Published: March 5, 2020

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Primary familial brain calcification (PFBC \[MIM: [213600](213600){#intref0010}\]), often referred as Fahr disease, constitutes a heterogeneous neurodegenerative disorder that presents with mineral calcium deposits in the brain. The clinical manifestations can include, but are not restricted to, movement disorders such as parkinsonism and ataxia, seizures, migraine, and neuropsychiatric symptoms. Both autosomal-dominant and -recessive inheritance patterns have been reported.[@bib1] The clinical picture and severity are often variable between and within families, with some family members being clinically asymptomatic. The advent of widespread brain imaging for individuals presenting with parkinsonism, who were previously clinically diagnosed, has significantly increased the number of PFBC-affected families identified.[@bib2]

There are two main pathogenic mechanisms described so far in PFBC. On the one hand, the calcium and phosphate homeostasis dysfunction via dominant mutations in *SLC20A2* (MIM: [158378](158378){#intref0015}) and *XPR1* (MIM: [605237](605237){#intref0020}).[@bib1] The inhibition of phosphate uptake by mutations in *SLC20A2* encoding for sodium-dependent phosphate transporter 2 (PiT-2) leads to deposition of calcium in the vascular extracellular matrix, and inhibition of phosphate export associated with *XPR1* mutations is expected to increase intracellular phosphate concentration and provoke calcium phosphate precipitation.[@bib3]

On the other hand, the second mechanism causes PFBC through disruption of the neurovascular unit (NVU). Endothelial integrity and function affecting the blood-brain barrier (BBB) is altered via dominant mutations in *PDGFB* (MIM: [190040](190040){#intref0025}) and *PDGFRB* (MIM: [173410](173410){#intref0030}) encoding for the platelet-derived growth factor B and its receptor, that lead to the impairment of pericytes recruitment and BBB integrity, causing vascular and perivascular calcium accumulation.[@bib2] The recessive brain calcification phenotype due to *MYORG*[@bib4] (MIM: [618255](618255){#intref0035}) mutations, has been shown to present specific *myorg* mRNA expression in mouse astrocytes and disturb the normal function of the NVU. Mutations in *JAM3*[@bib5] (MIM: [613730](613730){#intref0040}) and *OCLN*[@bib6] (MIM: [602876](602876){#intref0045}) encoding for tight junction proteins lead to excess solutes crossing the BBB causing CNS calcification and hemorrhage.

Interestingly, a study on the *Slc20a2* null mouse suggested that the calcified nodules present in the brain initiated in pericytes and astrocytes and found endogenous IgG around nodules proposing that there was increased BBB permeability[@bib7] and identifying a possible link between different PFBC causative genes.

Pathologically, human brains exhibit calcium salt deposits predominantly distributed around small blood vessels,[@bib2] and a reported subject with a *SLC20A2* mutation presented calcification in the tunica media of small arteries, arterioles, and capillaries, but not in veins distributed in the basal ganglia, thalamus, cerebellar white matter, and deeper layers of the cerebral cortex.[@bib8]

Despite important progress in discovering the genetic architecture of PFBC, more than half of the case subjects remain genetically unsolved.[@bib2]

In this study we report four unrelated families with seven individuals affected by autosomal-recessive primary familial brain calcification. In the families described here, we used a combination of homozygosity mapping, exome sequencing (ES), functional studies, and mouse model to identify and characterize the causal variants in *JAM2* (MIM: [606870](606870){#intref0050}) encoding for the junctional-adhesion-molecule-2, a tight-junction protein as a cause of PFBC.

