Generation of three induced pluripotent stem cell lines from individuals with Aicardi-Goutières syndrome caused by a c.3019G>A (p.G1007R) autosomal dominant pathogenic variant in ADAR1.

Mutations in Adenosine deaminase acting on RNA 1 (ADAR1) gene encoding RNA editing enzyme ADAR1 results in the neuroinflammatory leukodystrophy Aicardi Goutières Syndrome (AGS). AGS is an early onset leukoencephalopathy with an exacerbated interferon response leading to neurological regression with intellectual disability, spasticity, and motor deficits. We have generated three induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of individuals with ADAR1G1007R mutation. The generated iPSCs were investigated to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The reprogrammed iPSCs will allow us to model AGS, dissect the cellular mechanisms and testing different treatment targets.

Generation of three induced Pluripotent Stem Cell lines from individuals with Hypomyelination with Atrophy of Basal Ganglia and Cerebellum caused by a c.745G>A (p.D249N) autosomal dominant mutation in TUBB4A.

Mutations in tubulin alpha 4a (TUBB4A) result in a spectrum of leukodystrophies, including Hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC), resulting from a recurring mutation p.Asp249Asn (TUBB4AD249N). H-ABC presents with dystonia, motor and cognitive impairment and pathological features of hypomyelination and loss of cerebellar and striatal neurons. We have generated three induced pluripotent stem cell (iPSC) lines from fibroblast and peripheral blood mononuclear cells (PBMCs) of individuals with TUBB4AD249N mutation. The iPSCs were assessed to confirm a normal karyotype, pluripotency, and trilineage differentiation potential. The iPSCs will allow for disease modeling, understanding mechanisms and testing of therapeutic targets.

SARS-CoV-2 mRNA-based vaccines in the Aicardi Goutières Syndrome.

Aicardi Goutières Syndrome (AGS) is an autoinflammatory disorder resulting in sustained interferon activation through defects in nucleic acid modification and sensing pathways. Thus, mRNA-based vaccination used against SARS-CoV-2, raise disease-specific safety concerns. To assess interferon signaling, we tested mRNA SARS-CoV-2 vaccines in AGS whole blood samples. Interferon activation is measured through quantitation of interferon signaling gene (ISG) expression and is increased in AGS patients. There was no increase in ISG scores from baseline following treatment with the nucleoside modified mRNA formulation compared to an increase with unmodified. A patient-family survey reported that the vaccines were well tolerated. These findings suggest that COVID vaccination using nucleoside-modified forms of mRNA vaccines are unlikely to directly stimulate ISG expression in response to mRNA internalization in AGS tissues. With continued community spread, we recommend vaccination using nucleoside-modified mRNA vaccines in this rare disease group in individuals for whom vaccines were previously well tolerated.

Generation of human induced pluripotential stem cells from individuals with complex heterozygous, isogenic corrected, and homozygous Bloc1s1 mutations.

Genetic studies show that BLOC1S1 modulates mitochondrial and endosome-lysosome function (Wu et al., 2021a). Furthermore, Bloc1s1 mutations are linked to leukodystrophy (Bertoli-Avella et al., 2021). The Vanderver laboratory identified additional individuals with leukodystrophy that harbored either complex heterozygous (Bloc1s1 c.206A > C and c.359G > A), or homozygous (Bloc1s1 c.185 T > C) point mutations. We generated induced pluripotential stem cell (iPSC) lines from these subjects, from parents of the complex heterozygous mutations patient, and from CRISPR isogenic (c.206A > C and c.359G > A) corrected iPSC-line. These complex heterozygous, homozygous, and isogenic-corrected Bloc1s1 lines were phenotypically normal and were capable of differentiation towards the three germ layers.

Hepatic Involvement in Aicardi-Goutières Syndrome.

