Genetic and pathophysiological insights from autopsied patient with primary familial brain calcification: novel MYORG variants and astrocytic implications.

Primary familial brain calcification (PFBC) is a genetic neurological disorder characterized by symmetric brain calcifications that manifest with variable neurological symptoms. This study aimed to explore the genetic basis of PFBC and elucidate the underlying pathophysiological mechanisms. Six patients from four pedigrees with brain calcification were enrolled. Whole-exome sequencing identified two novel homozygous variants, c.488G > T (p.W163L) and c.2135G > A (p.W712*), within the myogenesis regulating glycosidase (MYORG) gene. Cerebellar ataxia (n = 5) and pyramidal signs (n = 4) were predominant symptoms, with significant clinical heterogeneity noted even within the same family. An autopsy of one patient revealed extensive brainstem calcifications, sparing the cerebral cortex, and marked by calcifications predominantly in capillaries and arterioles. The pathological study suggested morphological alterations characterized by shortened foot processes within astrocytes in regions with pronounced calcification and decreased immunoreactivity of AQP4. The morphology of astrocytes in regions without calcification remains preserved. Neuronal loss and gliosis were observed in the basal ganglia, thalamus, brainstem, cerebellum, and dentate nucleus. Notably, olivary hypertrophy, a previously undescribed feature in MYORG-PFBC, was discovered. Neuroimaging showed reduced blood flow in the cerebellum, highlighting the extent of cerebellar involvement. Among perivascular cells constituting the blood-brain barrier (BBB) and neurovascular unit, MYORG is most highly expressed in astrocytes. Astrocytes are integral components of the BBB, and their dysfunction can precipitate BBB disruption, potentially leading to brain calcification and subsequent neuronal loss. This study presents two novel homozygous variants in the MYORG gene and highlights the pivotal role of astrocytes in the development of brain calcifications, providing insights into the pathophysiological mechanisms underlying PFBC associated with MYORG variants.

A novel splicing mutation DNAH5 c.13,338 + 5G > C is involved in the pathogenesis of primary ciliary dyskinesia in a family with primary familial brain calcification.

BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76. CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.

Novel findings in a Swedish primary familial brain calcification cohort.

INTRODUCTION: Brain calcifications are frequent findings on imaging. In a small proportion of cases, these calcifications are associated with pathogenic gene variants, hence termed primary familial brain calcification (PFBC). The clinical penetrance is incomplete and phenotypic variability is substantial. This paper aims to characterize a Swedish PFBC cohort including 25 patients: 20 from seven families and five sporadic cases. METHODS: Longitudinal clinical assessment and CT imaging were conducted, abnormalities were assessed using the total calcification score (TCS). Genetic analyses, including a panel of six known PFBC genes, were performed in all index and sporadic cases. Additionally, three patients carrying a novel pathogenic copy number variant in SLC20A2 had their cerebrospinal fluid phosphate (CSF-Pi) levels measured. RESULTS: Among the 25 patients, the majority (76%) displayed varying symptoms during the initial assessment including motor (60%), psychiatric (40%), and/or cognitive abnormalities (24%). Clinical progression was observed in most patients (78.6%), but there was no significant difference in calcification between the first and second scans, with mean scores of 27.3 and 32.8, respectively. In three families and two sporadic cases, pathogenic genetic variants were identified, including a novel finding, in the SLC20A2 gene. In the three tested patients, the CSF-Pi levels were normal. CONCLUSIONS: This report demonstrates the variable expressivity seen in PFBC and includes a novel pathogenic variant in the SLC20A2 gene. In four families and three sporadic cases, no pathogenic variants were found, suggesting that new PFBC genes remain to be discovered.

JAM2 variants can be more common in primary familial brain calcification (PFBC) cases than those appear; may be due to a founder mutation.

INTRODUCTION: Mutations in JAM2 have been linked to ~ 2% of primary familial brain calcification (PFBC) cases. PFBC is a rare neurological disorder characterized by excessive calcium deposition in the brain. It causes movement disorders and psychiatric problems. Six other genes were identified as causing PFBC. However, the genetic basis of ~ 50% of PFBC cases remains unknown. This study presented the results of a comprehensive analysis of five unrelated Iranian PFBC families. METHODS: Clinical and paraclinical features of all patients were recorded. Whole-exome sequencing (WES) was done on the DNAs of probands. Data was analyzed, and haplotypes were determined. RESULTS: WES identified two homozygous variants in JAM2 across four families: a novel variant, c.426dup:p.Ser143Leufs*23, in one family and a known mutation, c.685C > T:p.Arg229*, in the remaining three families. Haplotype analysis using six intragenic single-nucleotide polymorphisms (SNPs) in JAM2 revealed an identical haplotype in probands who carried the same mutation, whereas two other probands presented diverse haplotypes. CONCLUSION: Based on our results, p.Arg229* may be a founder mutation in the Iranian population. The variant has been detected in two out of seven other reported JAM2-related families who may originate from the Middle East and exhibit an identical haplotype. Even though this particular mutation may not be classified as a founder mutation, it does appear to be a hotspot, given that it has been observed in 45% of the 11 JAM2-associated families. Our study expanded the clinical features and mutation spectrum of JAM2 and revealed that mutations in JAM2 may be more common than previously reported.