Two unrelated consanguineous families from traveller communities in England (family 1) and Northern Ireland (family 2), one non-consanguineous family from the United States (family 3) identified using GeneMatcher, and one Turkish consanguineous family (family 4) were included in this study ([Figure 1](#fig1){ref-type="fig"}A). Clinical features of affected individuals are presented in [Table 1](#tbl1){ref-type="table"}. The proband in family 1 (F1-II:2) had a normal birth and early milestones. He presented with childhood-onset cerebellar ataxia and learning difficulties. His symptoms progressed and were associated with additional behavioral problems and worsening cognitive impairment. He was examined by a neurologist at the age of 23 years old. At that stage, he already had difficulties following commands and had alternate exotropia with left eye preference for fixation, slow and jerky pursuit, and ophthalmoplegia. He had reduced ability to control tongue movements and was unable to protrude his tongue. He was dysarthric with dysphagia and a percutaneous endoscopic gastrostomy (PEG) insertion at the age of 22 years. He had increased tone in the upper and lower limbs with ankle contractures, brisk reflexes throughout, and upgoing plantars. There was upper and lower limb ataxia, bradykinesia, and generalized dystonia, worse in the upper limbs ([Video S1](#mmc2){ref-type="supplementary-material"}). Occasional seizures were seen later in the disease course. The interictal electroencephalography (EEG) showed moderate generalized slowing of cortical rhythms.Figure 1Clinical and Neuroimaging Features of *JAM2*-Related Disease(A) Pedigrees of the four families with bi-allelic *JAM2* mutations.(B) Brain images of *JAM2*-related disease. B1 to B3 are CT scans acquired from case subject F1-II:2 from family 1 and B4 to B6 are CT acquired from case subject F2-III:3 from family 2. In both individuals there is extensive, symmetrical, bilateral calcification involving the basal ganglia, deep cortical gray matter and cerebellum. B7, 8, and 9 are CT from case subject F3-II:1 demonstrating calcification in the basal ganglia and cortical gyri but not in the cerebellum. B10, 11, and 12 are axial MRI scans from individual F3-II:1 from family 3 showing basal ganglia and frontal calcification (B10 is T1 MRI, B11 is T2 MRI, and B12 is Axial Ven Bold reconstruction MRI that is sensitive to calcium shown as hypointense regions). Asterisk (^∗^) areas of calcification on MRI.Table 1Clinical Features of Affected Individuals with *JAM2* Bi-allelic Variants**Individual F1-II:2Individual F2-III:2Individual F2-III:3Individual F2-III:4Individual F2-III:5Individual F3-II:1Individual F4-II:3**cDNA sequencec.685C\>Tc.685C\>Tc.685C\>Tc.685C\>Tc.685C\>Tc.395−1dupG, c.323G\>Ac.177_180delCAGAAmino acid changep.Arg229Terp.Arg229Terp.Arg229Terp.Arg229Terp.Arg229TerIVS4-1dupG, p.Arg108Hisp.Arg60TerZygosityhomozygoushomozygoushomozygoushomozygoushomozygouscompound heterozygoushomozygousGender (male/female)malemalefemalemalemalemalefemaleBirth and early milestonesnormalnormalnormalnormalnormalnormalnormalOnset of symptomschildhoodlate 20slate 30steenageteenagechildhoodearly childhoodSymptom at onsetcerebellar ataxia and cognitive declinecognitive decline, depressiondifficulty walkingdepression, dysarthriadepression, dysarthriaautism spectrum disorderseizuresAge at examination (in years)2441394049157**Phenotype at Last Examination**Pyramidal syndromeyes; increased tone, brisk reflexes, upgoing plantarsyes; increased tone, brisk reflexes, upgoing plantarsyes; increased tone, brisk reflexes, upgoing plantarsyes; increased tone, brisk reflexes, upgoing plantarsyes; increased tone, brisk reflexes, upgoing plantarsnonoCerebellar syndromeyes; upper and lower limb ataxia, dysarthria, nystagmusyes; upper and lower limb ataxiayes; upper and lower limb ataxiayes; upper and lower limb ataxia, dysarthriayes; upper and lower limb ataxia, dysarthriayes; upper and lower limb mild ataxia, nystagmusnoParkinsonismyes; rigidity, bradykinesia.yes; hypophonia, hypomimia, bradykinesiayes; hypophonia, hypomimia, bradykinesiayes; rigidity, bradykinesiayes; rigidity, bradykinesianonoDystoniayes; generalizedyes; limb dystonia and orofacial dyskinesiasyes; limb dystonia and orofacial dyskinesiasnonononoOtherseizures, ophthalmoplegia, PEG inserted in advance stagePEG inserted in advance stagebecame anarthric in advanced stage----autism spectrum disorder--Cognitive functionsevere cognitive declinememory decline with severe impaired recallunable to comment on cognition due to anarthria.severe cognitive declinesevere cognitive declinedecline in academic performancenormal for her ageBrain imaging calcification patternbasal ganglia, thalamus, cerebellum, deep gray matterbasal ganglia, thalamus, cerebellum, deep gray matterbasal ganglia, thalamus, cerebellum, deep gray matterbasal ganglia, thalamus, cerebellum, deep gray matterbasal ganglia, thalamus, cerebellum, deep gray matterbasal ganglia, and frontal cortexbasal ganglia, dentate nucleus and cerebellar hemispheres