Aicardi-Goutières syndrome (AGS) is a monogenic type-I interferonopathy that results in neurologic injury. The systemic impact of sustained interferon activation is less well characterized. Liver inflammation is known to be associated with the neonatal form of AGS, but the incidence of AGS-related hepatitis across lifespan is unknown.We compared natural history data including liver enzyme levels with markers of inflammation, (liver-specific autoantibodies and interferon signaling gene expression[ISG] scores). Liver enzymes were classified as normal or elevated by the fold increase over the upper limit of normal (ULN). The highest increases were designated as hepatitis, defined as aspartate-aminotransferase or alanine-aminotransferase threefold ULN, or gamma-glutamyl transferase 2.5-fold ULN. A larger cohort was used to further characterize the longitudinal incidence of liver abnormalities and the association with age and genotype.Across the AGS cohort (n = 102), elevated liver enzymes were identified in 76 individuals (74.5%) with abnormalities at a level consistent with hepatitis in 29 individuals (28.4%). SAMHD1 mutations were less likely to be associated with hepatitis (log-rank test; p = 0.011). Hepatitis was associated with early-onset disease and microcephaly (log-rank test; microcephaly p = 0.0401, age onset p = 0.0355). While most subjects (n = 20/33) were found to have liver-specific autoantibodies, there was no association between the presence of autoantibodies or ISG scores with hepatitis-level enzyme elevations.In conclusion, all genotypes of AGS are associated with transient elevations of liver enzymes and the presence of liver-associated autoantibodies. This adds to our growing understanding of the systemic pathology AGS.

Cerebral Microangiopathy in Leukoencephalopathy With Cerebral Calcifications and Cysts: A Pathological Description.

Leukoencephalopathy with calcifications and cysts (LCC) is a neurological syndrome recently associated with pathogenic variants in SNORD118. We report autopsy neuropathological findings from an individual with genetically confirmed LCC. Histologic studies included staining of formalin-fixed paraffin-embedded tissue sections by hematoxylin and eosin, elastic van Gieson, and luxol fast blue. Immunohistochemistry stains against glial fibrillary acidic protein, proteolipid protein, phosphorylated neurofilament, CD31, alpha-interferon, LN3, and inflammatory markers were performed. Gross examination revealed dark tan/gray appearing white matter with widespread calcifications. Microscopy revealed a diffuse destructive process due to a vasculopathy with secondary ischemic lesions and mineralization. The vasculopathy involved clustered small vessels, resembling vascular malformations, and sporadic lymphocytic infiltration of vessel walls. The white matter was also diffusely abnormal, with concurrent loss of myelin and axons, tissue rarefaction with multifocal cystic degeneration, and the presence of foamy macrophages, secondary calcifications, and astrogliosis. The midbrain, pons, and cerebellum were diffusely involved. It is not understood why variants in SNORD118 result in a disorder that predominantly causes neurological disease and significantly disrupts the cerebral vasculature. Clinical and radiological benefit was recently reported in an LCC patient treated with Bevacizumab; it is important that these patients are rapidly diagnosed and trial of this treatment modality is considered in appropriate circumstances.

TUBB4A mutations result in both glial and neuronal degeneration in an H-ABC leukodystrophy mouse model.

Mutations in TUBB4A result in a spectrum of leukodystrophy including Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC), a rare hypomyelinating leukodystrophy, often associated with a recurring variant p.Asp249Asn (D249N). We have developed a novel knock-in mouse model harboring heterozygous (Tubb4aD249N/+) and the homozygous (Tubb4aD249N/D249N) mutation that recapitulate the progressive motor dysfunction with tremor, dystonia and ataxia seen in H-ABC. Tubb4aD249N/D249N mice have myelination deficits along with dramatic decrease in mature oligodendrocytes and their progenitor cells. Additionally, a significant loss occurs in the cerebellar granular neurons and striatal neurons in Tubb4aD249N/D249N mice. In vitro studies show decreased survival and dysfunction in microtubule dynamics in neurons from Tubb4aD249N/D249N mice. Thus Tubb4aD249N/D249N mice demonstrate the complex cellular physiology of H-ABC, likely due to independent effects on oligodendrocytes, striatal neurons, and cerebellar granule cells in the context of altered microtubule dynamics, with profound neurodevelopmental deficits.

