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

Lucia Schottlaender, Rosella Abeti, Zane Jaunmuktane, Carol Macmillan, Viorica Chelban, Benjamin O'callaghan, John Mckinley, Reza Maroofian, Stephanie Efthymiou, Alkyoni Athanasiou-Fragkouli, et al.

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## To cite this version:

Lucia Schottlaender, Rosella Abeti, Zane Jaunmuktane, Carol Macmillan, Viorica Chelban, et al. Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification. American Journal of Human Genetics, 2020, 106 (3), pp.412-421. 10.1016/j.ajhg.2020.02.007. hal-03155754

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

Lucia V. Schottlaender,<fnref n="1" />,²,³ Rosella Abeti,⁴ Zane Jaunmuktane,⁴,⁵ Carol Macmillan,⁶ Viorica Chelban,<fnref n="1" />

Benjamin O'Callaghan,<fnref n="1" /> John McKinley,⁷,⁸ Reza Maroofian,<fnref n="1" /> Stephanie Efthymiou,<fnref n="1" />

Alkyoni Athanasiou-Fragkouli,<fnref n="1" /> Raeburn Forbes,⁹ Marc P.M. Soutar,¹⁰ John H. Livingston,¹¹

Bernardett Kalmar,<fnref n="1" /> Orlando Swayne,⁴,¹² Gary Hotton,⁴,¹² SYNAPS Study Group,<fnref n="1" /> Alan Pittman,<fnref n="1" />

João Ricardo Mendes de Oliveira,¹³ Maria de Grandis,¹⁴ Angela Richard-Loendt,¹⁰

Francesca Launchbury,¹⁰ Juri Althonayan,⁴ Gavin McDonnell,⁸ Aisling Carr,<fnref n="1" />,¹² Suliman Khan,¹⁵

Christian Beetz,¹⁵ Atil Bisgin,¹⁶ Sevcan Tug Bozdogan,¹⁶ Amber Begtrup,¹⁷ Erin Torti,¹⁷

Linda Greensmith,<fnref n="1" /> Paola Giunti,⁴ Patrick J. Morrison,¹⁸ Sebastian Brandner,⁵ Michel Aurrand-Lions,¹⁴ and Henry Houlden<fnref n="1" />,¹²,¹⁹,*

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.<fnref n="1" /> 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 parkin-

sonism, who were previously clinically diagnosed, has significantly increased the number of PFBC-affected families identified.²

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).<fnref n="1" /> 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

<footnote n="1">¹Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, Queen Square, WC1N3BG London, UK; ²Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health, WC1N 1EH London, UK; ³Argentine National Scientific and Technological Research Council (CONICET), C1425FQB Buenos Aires, Argentina; FLENI Neurological Research Institute, C1428 AQK Buenos Aires, Argentina; ⁴Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, WC1N3BG London, UK; ⁵Division of Neuropathology, The National Hospital for Neurology and Neurosurgery, University College London Hospitals NHS Foundation Trust, Queen Square, London WC1N 3BG, UK; ⁶Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA; ⁷Department of Neurology, Dublin Neurological Institute at the Mater Misericordiae University Hospital, 57 Eccles St, Dublin 7 DO7W7XF, Ireland; ⁸Regional Neurosciences Centre, Royal Victoria Hospital, Belfast BT12 6BA, UK; ⁹Neurology Centre, Southern HSC Trust, Craigavon Area Hospital, Portadown BT63 5QQ, UK; ¹⁰Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, UK; ¹¹Paediatric Neurology, The Leeds Teaching Hospitals NHS Trust, Leeds General Infirmary, Leeds LS1 3EX, UK; ¹²The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK; ¹³Universidade Federal de Pernambuco, Departamento de Neuropsiquiatria, Recife 50670-901, Brazil; ¹⁴Aix-Marseille University, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, 13009 Marseille, France; ¹⁵CENTOGENE AG, Rostock 18055, Germany; ¹⁶Medical Genetics Department of Medical Faculty & AGENTEM (Adana Genetic Diseases Diagnosis and Treatment Center), Çukurova University, Adana 01330, Turkey; ¹⁷GeneDx, 207 Perry Parkway, Gaithersburg, MD 20877, USA; ¹⁸Centre for Cancer Research and Cell Biology, Queens University, Belfast BT9 7AE, UK; ¹⁹Neurogenetics Laboratory and Clinical Service, The National Hospital for Neurology and Neurosurgery, Queen Square, London WC1N 3BG, UK</footnote>

<footnote>*Correspondence: h.houlden@ucl.ac.uk</footnote>

<footnote>https://doi.org/10.1016/j.ajhg.2020.02.007.</footnote>

<footnote>© 2020 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Clinical and Neuroimaging Features of JAM2-Related Disease</figcaption></figure>

(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

(legend continued on next page)
increase intracellular phosphate concentration and pro-
voke calcium phosphate precipitation.³

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.² The recessive brain calcification phenotype due to *MYORG*⁴ (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 *JAM3*⁵ (MIM: 613730) and *OCLN*⁶ (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 permeability⁷ and identifying a possible link between different PFBC causative genes.

