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Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification
2020; Elsevier BV; Volume: 106; Issue: 3 Linguagem: Inglês
10.1016/j.ajhg.2020.02.007
ISSN1537-6605
AutoresLucía Schottlaender, Rosella Abeti, Zane Jaunmuktane, Carol Macmillan, Viorica Chelban, Benjamin O’Callaghan, John McKinley, Reza Maroofian, Stéphanie Efthymiou, Alkyoni Athanasiou‐Fragkouli, Raeburn Forbes, Marc P. M. Soutar, John H. Livingston, Bernardett Kalmar, Orlando Swayne, Gary Hotton, Alan Pittman, João Ricardo Mendes de Oliveira, Maria De Grandis, Angela Richard-Loendt, Francesca Launchbury, Juri Althonayan, Gavin McDonnell, Aisling Carr, Suliman Khan, Christian Beetz, Atıl Bişgin, Sevcan Tuğ Bozdoğan, Amber Begtrup, Erin Torti, Linda Greensmith, Paola Giunti, Patrick J. Morrison, Sebastian Brandner, Michel Aurrand‐Lions, Henry Houlden, Stanislav Groppa, Blagovesta Marinova Karashova, Wolfgang Nachbauer, Sylvia Boesch, Larissa Arning, Dagmar Timmann, Bru Cormand, Belén Pérez‐Dueñas, Gabriella Di Rosa, Jatinder S. Goraya, Tipu Sultan, Jun Mine, Daniela Avdjieva, Hadil Kathom, Radka Tincheva, Selina Banu, Mercedes Pineda-Marfa, Pierangelo Veggiotti, Michel D. Ferrari, Alberto Verrotti, Gian Luigi Marseglia, Salvatore Savasta, Mayte García-Silva, Alfons Macaya Ruiz, Barbara Garavaglia, Eugenia Borgione, Simona Portaro, Benigno Monteagudo Sanchez, Richard G. Boles, Savvas Papacostas, Michail Vikelis, Eleni Zamba Papanicolaou, Efthimios Dardiotis, Shazia Maqbool, Shahnaz Ibrahim, Salman Kirmani, Nuzhat Rana, Osama Atawneh, Georgios Koutsis, Marianthi Breza, Salvatore Mangano, Carmela Scuderi, Eugenia Borgione, Giovanna Morello, Tanya Stojkovic, Massimi Zollo, Gali Heimer, Yves Dauvilliers, Pasquale Striano, Issam Al-Khawaja, Fuad Al-Mutairi, Sherifa A. Hamed,
Tópico(s)Parathyroid Disorders and Treatments
ResumoPrimary 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. 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. Primary familial brain calcification (PFBC [MIM: 213600]), 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.1Batla A. Tai X.Y. Schottlaender L. Erro R. Balint B. Bhatia K.P. Deconstructing Fahr's disease/syndrome of brain calcification in the era of new genes.Parkinsonism Relat. Disord. 2017; 37: 1-10Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar 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.2Taglia I. Bonifati V. Mignarri A. Dotti M.T. Federico A. Primary familial brain calcification: update on molecular genetics.Neurol. Sci. 2015; 36: 787-794Crossref PubMed Scopus (25) Google Scholar 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) and XPR1 (MIM: 605237).1Batla A. Tai X.Y. Schottlaender L. Erro R. Balint B. Bhatia K.P. Deconstructing Fahr's disease/syndrome of brain calcification in the era of new genes.Parkinsonism Relat. Disord. 2017; 37: 1-10Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar 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.3Legati A. Giovannini D. Nicolas G. López-Sánchez U. Quintáns B. Oliveira J.R.M. Sears R.L. Ramos E.M. Spiteri E. Sobrido M.-J. et al.Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export.Nat. Genet. 2015; 47: 579-581Crossref PubMed Scopus (163) Google Scholar 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) and PDGFRB (MIM: 173410) 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.2Taglia I. Bonifati V. Mignarri A. Dotti M.T. Federico A. Primary familial brain calcification: update on molecular genetics.Neurol. Sci. 2015; 36: 787-794Crossref PubMed Scopus (25) Google Scholar The recessive brain calcification phenotype due to MYORG4Yao X.-P. Cheng X. Wang C. Zhao M. Guo X.-X. Su H.-Z. Lai L.-L. Zou X.-H. Chen X.-J. Zhao Y. et al.Biallelic Mutations in MYORG Cause Autosomal Recessive Primary Familial Brain Calcification.Neuron. 2018; 98: 1116-1123.e5Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar (MIM: 618255) mutations, has been shown to present specific myorg mRNA expression in mouse astrocytes and disturb the normal function of the NVU. Mutations in JAM35Mochida G.H. Ganesh V.S. Felie J.M. Gleason D. Hill R.S. Clapham K.R. Rakiec D. Tan W.-H. Akawi N. Al-Saffar M. et al.A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts.Am. J. Hum. Genet. 2010; 87: 882-889Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar (MIM: 613730) and OCLN6O'Driscoll M.C. Daly S.B. Urquhart J.E. Black G.C.M. Pilz D.T. Brockmann K. McEntagart M. Abdel-Salam G. Zaki M. Wolf N.I. et al.Recessive mutations in the gene encoding the tight junction protein occludin cause band-like calcification with simplified gyration and polymicrogyria.Am. J. Hum. Genet. 2010; 87: 354-364Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar (MIM: 602876) 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 permeability7Jensen N. Schrøder H.D. Hejbøl E.K. Thomsen J.S. Brüel A. Larsen F.T. Vinding M.C. Orlowski D. Füchtbauer E.-M. Oliveira J.R.M. Pedersen L. Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.Am. J. Pathol. 2018; 188: 1865-1881Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar and identifying a possible link between different PFBC causative genes. Pathologically, human brains exhibit calcium salt deposits predominantly distributed around small blood vessels,2Taglia I. Bonifati V. Mignarri A. Dotti M.T. Federico A. Primary familial brain calcification: update on molecular genetics.Neurol. Sci. 2015; 36: 787-794Crossref PubMed Scopus (25) Google Scholar 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.8Kimura T. Miura T. Aoki K. Saito S. Hondo H. Konno T. Uchiyama A. Ikeuchi T. Takahashi H. Kakita A. Familial idiopathic basal ganglia calcification: Histopathologic features of an autopsied patient with an SLC20A2 mutation.Neuropathology. 2016; 36: 365-371Crossref PubMed Scopus (31) Google Scholar Despite important progress in discovering the genetic architecture of PFBC, more than half of the case subjects remain genetically unsolved.2Taglia I. Bonifati V. Mignarri A. Dotti M.T. Federico A. Primary familial brain calcification: update on molecular genetics.Neurol. Sci. 2015; 36: 787-794Crossref PubMed Scopus (25) Google Scholar 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) 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 1A). Clinical features of affected individuals are presented in Table 1. 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). Occasional seizures were seen later in the disease course. The interictal electroencephalography (EEG) showed moderate generalized slowing of cortical rhythms.Table 1Clinical Features of Affected Individuals with JAM2 Bi-allelic VariantsIndividual F1-II:2Individual F2-III:2Individual F2-III:3Individual F2-III:4Individual F2-III:5Individual F3-II:1Individual F4-II:3cDNA 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)2441394049157Phenotype at Last ExaminationPyramidal 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 Open table in a new tab eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiM2RlMTFkNTk5NGMyZGJhNWRkOTA2ZGE1NWVkY2Q5YyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQxODY3MDUyfQ.lrQeP_HyN0c0bkZvKFw8jnAEtLRvCEwcMpP_wOvyjCkmltYEBefuNBjJngheAsqKEqKUWOFx2rKI9lYffsUYACddNzRdWPH_CI7inLTTH-g1yuRvOvsf6uuHVDQBEKprr1IZdKixasrUcmTfvfSn6QBe6s1rMQgADxfYo1wkK1XZE3Nbl71MIN5360rYNA-tNhzoAamKPvuMi0zms2arfhQj2-p54MoHUuMsk-JRlm5KJ7uExSwZN_F4a8hHup7QuizP480h79WWsO4Cyt-gJqNv4msrhbEqzaaVfXuTX6ktLuTaErjHAeGJrEvOWCOjSN2iSEg4-f0JqTP5eqOhQA Download .mp4 (93.39 MB) Help with .mp4 files 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 1B). 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; c.685C>T [p.Arg229Ter]) in JAM2 residing within a 5 Mb region of homozygosity on chr21q21.3 (Figure S1). Sanger sequencing verified the correct segregation of the variant on available samples of both families (Figures 1A, 2A, 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 2C). Furthermore, western blot analysis confirmed the absence of JAM2 protein in the homozygous proband (Figure 2D). 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). In family 3, Trio-ES revealed compound heterozygous variants in JAM2 (GenBank: NM_021219; 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 1A, 2A, and 2B). In family 4, clinical ES uncovered a homozygous 4 bp deletion in JAM2 (GenBank: NM_021219; c.177_180delCAGA [p.Arg60Ter]) (Figures 1A, 2A, 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). 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 3A). 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 3B and 3C, Video S2). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMzEwZTBmYjA0Y2NiYTkwOTVjNTYxMGNiYjA3ZTNiNCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQxODY3MDUyfQ.BeueLFomGeoGzfDoq00EvgyQPQDdiCOJIQtmsi-RX3W9Ci5FdIiuIWJfJrQjnLoFf1C_YoEjENVXAg8ZKHBtO04nZhgT505PNTea6gsA5prsIRvkhmPjZzgVibiWm1Ie-OXh14pqVtBPjbUIlTfGqp6s1lGO6pWCu6aeF4GgFqXnTDMYxrPDz6W1kWIfYWksVsSlMhmbITcl-vYkj1uy_go0K5V13R9becqkTwArgHY8JuSvNMqYllYCgd_VIS0N2j_8n11wsCcc3DrSCiMsa7dEz5JTH1naQ-L3xPffkXVHIDYiniFt57ZjFi7yaZtSUnMV-44PBYVNuBQZ6WjfdQ Download .mp4 (44.65 MB) Help with .mp4 files 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). 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). 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. 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). 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 and S6). Mouse models of other brain calcification genes have shown brain calcification in similar areas to those found in humans. 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