Inside human and other animal cells, filaments known as microtubules help support the shape of the cell and move proteins to where they need to be. Defects in microtubules may lead to disease. For example, genetic mutations affecting a microtubule component called TUBB4A cause a rare brain disease in humans known as H-ABC. Individuals with H-ABC display many symptoms including abnormal walking, speech defects, impaired swallowing, and several cognitive defects. Abnormalities in several areas of the brain, including the cerebellum and striatum contribute to these defects. . In these structures, the neurons that carry messages around the brain and their supporting cells, known as oligodendrocytes, die, which causes these parts of the brain to gradually waste away. At this time, there are no therapies available to treat H-ABC. Furthermore, research into the disease has been hampered by the lack of a suitable "model" in mice or other laboratory animals. To address this issue, Sase, Almad et al. generated mice carrying a mutation in a gene which codes for the mouse equivalent of the human protein TUBB4A. Experiments showed that the mutant mice had similar physical symptoms to humans with H-ABC, including an abnormal walking gait, poor coordination and involuntary movements such as twitching and reduced reflexes. H-ABC mice had smaller cerebellums than normal mice, which was consistent with the wasting away of the cerebellum observed in individuals with H-ABC. The mice also lost neurons in the striatum and cerebellum, and oligodendrocytes in the brain and spinal cord. Furthermore, the mutant TUBB4A protein affected the behavior and formation of microtubules in H-ABC mice. The findings of Sase, Almad et al. provide the first mouse model that shares many features of H-ABC disease in humans. This model provides a useful tool to study the disease and develop potential new therapies.

Type II Alexander disease caused by splicing errors and aberrant overexpression of an uncharacterized GFAP isoform.

Alexander disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander disease.

Genome sequencing in persistently unsolved white matter disorders.

Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease-associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

Bi-allelic CSF1R Mutations Cause Skeletal Dysplasia of Dysosteosclerosis-Pyle Disease Spectrum and Degenerative Encephalopathy with Brain Malformation.

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

Aicardi goutières syndrome is associated with pulmonary hypertension.

While pulmonary hypertension (PH) is a potentially life threatening complication of many inflammatory conditions, an association between Aicardi Goutières syndrome (AGS), a rare genetic cause of interferon (IFN) overproduction, and the development of PH has not been characterized to date. We analyzed the cardiac function of individuals with AGS enrolled in the Myelin Disorders Bioregistry Project using retrospective chart review (n = 61). Additional prospective echocardiograms were obtained when possible (n = 22). An IFN signature score, a marker of systemic inflammation, was calculated through the measurement of mRNA transcripts of type I IFN-inducible genes (interferon signaling genes or ISG). Pathologic analysis was performed as available from autopsy samples. Within our cohort, four individuals were identified to be affected by PH: three with pathogenic gain-of-function mutations in the IFIH1 gene and one with heterozygous TREX1 mutations. All studied individuals with AGS were noted to have elevated IFN signature scores (Mann-Whitney p < .001), with the highest levels in individuals with IFIH1 mutations (Mann-Whitney p < .0001). We present clinical and histologic evidence of PH in a series of four individuals with AGS, a rare interferonopathy. Importantly, IFIH1 and TREX1 may represent a novel cause of PH. Furthermore, these findings underscore the importance of screening all individuals with AGS for PH.