The effects of etidronate on brain calcifications in Fahr's disease or syndrome: rationale and design of the randomised, placebo-controlled, double-blind CALCIFADE trial.

BACKGROUND: Fahr's disease and syndrome are rare disorders leading to calcification of the small arteries in the basal ganglia of the brain, resulting in a wide range of symptoms comprising cognitive decline, movement disorders and neuropsychiatric symptoms. No disease-modifying therapies are available. Studies have shown the potential of treatment of ectopic vascular calcifications with bisphosphonates. This paper describes the rationale and design of the CALCIFADE trial which evaluates the effects of etidronate in patients with Fahr's disease or syndrome. METHODS: The CALCIFADE trial is a randomised, placebo-controlled, double-blind trial which evaluates the effects of etidronate 20 mg/kg during 12 months follow-up in patients aged ≥ 18 years with Fahr's disease or syndrome. Etidronate and placebo will be administered in capsules daily for two weeks on followed by ten weeks off. The study will be conducted at the outpatient clinic of the University Medical Center Utrecht, the Netherlands. The primary endpoint is the change in cognitive functioning after 12 months of treatment. Secondary endpoints are the change in mobility, neuropsychiatric symptoms, volume of brain calcifications, dependence in activities of daily living, and quality of life. RESULTS: Patient recruitment started in April 2023. Results are expected in 2026 and will be disseminated through peer-reviewed journals as well as presentations at national and international conferences. CONCLUSIONS: Fahr's disease and syndrome are slowly progressive disorders with a negative impact on a variety of health outcomes. Etidronate might be a new promising treatment for patients with Fahr's disease or syndrome. TRIAL REGISTRATION: ClinicalTrials.gov, NCT05662111. Registered 22 December 2022, https://clinicaltrials.gov/ct2/show/NCT01585402 .

The Genetics of Primary Familial Brain Calcification: A Literature Review.

Primary familial brain calcification (PFBC), also known as Fahr's disease, is a rare inherited disorder characterized by bilateral calcification in the basal ganglia according to neuroimaging. Other brain regions, such as the thalamus, cerebellum, and subcortical white matter, can also be affected. Among the diverse clinical phenotypes, the most common manifestations are movement disorders, cognitive deficits, and psychiatric disturbances. Although patients with PFBC always exhibit brain calcification, nearly one-third of cases remain clinically asymptomatic. Due to advances in the genetics of PFBC, the diagnostic criteria of PFBC may need to be modified. Hitherto, seven genes have been associated with PFBC, including four dominant inherited genes (SLC20A2, PDGFRB, PDGFB, and XPR1) and three recessive inherited genes (MYORG, JAM2, and CMPK2). Nevertheless, around 50% of patients with PFBC do not have pathogenic variants in these genes, and further PFBC-associated genes are waiting to be identified. The function of currently known genes suggests that PFBC could be caused by the dysfunction of the neurovascular unit, the dysregulation of phosphate homeostasis, or mitochondrial dysfunction. An improved understanding of the underlying pathogenic mechanisms for PFBC may facilitate the development of novel therapies.

PSEN1/SLC20A2 double mutation causes early-onset Alzheimer's disease and primary familial brain calcification co-morbidity.

Primary familial brain calcification (PFBC; formerly Fahr's disease) and early-onset Alzheimer's disease (EOAD) may share partially overlapping pathogenic principles. Although the heterozygous loss-of-function mutation c.1523 + 1G > T in the PFBC-linked gene SLC20A2 was detected in a patient with asymmetric tremor, early-onset dementia, and brain calcifications, CSF ß-amyloid parameters and FBB-PET suggested cortical ß-amyloid pathology. Genetic re-analysis of exome sequences revealed the probably pathogenic missense mutation c.235G > A/p.A79T in PSEN1. The SLC20A2 mutation segregated with mild calcifications in two children younger than 30 years. We thus describe the stochastically extremely unlikely co-morbidity of genetic PFBC and genetic EOAD. The clinical syndromes pointed to additive rather than synergistic effects of the two mutations. MRI data revealed the formation of PFBC calcifications decades before the probable onset of the disease. Our report furthermore exemplifies the value of neuropsychology and amyloid PET for differential diagnosis.

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.

Coexistent meningioma in a patient with primary familial brain calcification: A case report.

Primary familial brain calcification (PFBC) is an idiopathic pathology characterized by the development of calcific deposits in the supratentorial region such as telencephalon and diencephalon but also, in more extensive forms, in the cerebellum. Meningiomas are among the most common central nervous system (CNS) tumors generally related to a good prognosis. The simultaneous presence of intracerebral or extra-axial tumors and PFBC represents an exceedingly rare occurrence. A 72-year-old female patient was admitted to our department because of anoculogyric crisis followed by generalized seizures. She performed a brain CT scan which showed widespread hyperdensities in the paraventricular supratentorial region, basal ganglia and at the level of bilateral cerebellar dentate nuclei, characteristics of PFBC. Concomitant left frontal and smaller right temporal extra-axial lesions were identified and then confirmed in a brain MRI. The patient underwent a microsurgical resection of the lesion and subsequent histological examination reported a meningothelial meningioma (WHO Grade I). According to our literature review, this is the first paper that reports the coexistence of both intracranial meningiomas and PFBC. To date, it is not possible to provide an exact correlation between pathogenesis and genetic mechanism underlying this association.