Video S1. Clinical Presentation of the Affected Individual from Family Carrying Bi-allelic *JAM2* Variants

There were four affected individuals in the second family. Two siblings (F2-III:2 and F2-III:3) presented with borderline low IQ in childhood but had no definite physical limitations in early life. In their twenties they had social withdrawal and severe depression requiring treatment. The disease progressed and examination at 41 and 39 years revealed severe speech hypophonia, dysphagia, hypomimia, reduced vertical up gaze, orofacial dyskinesias, slow and reduced tongue movements with bradykinesia, and dystonic limb posturing in both affected individuals. Tone was increased in an extrapyramidal pattern with lower limb hyperreflexia and extensor plantar responses, grasp reflexes, positive glabellar tap, and brisk jaw jerk. They both had memory decline with impaired recall. Treatment with ropinirole did not lead to any significant improvement in symptoms. The disease progressed, they became bedridden and case F2-III:2 needed a PEG insertion 10 years after the onset of movement problems due to recurrent aspiration pneumonia, and the proband died in the late 40s. Case subjects F2-III:4 and F2-III:5 are maternal first cousins of the index case subject in family 2. They presented an almost identical phenotype. On a background of depression, both brothers noted progressive "slurring" of speech, slowing of all movements, difficulty with walking, recurrent falls, and poor memory. Examination demonstrated a similar phenotype with dysarthria, abnormal pursuit with frequent saccadic intrusions, pronounced bradykinesia, extrapyramidal rigidity, and bilaterally extensor plantar responses.

The proband in family 3 (F3-II:1) had normal early development but later developed mild delay in fine motor and language milestones that were progressive. He also developed mild coordination problems and autism spectrum disorder (ASD) and received special education services at school age. At age 11 years, repeat neuropsychologic evaluation showed a continuous decline in academic performance. Parents were asymptomatic, an older brother had some anxiety and hyperactivity, and a younger brother aged 13 years had mild autistic features. At the last examination of the proband aged 15 years, he had autism spectrum features, hyperactivity, developmental delay, and coordination problems. The coordination difficulties were mild but affected both fine motor skills (buttons, zippers, hooks) and complex gross motor tasks. His learning difficulties were progressive.

The affected member of family 4 (F4-II:3) was born to Turkish consanguineous parents and presented with seizures when she was 18 months of age; current age is 7.5 years and she has had a total of 3 seizures. Her development so far has been in keeping with her peers and her last examination did not reveal any neurological signs. Brain calcification was identified on MRI imaging with bilateral symmetric calcification of the basal ganglia, dentate nucleus, and subcortical white matter of cerebellar hemispheres. Prior to exome sequencing, the known Fahr's genes was sequenced and negative.

All seven case subjects reported here had brain calcification identified on brain CT and/or MRI imaging. They had a consistent pattern of bilateral symmetric calcification of the basal ganglia and deep cortical gray matter. In addition, the older individuals from families 1 and 2 had severe calcification in the cerebellum folia and the thalamus ([Figure 1](#fig1){ref-type="fig"}B). All families had extensive genetic, metabolic, and mitochondrial investigations carried out that excluded acquired and other inherited causes of brain calcification.