Pathologically, human brains exhibit calcium salt de-
posits predominantly distributed around small blood ves-
sels,² and a reported subject with a *SLC20A2* mutation pre-
sented 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.⁸

Despite important progress in discovering the genetic ar-
chitecture of PFBC, more than half of the case subjects
remain genetically unsolved.²

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 mile-
stones. 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 un-
able 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 general-
ized slowing of cortical rhythms.

There were four affected individuals in the second fam-
ily. Two siblings (F2-III:2 and F2-III:3) presented with
borderline low IQ in childhood but had no definite phys-
ical limitations in early life. In their twenties they had so-
sial withdrawal and severe depression requiring treatment.
The disease progressed and examination at 41 and 39 years
revealed severe speech hypophonia, dysphagia, hypomi-
mia, 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. Treat-
ment 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 pre-
sented 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 demon-
strated a similar phenotype with dysarthria, abnormal pur-
suit with frequent saccadic intrusions, pronounced brady-
kinesia, extrapyramidal rigidity, and bilaterally extensor
plantar responses.

The proband in family 3 (F3-II:1) had normal early devel-
opment but later developed mild delay in fine motor and
language milestones that were progressive. He also devel-
oped mild coordination problems and autism spectrum
disorder (ASD) and received special education services at
school age. At age 11 years, repeat neuropsychologic

<footnote>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.</footnote>
<table><caption>Table 1. Clinical Features of Affected Individuals with JAM2 Bi-allelic Variants</caption><thead><tr><th></th><th>Individual F1-II:2</th><th>Individual F2-III:2</th><th>Individual F2-III:3</th><th>Individual F2-III:4</th><th>Individual F2-III:5</th><th>Individual F3-II:1</th><th>Individual F4-II:3</th></tr></thead><tbody><tr><td>cDNA sequence</td><td>c.685C>T</td><td>c.685C>T</td><td>c.685C>T</td><td>c.685C>T</td><td>c.685C>T</td><td>c.395-1dupG, c.323G>A</td><td>c.177_180delCAGA</td></tr><tr><td>Amino acid change</td><td>p.Arg229Ter</td><td>p.Arg229Ter</td><td>p.Arg229Ter</td><td>p.Arg229Ter</td><td>p.Arg229Ter</td><td>IVS4-1dupG, p.Arg108His</td><td>p.Arg60Ter</td></tr><tr><td>Zygosity</td><td>homozygous</td><td>homozygous</td><td>homozygous</td><td>homozygous</td><td>homozygous</td><td>compound heterozygous</td><td>homozygous</td></tr><tr><td>Gender (male/female)</td><td>male</td><td>male</td><td>female</td><td>male</td><td>male</td><td>male</td><td>female</td></tr><tr><td>Birth and early milestones</td><td>normal</td><td>normal</td><td>normal</td><td>normal</td><td>normal</td><td>normal</td><td>normal</td></tr><tr><td>Onset of symptoms</td><td>childhood</td><td>late 20s</td><td>late 30s</td><td>teenage</td><td>teenage</td><td>childhood</td><td>early childhood</td></tr><tr><td>Symptom at onset</td><td>cerebellar ataxia and cognitive decline</td><td>cognitive decline, depression</td><td>difficulty walking</td><td>depression, dysarthria</td><td>depression, dysarthria</td><td>autism spectrum disorder</td><td>seizures</td></tr><tr><td>Age at examination (in years)</td><td>24</td><td>41</td><td>39</td><td>40</td><td>49</td><td>15</td><td>7</td></tr><tr><td colspan="8" style="text-align: left; font-weight: bold;">Phenotype at Last Examination</td></tr><tr><td>Pyramidal syndrome</td><td>yes; increased tone, brisk reflexes, upgoing plantars</td><td>yes; increased tone, brisk reflexes, upgoing plantars</td><td>yes; increased tone, brisk reflexes, upgoing plantars</td><td>yes; increased tone, brisk reflexes, upgoing plantars</td><td>yes; increased tone, brisk reflexes, upgoing plantars</td><td>no</td><td>no</td></tr><tr><td>Cerebellar syndrome</td><td>yes; upper and lower limb ataxia, dysarthria, nystagmus</td><td>yes; upper and lower limb ataxia</td><td>yes; upper and lower limb ataxia</td><td>yes; upper and lower limb ataxia, dysarthria</td><td>yes; upper and lower limb ataxia, dysarthria</td><td>yes; upper and lower limb mild ataxia, nystagmus</td><td>no</td></tr><tr><td>Parkinsonism</td><td>yes; rigidity, bradykinesia.</td><td>yes; hypophonia, hypomimia, bradykinesia</td><td>yes; hypophonia, hypomimia, bradykinesia</td><td>yes; rigidity, bradykinesia</td><td>yes; rigidity, bradykinesia</td><td>no</td><td>no</td></tr><tr><td>Dystonia</td><td>yes; generalized</td><td>yes; limb dystonia and orofacial dyskinesias</td><td>yes; limb dystonia and orofacial dyskinesias</td><td>no</td><td>no</td><td>no</td><td>no</td></tr><tr><td>Other</td><td>seizures, ophthalmoplegia, PEG inserted in advance stage</td><td>PEG inserted in advance stage</td><td>became anarthric in advanced stage</td><td>-</td><td>-</td><td>autism spectrum disorder</td><td>-</td></tr><tr><td>Cognitive function</td><td>severe cognitive decline</td><td>memory decline with severe impaired recall</td><td>unable to comment on cognition due to anarthria.</td><td>severe cognitive decline</td><td>severe cognitive decline</td><td>decline in academic performance</td><td>normal for her age</td></tr><tr><td>Brain imaging calcification pattern</td><td>basal ganglia, thalamus, cerebellum, deep gray matter</td><td>basal ganglia, thalamus, cerebellum, deep gray matter</td><td>basal ganglia, thalamus, cerebellum, deep gray matter</td><td>basal ganglia, thalamus, cerebellum, deep gray matter</td><td>basal ganglia, thalamus, cerebellum, deep gray matter</td><td>basal ganglia, and frontal cortex</td><td>basal ganglia, dentate nucleus and cerebellar hemispheres</td></tr></tbody></table>
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 (butons, 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.Val132-GlyfsX9). 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
<figure><img src="image_4.png" /></figure>