Astrocytes, an active player in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is an early-onset, autoimmune and genetically heterogeneous disorder with severe neurologic injury. Molecular studies have established that autosomal recessive mutations in one of the following genes are causative: TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1 and IFIH1/MDA5. The phenotypic presentation and pathophysiology of AGS is associated with over-production of the cytokine Interferon-alpha (IFN-α) and its downstream signaling, characterized as type I interferonopathy. Astrocytes are one of the major source of IFN in the central nervous system (CNS) and it is proposed that they could be key players in AGS pathology. Astrocytes are the most ubiquitous glial cell in the CNS and perform a number of crucial and complex functions ranging from formation of blood-brain barrier, maintaining ionic homeostasis, metabolic support to synapse formation and elimination in healthy CNS. Involvement of astrocytic dysfunction in neurological diseases-Alexander's disease, Epilepsy, Alzheimer's and amyotrophic lateral sclerosis (ALS)-has been well-established. It is now known that compromised astrocytic function can contribute to CNS abnormalities and severe neurodegeneration, nevertheless, its contribution in AGS is unclear. The current review discusses known molecular and cellular pathways for AGS mutations and how it stimulates IFN-α signaling. We shed light on how astrocytes might be key players in the phenotypic presentations of AGS and emphasize the cell-autonomous and non-cell-autonomous role of astrocytes. Understanding the contribution of astrocytes will help reveal mechanisms underlying interferonopathy and develop targeted astrocyte specific therapeutic treatments in AGS.

TUBB4A mutations result in specific neuronal and oligodendrocytic defects that closely match clinically distinct phenotypes.

Hypomyelinating leukodystrophies are heritable disorders defined by lack of development of brain myelin, but the cellular mechanisms of hypomyelination are often poorly understood. Mutations in TUBB4A, encoding the tubulin isoform tubulin beta class IVA (Tubb4a), result in the symptom complex of hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC). Additionally, TUBB4A mutations are known to result in a broad phenotypic spectrum, ranging from primary dystonia (DYT4), isolated hypomyelination with spastic quadriplegia, and an infantile onset encephalopathy, suggesting multiple cell types may be involved. We present a study of the cellular effects of TUBB4A mutations responsible for H-ABC (p.Asp249Asn), DYT4 (p.Arg2Gly), a severe combined phenotype with hypomyelination and encephalopathy (p.Asn414Lys), as well as milder phenotypes causing isolated hypomyelination (p.Val255Ile and p.Arg282Pro). We used a combination of histopathological, biochemical and cellular approaches to determine how these different mutations may have variable cellular effects in neurons and/or oligodendrocytes. Our results demonstrate that specific mutations lead to either purely neuronal, combined neuronal and oligodendrocytic or purely oligodendrocytic defects that closely match their respective clinical phenotypes. Thus, the DYT4 mutation that leads to phenotypes attributable to neuronal dysfunction results in altered neuronal morphology, but with unchanged tubulin quantity and polymerization, with normal oligodendrocyte morphology and myelin gene expression. Conversely, mutations associated with isolated hypomyelination (p.Val255Ile and p.Arg282Pro) and the severe combined phenotype (p.Asn414Lys) resulted in normal neuronal morphology but were associated with altered oligodendrocyte morphology, myelin gene expression, and microtubule dysfunction. The H-ABC mutation (p.Asp249Asn) that exhibits a combined neuronal and myelin phenotype had overlapping cellular defects involving both neuronal and oligodendrocyte cell types in vitro. Only mutations causing hypomyelination phenotypes showed altered microtubule dynamics and acted through a dominant toxic gain of function mechanism. The DYT4 mutation had no impact on microtubule dynamics suggesting a distinct mechanism of action. In summary, the different clinical phenotypes associated with TUBB4A reflect the selective and specific cellular effects of the causative mutations. Cellular specificity of disease pathogenesis is relevant to developing targeted treatments for this disabling condition.

Neonatal detection of Aicardi Goutières Syndrome by increased C26:0 lysophosphatidylcholine and interferon signature on newborn screening blood spots.

