A Homozygous Variant in NAA60 Is Associated with Primary Familial Brain Calcification.

BACKGROUND: Primary familial brain calcification (PFBC) is a monogenic disorder characterized by bilateral calcifications in the brain. The genetic basis remains unknown in over half of the PFBC patients, indicating the existence of additional novel causative genes. NAA60 was a recently reported novel causative gene for PFBC. OBJECTIVE: The aim was to identify the probable novel causative gene in an autosomal recessive inherited PFBC family. METHODS: We performed a comprehensive genetic study on a consanguineous Chinese family with 3 siblings diagnosed with PFBC. We evaluated the effect of the variant in a probable novel causative gene on the protein level using Western blot, immunofluorescence, and coimmunoprecipitation. Possible downstream pathogenic mechanisms were further explored in gene knockout (KO) cell lines and animal models. RESULTS: We identified a PFBC co-segregated homozygous variant of c.460_461del (p.D154Lfs*113) in NAA60. Functional assays showed that this variant disrupts NAA60 protein localization to Golgi and accelerated protein degradation. The mutant NAA60 protein alters its interaction with the PFBC-related proteins PiT2 and XPR1, affecting intracellular phosphate homeostasis. Further mass spectrometry analysis in NAA60 KO cell lines revealed decreased expression of multiple brain calcification-associated proteins, including reduced folate carrier (RFC), a folate metabolism-related protein. CONCLUSIONS: Our study replicated the identification of NAA60 as a novel causative gene for autosomal recessive PFBC, demonstrating our causative variant leads to NAA60 loss of function. The NAA60 loss of function disrupts not only PFBC-related proteins (eg, PiT2 and XPR1) but also a wide range of other brain calcification-associated membrane protein substrates (eg, RFC), and provided a novel probable pathogenic mechanism for PFBC. © 2024 International Parkinson and Movement Disorder Society.

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.

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.

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.

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.

Aneurysmal subarachnoid hemorrhage with PFBC and beta thalassemia: a case report.

BACKGROUND: Primary familial brain calcification (PFBC), habitually called Fahr's disease, is characterized by bilateral calcification of the basal ganglia, accompanied by extensive calcification of the cerebellar dentate nucleus, brainstem cerebrum, and cerebellum at the grey-white matter junction. However, there are few reports about PFBC with aneurysmal subarachnoid hemorrhage (aSAH) and thalassemia. CASE PRESENTATION: We describe a patient admitted to the hospital with an acute deterioration in the level of consciousness with no history of neuropsychiatric features or movement disorders. After computed tomography (CT) and CT angiography (CTA), the patient was diagnosed with PFBC, accompanied by aneurysmal subarachnoid haemorrhage (aSAH), intracranial haemorrhage (ICH), and hemoglobin electrophoresis suggested beta-thalassemia. This patient underwent craniotomy aneurysm clipping and intracranial hematoma removal. CONCLUSIONS: For patients with PFBC, we should pay attention to their blood pressure and intracranial vascular conditions. The CTA is necessary to clarify the cerebrovascular conditions of the patient, especially when combined with hypertension and persistent headache or other related prodromal symptoms of cerebrovascular disease.

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.

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-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.

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.

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.

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.

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.

Biallelic loss-of-function mutations in JAM2 cause primary familial brain calcification.

Primary familial brain calcification is a monogenic disease characterized by bilateral calcifications in the basal ganglia and other brain regions, and commonly presents motor, psychiatric, and cognitive symptoms. Currently, four autosomal dominant (SLC20A2, PDGFRB, PDGFB, XPR1) and one autosomal recessive (MYORG) causative genes have been identified. Compared with patients with autosomal dominant primary familial brain calcification, patients with the recessive form of the disease present with more severe clinical and imaging phenotypes, and deserve more clinical and research attention. Biallelic mutations in MYORG cannot explain all autosomal recessive primary familial brain calcification cases, indicating the existence of novel autosomal recessive genes. Using homozygosity mapping and whole genome sequencing, we detected a homozygous frameshift mutation (c.140delT, p.L48*) in the JAM2 gene in a consanguineous family with two affected siblings diagnosed with primary familial brain calcification. Further genetic screening in a cohort of 398 probands detected a homozygous start codon mutation (c.1A>G, p.M1?) and compound heterozygous mutations [c.504G>C, p.W168C and c.(67+1_68-1)_(394+1_395-1), p.Y23_V131delinsL], respectively, in two unrelated families. The clinical phenotypes of the four patients included parkinsonism (3/4), dysarthria (3/4), seizures (1/4), and probable asymptomatic (1/4), with diverse onset ages. All patients presented with severe calcifications in the cortex in addition to extensive calcifications in multiple brain areas (lenticular nuclei, caudate nuclei, thalamus, cerebellar hemispheres, ± brainstem; total calcification scores: 43-77). JAM2 encodes junctional adhesion molecule 2, which is highly expressed in neurovascular unit-related cell types (endothelial cells and astrocytes) and is predominantly localized on the plasma membrane. It may be important in cell-cell adhesion and maintaining homeostasis in the CNS. In Chinese hamster ovary cells, truncated His-tagged JAM2 proteins were detected by western blot following transfection of p.Y23_V131delinsL mutant plasmid, while no protein was detected following transfection of p.L48* or p.1M? mutant plasmids. In immunofluorescence experiments, the p.W168C mutant JAM2 protein failed to translocate to the plasma membrane. We speculated that mutant JAM2 protein resulted in impaired cell-cell adhesion functions and reduced integrity of the neurovascular unit. This is similar to the mechanisms of other causative genes for primary familial brain calcification or brain calcification syndromes (e.g. PDGFRB, PDGFB, MYORG, JAM3, and OCLN), all of which are highly expressed and functionally important in the neurovascular unit. Our study identifies a novel causative gene for primary familial brain calcification, whose vital function and high expression in the neurovascular unit further supports impairment of the neurovascular unit as the root of primary familial brain calcification pathogenesis.