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).

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 cere-

<caption>**Figure 2. Validation of JAM2 Variants Identified in This Study**</caption>

(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.

bellar 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 promi-

nent 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
A

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Behavioral Study on *jam2* KO Mice and Wild-Type (WT)</figcaption></figure>

(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 for example beam walking test of a *jam2* KO mouse and a control (wild-type).

B

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

rior 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. 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 pericytes⁷,⁹,¹⁰ and for *myo*-null mice developing calcifications at 9 months.⁴ 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.¹¹ *Ocln*-null mice present with similar calcification surrounding blood vessels as seen in an autopsied case,⁶ 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.⁵ 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

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 poste-
<figure><img src="image_8.png" /><figcaption>Figure 4. Brain Pathology in Control and *jam2* KO Mice of Young (6 Months Old) and Old Age (18 Months Old)</figcaption></figure>

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

*(legend continued on next page)*
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.<sup>12,13</sup> 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.<sup>14</sup>

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,<sup>5,6</sup> 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,<sup>13</sup> 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*.<sup>15</sup> 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).

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.<sup>16</sup> It was indistinguishable from *SLC20A2*, *PDGFB*, *PDGFRB*, and *XPR1* case subjects and there was no calcification in the pons as seen in *MYORG* subjects.<sup>1,17</sup> 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<sup>6</sup> and *JAM3* subjects present multifocal intraparenchymal hemorrhage, massive cysts, and subependymal calcification.<sup>5</sup>

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.

## Accession Numbers

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

## Supplemental Data

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

## Acknowledgments

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, Ataxia UK, The MSA Trust, Brain Research UK, Sparks GOSH Charity, Muscular Dystrophy UK (MDUK), and Muscular 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, CAPES, and DECIT-MS. We thank Joanne Lau for technical support. This research was

<footnote>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.</footnote>
also conducted as part of the Queen Square Genomics group at UCL, supported by the National Institute for Health Research (NIHR) award to UCLH NHS Trust/UCL Biomedical Research Centre (BRC).

Declaration of Interests

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

Received: October 9, 2019

Accepted: February 10, 2020

Published: March 5, 2020

Web Resources

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/

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