In order to localize the chromosomal location of the pathogenic variant, we genotyped three affected and two unaffected individuals from extended family 2 genome-wide by using Illumina HumanCytoSNP-12v2-1 Beadchip array incorporating ∼200,000 genetic markers. Three regions of homozygosity were detected on chr10:37,414,883--43,132,376, chr13:88,327,643--93,518,692, and chr21:22,370,881--28,338,710. Next, we performed exome sequencing on probands of families 1 and 2 to identify the causative variant(s). On the assumption that the disease follows an autosomal-recessive pattern of inheritance in the families as well as presence of consanguinity in two families, we prioritized the bi-allelic potentially functional variants residing within the runs of homozygosity. These variants were screened through all publicly available population databases and our in-house database. We excluded synonymous variants, intronic variants (\>7 bp from exon boundaries) and common variants (minor allele frequency \> 0.001%). The selected variants were validated, and segregation analysis was performed using Sanger sequencing.

In families 1 and 2, filtered exome-sequencing data narrowed down the variants to the same homozygous nonsense variant (GenBank: [NM_021219](ncbi-n:NM_021219){#intref0055}; c.685C\>T \[p.Arg229Ter\]) in *JAM2* residing within a 5 Mb region of homozygosity on chr21q21.3 ([Figure S1](#mmc1){ref-type="supplementary-material"}). Sanger sequencing verified the correct segregation of the variant on available samples of both families ([Figures 1](#fig1){ref-type="fig"}A, [2](#fig2){ref-type="fig"}A, and 2B). This variant was absent in our in-house exome database of more than 10,000 exomes, absent in homozygous state in all publicly available databases, and present in heterozygous state with a minor allele frequency (MAF) of 0.00002 in gnomAD (6/280662). The early stop codon introduced by c.685C\>T is predicted to result in production of a truncated protein lacking the transmembrane and cytoplasmic regions of *JAM2* and cause reduction in total *JAM2* as a consequence of nonsense-mediated decay (NMD) of the mutant transcript. Indeed, RT-PCR analysis confirmed a reduction of *JAM2* mRNA expression levels in the proband in family 1 compared to the heterozygous carrier and unrelated control subject ([Figure 2](#fig2){ref-type="fig"}C). Furthermore, western blot analysis confirmed the absence of JAM2 protein in the homozygous proband ([Figure 2](#fig2){ref-type="fig"}D). The reduction of RNA expression and absent JAM2 protein in the fibroblast cell lines of the proband support the loss-of-function role of this variant. As JAM2, JAM3, and TJP1 proteins are all junctional components and associated with brain calcification phenotype, we investigated whether *JAM2* c.685C\>T variant affected the localization of the other two proteins. We show that there was no difference in the localization of JAM3 and TJP1 in primary dermal fibroblasts from *JAM2* homozygous affected individuals ([Figure S2](#mmc1){ref-type="supplementary-material"}).Figure 2Validation of *JAM2* Variants Identified in This Study(A) Validation by Sanger sequencing of the stop variant c.685C\>T (p.Arg229Ter) in families 1 and 2, the bi-allelic variants in family 3 (IVS4-1dupG and p.Arg108His), and the homozygous change in family 4 c.177_180delCAGA (p.Arg60Ter).(B) Protein structure of JAM2 showing the 2 main domains (Ig-like V-type and Ig-like C2-type) as well as the variants identified in the 4 families.(C) Total RNA and protein extracts were prepared from fibroblast cell lines isolated from the proband of family 1 (HOM), his unaffected mother (HET), and unrelated control subjects (Con) to assess the c.685C\>T (p.Arg229Ter) *JAM2* variant. Agarose gel separation of RT-PCR products shows a reduction in *JAM2* mRNA expression in the homozygous proband (HOM) compared to the proband's mother (HET) and unrelated control subject (Con). *GAPDH* RT-PCR served as a loading control.(D) Western blot analysis of total protein lysates using anti-JAM2 and GAPDH (loading control) antibodies revealed presence of JAM2 protein in control (Control) and unaffected carrier (HET) cells but complete loss of JAM2 protein in the homozygous proband of family 1 (HOM). NS, non-specific band; asterisk, JAM2.

In family 3, Trio-ES revealed compound heterozygous variants in *JAM2* (GenBank: [NM_021219](ncbi-n:NM_021219){#intref0060}; c.395−1dupG \[IVS4-1dupG\] and c.323G\>A \[p.Arg108His\]). The c.395−1dupG was inherited from the father and c.323G\>A was inherited from the mother. The c.323G\>A was reported once in gnomAD in heterozygous state (MAF 0.00003, 1/31408), three times in GeneDx in-house database (MAF 0.00002, 3/167854), and was absent in all databases as homozygous. It disrupts a highly conserved residue (CADD:34) and is predicted to be damaging to the protein function by *in silico* prediction tools (SIFT, PROVEAN, Mutation Taster, Mutation Assessor, and PolyPhen). The c.395−1dupG is predicted to cause the retention of the canonical splice acceptor site of intron 4 with insertion of the G nucleotide as the first base of exon 5 causing a frameshift and a truncated protein (p.Val132GlyfsX9). This variant was absent from all public databases and present five times in heterozygous state in GeneDx in-house database (MAF 0.00003, 5/171284). The variants segregated fully within the family ([Figures 1](#fig1){ref-type="fig"}A, [2](#fig2){ref-type="fig"}A, and 2B).

In family 4, clinical ES uncovered a homozygous 4 bp deletion in *JAM2* (GenBank: [NM_021219](ncbi-n:NM_021219){#intref0065}; c.177_180delCAGA \[p.Arg60Ter\]) ([Figures 1](#fig1){ref-type="fig"}A, [2](#fig2){ref-type="fig"}A, and 2B) that causes a frameshift and an early termination of the protein affecting the extracellular, transmembrane, and cytoplasmic domains of JAM2. The variant was not reported in any public databases and was predicted pathogenic by MutationTaster causing loss of function by NMD.

In order to characterize the link between *JAM2* variants and the human neurological phenotype, we developed *jam2* knockout (*jam2* KO) mice. Behavioral tests in the *jam2* KO mice showed significant difficulties in beam walking test and gait abnormalities when compared to wild-type mice ([Figure 3](#fig3){ref-type="fig"}). There was a significant reduction in stride length (wild-type: 8.14 ± 0.9, *jam2* KO: 6.3 ± 1; ^∗∗∗^p \< 0.0001) and increase in sway length (wild-type: 0.13 ± 0.14, *jam2* KO: 0.9 ± 0.4; ^∗∗^p = 0.002) when comparing *jam2* KO to wild-type littermates' controls ([Figure 3](#fig3){ref-type="fig"}A). Additionally, the number of missed steps (wild-type: 1.2 ± 1.3, *jam2* KO: 6.5 ± 3.6; ^∗^p = 0.017) in the beam-walking test was higher in *jam2* KO compared to controls ([Figure 3](#fig3){ref-type="fig"}B and 3C, [Video S2](#mmc1){ref-type="supplementary-material"}).Figure 3Behavioral Study on *jam2* KO Mice and Wild-Type (WT)(A) Representative images of wild-type (left) and *jam2* KO strides (right) in the gait test. Forepaw (blue) and hindpaw (red). Altered gait of the *jam2* KO mice was analyzed in stance length, stride, and sway compared to wild-type (^∗∗∗^p \< 0.0001, ^∗∗^p = 0.002; means ± SEM; n = 6 mice per genotype).(B and C) Walking beam performance on test day, showing elevated latency to cross the beam in *jam2* KO (^∗^p = 0.017; means ± SEM; n = 5 wild-type, n = 6 *jam2* KO). See [Video S2](#mmc3){ref-type="supplementary-material"} for example beam walking test of a *jam2* KO mouse and a control (wild-type).

Video S2. Video Presenting the Beam Walking Test of One Wild-Type (WT) and One *jam2* KO Mouse Showing an Increased Number of Missed Steps in the KO Mouse when Compared to Wild-Type

Brains of two *jam2* KO and two wild-type (C57BL/6) mice were examined at a young age (6 months old) and four *jam2* KO and four wild-type mice were examined at an old age (18 months old). In addition, spinal cord sections from one of the young *jam2* KO mice and from all old *jam2* KO and control mice were examined. We observed prominent widespread vacuolation in the midbrain and some in the thalamus and cerebral and cerebellar cortex of young *jam2* KO mice. In the midbrain, the vacuolar change was accompanied by prominent reactive astrogliosis, mild microglial activation, and mild reduction in the neuronal density compared to controls ([Figure 4](#fig4){ref-type="fig"}). Brains of aged *jam2* KO mice showed similar changes, with prominent widespread neuropil vacuolation in the midbrain accompanied by marked astrogliosis, mild microglial activation, and moderately reduced neuronal density. In contrast to young *jam2* KO mice, there was more prominent vacuolation in the cerebral cortex, thalamus, and cerebellar cortex and particularly widespread vacuolation in the cerebellar white matter. To a lesser extent, neuropil vacuolation in the same regions was also seen in the age-matched control wild-type mice, suggesting that *jam2* KO mice develop age-related changes at a much younger age, which in some areas, such as cerebellar white matter, midbrain, thalamus, and cerebellar cortex, increase in severity with age. In addition, we performed automated quantification of the neuropil vacuolation on H&E-stained sections, GFAP immunoreactive gliosis and Iba1-positive microglial activation in young and aged wild-type and *jam2* KO mice ([Figure S3](#mmc1){ref-type="supplementary-material"}). Automated quantification of the percentage of vacuolation, gliosis, and microglial activation in the cortex, midbrain, and cerebellum was performed on digitalized slides, using open source software QuPath. There was a significant increase in the degree of neuropil vacuolation (p \< 0.00007) and astrogliosis (p \< 0.0138) in the midbrain of old *jam2* KO mice when compared with age-matched wild-type mice, whereas Iba1-positive microglial activation in old *jam2* KO mice was less pronounced than in age-matched wild-type mice (p \< 0.035). No mineralization or calcification was observed in the brains of young or old *jam2* KO mice or controls at the time of examination.Figure 4Brain Pathology in Control and *jam2* KO Mice of Young (6 Months Old) and Old Age (18 Months Old)Littermate controls (wild-type) (a, a1--a6) show no significant pathology on (H&E) stained sections (a, cortex; a1, cerebellum; a2, midbrain) and on immunohistochemistry for astrocytes (GFAP) (a3, midbrain), microglia (Iba1) (a4, midbrain), neuropil (APP) (a5, midbrain), and neurones (NeuN) (a6, midbrain). In age-matched *jam2* KO mice, occasional cortical vacuolation is evident in the cerebral (b) and cerebellar (b1) cortex and widespread prominent vacuolation is seen in the midbrain (b2). In the midbrain region there is also marked reactive astrocytosis (b3) and mild microglial activation (b4). APP immunostaining highlights vacuolar change in the neuropil (b5) and NeuN shows mild reduction in the neuronal density (b6). Aged wild-type mice show occasional vacuolation in the cerebral cortex (c), cerebellar cortex (c1), and midbrain (c2) (H&E). There is mild patchy astrogliosis in the midbrain (c3) and mild microglial activation (c4). No significant disruption of the cytoarchitecture is seen on APP immunostaining (c5), but NeuN highlights some degree of neuronal loss (c6). In age-matched old *jam2* KO mice, there is increasingly prominent vacuolation in the cerebral cortex (d) and in the cerebellar white matter (d1), midbrain (d2), and thalamus (not shown). Similar to young *jam2* KO mice, there is widespread astrogliosis in the midbrain (d3), but microglial activation remains mildly increased (d4). Frequent vacuolation is highlighted in the neuropil with APP immunostaining (d5). Similar to age-matched littermate controls, there is reduction of the neuronal density in the midbrain of *jam2* KO mouse (d6). Scale bar: 100 μm in a--d, a2--a6, b2--b6, c2--c6, d2--d6; 200 μm in a1--d1.

The spinal cord morphology was similar in young and old *jam2* KO-deficient mice but remarkably differed from that of control mice. The main findings in *jam2* KO spinal cords were widespread neuronal, perivascular, and neuropil mineralization as well as widespread vacuolation in the gray matter. The mineralized deposits were negative for PAS and showed weak reactivity for Alcian blue, excluding calcification, cartilagination, or ossification stage. The mineralization and vacuolar change were evident across the gray matter in both anterior and posterior horns bilaterally and at all levels of the spinal cord. Although mineralization and calcification are known to occur with age in wild-type mice, in the four spinal cords from our control group (age-matched to old *jam2* KO mice), no mineralization or neuropil vacuolation at any of the spinal cord levels was observed ([Figure S4](#mmc1){ref-type="supplementary-material"}). JAM3 and TJP1 tight-junction proteins were investigated in the KO mice, and there was no difference in localization between affected and wild-type mice, in different areas of the brain ([Figures S5](#mmc1){ref-type="supplementary-material"} and [S6](#mmc1){ref-type="supplementary-material"}).

Mouse models of other brain calcification genes have shown brain calcification in similar areas to those found in humans. Such is the case of *Slc20a2*-null mice exhibiting progressive calcification of the thalamus, basal ganglia, and cortex beginning at 8 weeks and present in close proximity or within blood vessels and affecting astrocytes and perycites[@bib7]^,^[@bib9]^,^[@bib10] and for *myorg*-null mice developing calcifications at 9 months.[@bib4] A study on mice carrying hypomorphic *Pdgfb* alleles interestingly showed that the calcifications depend on the loss of endothelial PDGF-B and correlate with the degree of pericyte and BBB deficiency.[@bib11] *Ocln*-null mice present with similar calcification surrounding blood vessels as seen in an autopsied case,[@bib6] but there were no abnormalities reported in the brain of *jam3* KO mice, and there was absence of Jam3 in the vasculature of the adult mouse brain.[@bib5] In our study, we were not able to detect calcification in the brain of our *jam2* mouse model even though we studied these mice until the age of 18 months, an age at which other PFBC models already present calcification and thus it does not seem to be age related. However, our *jam2* KO mice did show prominent vacuolation, inflammatory response, and neuronal density reduction in the same areas affected by calcification in humans. Further studies are needed to understand better the disease mechanisms and the differences between humans and mice for *JAM2* and also *JAM3*.

Together, our results confirm that *JAM2* variants underlie the phenotype in the four families reported here. This implicates *JAM2* as a cause of human Mendelian disease. *JAM2* encodes for the junctional-adhesion-molecule-2 (JAM2), member of the junctional adhesion molecules family, localized in the tight junctions of endothelial cells and the NVU.[@bib12]^,^[@bib13] JAM2 is an adhesive ligand interacting with various immune cell types and regulating vascular function and was recently identified as an inhibitor of somatodendritic myelination in spinal cord neurons.[@bib14]

Junctional adhesion molecules are a family of proteins that play an important role in the regulation of cell polarity, endothelium permeability, and leukocyte migration and the blood-brain-barier (BBB) function. In addition to *JAM2*, recessive variants in *JAM3* and *OCLN* were linked to complex neurological disorders presenting with calcification in the brain,[@bib5]^,^[@bib6] suggesting that deregulation of the central NVU is important in pathogenesis of PFBC. Even though increased permeability of the BBB hasn't been confirmed in *jam2* KO mice so far,[@bib13] including this study, mutations have now been identified in three genes encoding tight junction proteins in humans (*JAM2*, *JAM3*, and *OCLN*), suggesting that loss of cell-to-cell adhesion with subsequent dysfunction of solute passage is an important cause of brain calcification. Furthermore, a multicenter collaboration from China recently described four patients from three families that presented with PFBC and bi-allelic mutations in *JAM2*.[@bib15] They interestingly show failure of JAM2 to translocate to the plasma membrane in JAM2 transfected mutants of hamster ovary cells, and they propose a cell-to-cell adhesion impairment as the mechanism causing failure of the NVU and consequent brain calcification phenotype. This work further supports our hypothesis and together with *PDGFB*, *PDGFRB*, and *MYORG* the tight junction genes solidify dysregulation of BBB integrity as a key PFBC pathomechanism ([Figure S7](#mmc1){ref-type="supplementary-material"}).

Importantly, we show that the human *JAM2*-related neurological phenotype seen is replicated in the *jam2* KO mouse. It is noteworthy that the older human individuals carrying *JAM2* variants, who had longer disease duration, presented impaired gait of variable severity including ataxic and/or parkinsonian gait illustrated in the mouse model. The main walking and behavioral findings in the affected mice were progressive gait abnormalities, similar to ataxic mouse, clearly seen on standard mouse phenotype assessment. Furthermore, neuropathology of *jam2* KO mouse model (prominent vacuolation in the cerebral cortex, thalamus, and cerebellar cortex and particularly widespread vacuolation in the midbrain) affected similar brain areas to those observed on brain imaging of the human phenotype. The pattern of calcification seen on the brain scans of our subjects was largely the same as that seen in PFBC-affected subjects where the brain is structurally normal and the calcification is almost exclusively in the gray matter affecting basal ganglia, thalamus, deep cortex, dental, and cerebellar folia.[@bib16] It was indistinguishable from *SLC20A2*, *PDGFB*, *PDGFRB*, and *XPR1* case subjects and there was no calcification in the pons as seen in *MYORG* subjects.[@bib1]^,^[@bib17] In contrast, the calcification pattern observed in *JAM2* subjects differs to that of other tight junction genes, as *OCLN* subjects have band-like calcification with simplified gyration[@bib6] and *JAM3* subjects present multifocal intraparenchymal hemorrhage, massive cysts, and subependymal calcification.[@bib5]

In summary, we show that *JAM2* is recurrently mutated in families with recessive PFBC presenting with a combination of movement disorder and/or cognitive and psychiatric manifestations. The human phenotype was replicated in a *jam2* KO mouse model. The presence of mutations in several genes involved in the central NVU presenting clinically with brain calcification suggests that the NVU is likely to represent a potential therapeutic target in this group of disorders.

Declaration of Interests {#sec2}
========================

The authors declare that A. Begtrup and E.T. are employees of GeneDx, Inc., USA, and S.K. and C.B. are employees of CENTOGENE AG, Rostock, Germany. The other authors declare no competing interests.

Accession Numbers {#app5}
=================

The variants reported in this paper have been submitted to the Leiden Open Variation Database and the accession numbers are 643210, 643209, 643163, 643162.

Web Resources {#app3}
=============

CADD, <https://cadd.gs.washington.edu/>Ensembl Genome Browser, <http://www.ensembl.org/index.html>ExAC Browser, <http://exac.broadinstitute.org/>GeneMatcher, <https://genematcher.org/>GERP, <http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html>gnomAD Browser, <https://gnomad.broadinstitute.org/>HomozygosityMapper software, <http://www.homozygositymapper.org/>Leiden Open Variation Database, <https://www.lovd.nl/>Mutation Assessor, <http://mutationassessor.org/>MutationTaster, <http://www.mutationtaster.org/>OMIM, <https://www.omim.org/>PolyPhen-2, <http://genetics.bwh.harvard.edu/pph2/>PROVEAN, <http://provean.jcvi.org>QuPath, <https://qupath.github.io/>SIFT, <https://sift.bii.a-star.edu.sg/>

Accession numbers {#app4}
=================

The variants reported in this paper have been submitted to the Leiden Open Variation Database and the accession numbers are 00286201, 00286202, 00286204, 00286250.

Supplemental Data {#app2}
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Document S1. Figures S1--S7Document S2. Article plus Supplemental Information

We are grateful to the patients for their essential help. We are grateful for the funding support from The Medical Research Council (MRC) (MR/S01165X/1, MR/S005021/1), The Wellcome Trust (Synaptopathies Strategic Award, WT093205MA, WT104033AIA), The Rosetree Trust, 10.13039/501100000346Ataxia UK, The MSA Trust, 10.13039/100013790Brain Research UK, Sparks GOSH Charity, Muscular Dystrophy UK (MDUK), and 10.13039/100005202Muscular Dystrophy Association (MDA USA). We also thank James Polke and Conceição Bettencourt for assistance with the text and interpretation. S.B. and H.H. are partly funded by the National Institute of Health Research (NIHR) UCLH/UCL Biomedical Research Centre and Dementia Biomedical Research Unit. J.R.M.deO. receives funding from CNP, 10.13039/501100002322CAPES, and DECIT-MS. We thank Joanne Lau for technical support. This research was also conducted as part of the Queen Square Genomics group at UCL, supported by the 10.13039/501100000272National Institute for Health Research (NIHR) award to UCLH NHS Trust/UCL Biomedical Research Centre (BRC).

Supplemental Data can be found online at <https://doi.org/10.1016/j.ajhg.2020.02.007>.