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.

The Pathology of Primary Familial Brain Calcification: Implications for Treatment.

Primary familial brain calcification (PFBC) is an inherited neurodegenerative disorder mainly characterized by progressive calcium deposition bilaterally in the brain, accompanied by various symptoms, such as dystonia, ataxia, parkinsonism, dementia, depression, headaches, and epilepsy. Currently, the etiology of PFBC is largely unknown, and no specific prevention or treatment is available. During the past 10 years, six causative genes (SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, and JAM2) have been identified in PFBC. In this review, considering mechanistic studies of these genes at the cellular level and in animals, we summarize the pathogenesis and potential preventive and therapeutic strategies for PFBC patients. Our systematic analysis suggests a classification for PFBC genetic etiology based on several characteristics, provides a summary of the known composition of brain calcification, and identifies some potential therapeutic targets for PFBC.

Mechanisms of PiT2-loop7 Missense Mutations Induced Pi Dyshomeostasis.

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.

Knockdown of myorg leads to brain calcification in zebrafish.

Primary familial brain calcification (PFBC) is a neurogenetic disorder characterized by bilateral calcified deposits in the brain. We previously identified that MYORG as the first pathogenic gene for autosomal recessive PFBC, and established a Myorg-KO mouse model. However, Myorg-KO mice developed brain calcifications until nine months of age, which limits their utility as a facile PFBC model system. Hence, whether there is another typical animal model for mimicking PFBC phenotypes in an early stage still remained unknown. In this study, we profiled the mRNA expression pattern of myorg in zebrafish, and used a morpholino-mediated blocking strategy to knockdown myorg mRNA at splicing and translation initiation levels. We observed multiple calcifications throughout the brain by calcein staining at 2-4 days post-fertilization in myorg-deficient zebrafish, and rescued the calcification phenotype by replenishing myorg cDNA. Overall, we built a novel model for PFBC via knockdown of myorg by antisense oligonucleotides in zebrafish, which could shorten the observation period and replenish the Myorg-KO mouse model phenotype in mechanistic and therapeutic studies.

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.

Primary Familial Brain Calcification Caused by a Novel Compound Heterozygous Mutation in the MYORG Gene.

BACKGROUND PURPOSE: To demonstrate a novel compound heterozygous mutation in MYORG-related recessive primary familial brain calcification. CASE REPORT: We report a case of primary familial brain calcification with newly-discovered compound heterozygous mutation in the MYORG gene presenting with progressive parkinsonism, cerebellar signs, and typical diffuse brain calcifications. CONCLUSION: Clinicians should consider MYORG testing in patients who have primary brain calcifications with either a negative or recessive family history.

A novel mutation in MYORG leads to primary familial brain calcification and cerebral infarction.

Primary familial brain calcification (PFBC) is a rare inherited disorder characterized by bilateral calcification mainly in the basal ganglia, thalamus, and cerebellar nuclei. Recently, the MYORG gene, as the first autosomal recessive causal gene for PFBC, was reported in six unrelated Chinese families. Patients with PFBC rarely present with cerebrovascular disease. Here, we report a young patient with PFBC who carried a novel homozygous mutation in the MYORG gene presenting with cerebral infarction involving the posterior limb of the right internal capsule. Brain computed tomography (CT) demonstrated symmetric calcifications in the basal ganglia, thalamus, midbrain, pons, cerebellum and frontal lobes. We found one homozygous mutation in the MYORG gene (NM_020702.3 exon2: c.830delC; p.P277Qfs*3) in this patient by Sanger sequencing. Currently, the association of PFBC and cerebral infarction, as well as the physiological role of the MYORG gene, is not clear and worth special attention and further investigation.

A heterozygous deletion of PDGFB gene causes paroxysmal kinesigenic dyskinesia with primary familial brain calcification.

BACKGROUND: Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized with calcium deposition in multiple brain regions. Mutations in PDGFB have been discovered in sporadic and familial PFBC cases. While several known variants displayed loss-of function, no complete deletion of platelet-derived growth factor B (PDGFB) has been reported. METHODS: For the diagnostic purpose, brain computerized tomography or magnetic resonance imaging scanning and whole-genome sequencing were performed on the proband and family members in the pedigree. RESULTS: We identified a heterozygous PDGFB complete deletion in a Chinese pedigree. The proband presented with paroxysmal kinesigenic dyskinesia (PKD), a rare symptom in PFBC. The proband's mother carrying the same mutation was asymptomatic. CONCLUSIONS: For the first time, we reported a PFBC with a heterozygous deletion of PDGFB, and provided evidence of haploinsufficiency in the pathogenesis of PFBC.

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.