Hereditary and non-hereditary etiologies associated with extensive brain calcification: case series.

Cerebral calcification may be caused by several potentially treatable conditions, however, in most cases it does not receive special attention in clinical practice. From the point of view of etiology, the diseases associated with cerebral calcification can be divided into two main groups: idiopathic (mostly incurable) and secondary (potentially treatable). The first group includes mainly the hereditary diseases identified before 2021 (primary familial brain calcification subtypes, previously known as Fahr's disease or Fahr's syndrome). In contrast, the second group includes diseases with cerebral calcification that develop generally as a consequence of metabolic/endocrine/autoimmune abnormalities. The aim of our research was to present hereditary and non-hereditary etiologies associated with extensive brain calcification. We compare the detailed clinical, radiological and laboratory results of 6 patients with prominent cerebral calcification identified in our clinic in the last 3 years (idiopathic and secondary etiologies as well). Our research draws attention to the complexity of the etiologies in the context of cerebral calcification. We recommend, beside NGS-based sequence analyses, the application of array comparative genomic hybridization as well, to identify potential genetic etiologies associated with brain calcification.

MYORG Mutation Heterozygosity Is Associated With Brain Calcification.

BACKGROUND: Biallelic mutations in the MYORG gene were first identified as the cause of recessively inherited primary familial brain calcification. Interestingly, some heterozygous carriers also exhibited brain calcifications. OBJECTIVES: To further investigate the role of single heterozygous MYORG mutations in the development of brain calcifications. METHODS: A nation-wide cohort of Chinese primary familial brain calcification probands was enrolled from March 2016 through September 2019. Mutational analysis of MYORG was performed in 435 primary familial brain calcification probands who were negative for mutations in the other four known primary familial brain calcification-causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1). RESULTS: Biallelic MYORG mutations were identified in 14 primary familial brain calcification patients from 10 unrelated families. Interestingly, 12 heterozygous carriers from seven of these families also exhibited mild-to-moderate brain calcifications. Moreover, single heterozygous mutations were detected in an additional 9 probands and in 7 of their family members affected with brain calcifications. In our cohort, clinical and imaging penetrance of individuals with biallelic mutations were 100%, whereas among individuals with heterozygous mutations, penetrance of imaging phenotype was reduced to 73.7% (28 of 38) and clinical penetrance was much lower. Most (34 of 38) remained asymptomatic whereas 4 carriers had symptoms of uncertain clinical significance (nonspecific depression, epilepsy and late-onset parkinsonism). Compared with individuals with biallelic MYORG mutations, individuals with heterozygous mutations had brain calcifications with much lower calcification scores (P < 2e-16). CONCLUSIONS: Presence of brain calcifications in individuals with heterozygous MYORG mutations suggested a semidominant inheritance pattern with incomplete penetrance. This finding further expanded the genotype-phenotype correlations of MYORG-related primary familial brain calcification. © 2020 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.

Brain hypoperfusion and nigrostriatal dopaminergic dysfunction in primary familial brain calcification caused by novel MYORG variants: case report.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare inherited disease characterized by multiple calcified foci in the brain parenchyma. MYORG is the first gene found to be associated with autosomal recessive PFBC. The precise pathogenic mechanism of neurodegeneration in PFBC remains unclear. The clinical phenotypes of PFBC are variable, and there is no clear correlation between clinical manifestations and radiological and pathological features of calcification. CASE PRESENTATION: Two sisters in a Taiwanese family presented with young-onset Parkinsonism and multifocal dystonia. Their brain CTs showed multiple intracerebral calcifications. The genetic study detected two heterozygous novel variants, c.104 T > A (p.Met35Lys) and c.850 T > C (p.Cys284Arg) in the MYORG gene. In both patients, MR susceptibility weighted images revealed calcification of the deep medullary veins. Tc99m ECD SPECT demonstrated a significant decrease of tracer uptake in the brain cortex and subcortical gray matter. Tc99m TRODAT-1 SPECT revealed decreased tracer uptake in the bilateral striatum. CONCLUSION: Two novel MYORG variants were identified in Taiwanese family members presenting with PFBC. Abnormalities in the brain perfusion and dopamine transporter SPECTs suggest that cerebral ischemia due to extensive calcified vasculopathy, disruption of the basal ganglia-thalamo-cortical circuit, and nigrostriatal dopaminergic dysfunction are plausible pathogenic mechanisms of neurodegeneration in PFBC patients. Further investigation into the correlations between the pathogenicity-implicated imaging findings and the clinical phenotype are recommended.

Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.

Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor ß (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients.