Brain magnetic resonance imaging (MRI) revealed acute ischemic stroke in bilateral basal ganglia and periventricular regions. Mutational analysis identified a SLC20A2 gene mutation c.344C > T (p.Thr115Met) in exon 3. One of his daughters had also suffered from brain calcification. MR perfusion imaging revealed hypoperfusion in bilateral basal ganglia, prefrontal and temporal lobe. After treatment, he discharged with a favorable functional outcome but cognitive impairment. CONCLUSIONS: Ischemic stroke can occur in PFBC patients, which may be associated with hypoperfusion and calcification of arteries. And hypoperfusion in frontotemporal lobar may be related with their cognitive impairment.

Anticipation in a family with primary familial brain calcification caused by an SLC20A2 variant.

AIM OF THE STUDY: To describe a family with primary familial brain calcification (PFBC) due to SLC20A2 variant showing possible genetic anticipation. MATERIALS AND METHODS: We conducted historical, genealogical, clinical, and radiologic studies of a family with PFBC. Clinical evaluations including neurological examination and head computed tomography (CT) scans of a proband and her father were performed. They provided additional information regarding other family members. To identify a causative gene variant, we performed whole-exome sequencing for the proband followed by segregation analysis in other affected members using direct sequencing. RESULTS: In this family, nine affected members were identified over four generations. The proband suffered from chronic daily headache including thunderclap headache. We identified an SLC20A2 (c.509delT, p.(Leu170*)) variant in three affected members over three generations. Interestingly, the age of onset became younger as the disease passed through successive generations, suggestive of genetic anticipation. CONCLUSIONS AND CLINICAL IMPLICATIONS: For clinical purpose, it is important to consider thunderclap headache and genetic anticipation in PFBC caused by SLC20A2 variants. Further investigation is required to validate our observation.

Estimation of minimal disease prevalence from population genomic data: Application to primary familial brain calcification.

Primary Familial Brain Calcification (PFBC) is a rare calcifying disorder of the brain with autosomal dominant inheritance, of unknown prevalence. Four causal genes have been identified so far: SLC20A2, PDGFB, PDGFRB, and XPR1, with pathogenic, probably pathogenic or missense variants of unknown significance found in 27.7% probands in the French PFBC series. Estimating PFBC prevalence from a clinical input is arduous due to a large diversity of symptoms and ages of onset and to incomplete clinical penetrance. Abnormal calcifications on CT scan can be used as a reliable diagnostic biomarker whatever the clinical status, but differential diagnoses should be ruled out including the challenging exclusion of common basal ganglia calcifications. Our primary aim was to estimate the minimal prevalence of PFBC due to a variant in one of the known genes. We extracted variants from the four known genes present in the gnomAD database gathering genomic data from 138,632 individuals. We interpreted all variants based on their predicted effect, their frequency, and previous studies on PFBC patients. Using the most conservative estimate, the minimal prevalence of PFBC related to a variant in one of the four known genes was 4.5 p. 10,000 (95%CI [3.4-5.5] p. 10,000). We then used variant detection rates in patients to extrapolate an overall minimal prevalence of PFBC to 2.1 p. 1,000 (95%CI [1.9-2.4] p. 1,000). The population-based genomic analysis indicates that PFBC is not an exceptionally rare disorder, still underestimated and underdiagnosed.