BACKGROUND: Aicardi Goutières Syndrome (AGS) is a heritable interferonopathy associated with systemic autoinflammation causing interferon (IFN) elevation, central nervous system calcifications, leukodystrophy and severe neurologic sequelae. An infant with TREX1 mutations was recently found to have abnormal C26:0 lysophosphatidylcholine (C26:0 Lyso-PC) in a newborn screening platform for X-linked adrenoleukodystrophy, prompting analysis of this analyte in retrospectively collected samples from individuals affected by AGS. METHODS: In this study, we explored C26:0 Lyso-PC levels and IFN signatures in newborn blood spots and post-natal blood samples in 19 children with a molecular and clinical diagnosis of AGS and in the blood spots of 22 healthy newborns. We used Nanostring nCounter™ for IFN-induced gene analysis and a high-performance liquid chromatography with tandem mass spectrometry (HPLC MS/MS) newborn screening platform for C26:0 Lyso-PC analysis. RESULTS: Newborn screening cards from patients across six AGS associated genes were collected, with a median disease presentation of 2months. Thirteen out of 19 (68%) children with AGS had elevations of first tier C26:0 Lyso-PC (>0.4µM), that would have resulted in a second screen being performed in a two tier screening system for X-linked adrenoleukodystrophy (X-ALD). The median (95%CI) of first tier C26:0 Lyso-PC values in AGS individuals (0.43µM [0.37-0.48]) was higher than that seen in controls (0.21µM [0.21-0.21]), but lower than X-ALD individuals (0.72µM [0.59-0.84])(p<0.001). Fourteen of 19 children had elevated expression of IFN signaling on blood cards relative to controls (Sensitivity 73.7%, 95%CI 51-88%, Specificity 95%, 95% CI 78-99%) including an individual with delayed disease presentation (36months of age). All five AGS patients with negative IFN signature at birth had RNASEH2B mutations. Consistency of agreement between IFN signature in neonatal and post-natal samples was high (0.85). CONCLUSION: This suggests that inflammatory markers in AGS can be identified in the newborn period, before symptom onset. Additionally, since C26:0 Lyso-PC screening is currently used in X-ALD newborn screening panels, clinicians should be alert to the fact that AGS infants may present as false positives during X-ALD screening.

Whole exome sequencing in patients with white matter abnormalities.

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.

Disease specific therapies in leukodystrophies and leukoencephalopathies.

Leukodystrophies are a heterogeneous, often progressive group of disorders manifesting a wide range of symptoms and complications. Most of these disorders have historically had no etiologic or disease specific therapeutic approaches. Recently, a greater understanding of the pathologic mechanisms associated with leukodystrophies has allowed clinicians and researchers to prioritize treatment strategies and advance research in therapies for specific disorders, some of which are on the verge of pilot or Phase I/II clinical trials. This shifts the care of leukodystrophy patients from the management of the complex array of symptoms and sequelae alone to targeted therapeutics. The unmet needs of leukodystrophy patients still remain an overwhelming burden. While the overwhelming consensus is that these disorders collectively are symptomatically treatable, leukodystrophy patients are in need of advanced therapies and if possible, a cure.

Aicardi-Goutières syndrome harbours abundant systemic and brain-reactive autoantibodies.

OBJECTIVES: Aicardi-Goutières syndrome (AGS) is an autoimmune disorder that shares similarities with systemic lupus erythematous. AGS inflammatory responses specially target the cerebral white matter. However, it remains uncertain why the brain is the most affected organ, and little is known about the presence of autoantibodies in AGS. Here, we aim to profile specific autoantibodies in AGS and to determine whether these autoantibodies target cerebral epitopes. METHODS: Using a multiplex microarray, we assessed the spectrum of serum autoantibodies in 56 genetically confirmed patients with AGS. We investigated the presence of immunoglobulins in AGS brain specimens using immunohistochemistry and studied the reactivity of sera against brain epitopes with proteomics. RESULTS: Serum from patients exhibited high levels of IgGs against nuclear antigens (gP210, Nup62, PCNA, Ro/SSA, Sm/RNP complex, SS-A/SS-B), components of the basement membrane (entactin, laminin), fibrinogen IV and gliadin. Upon testing whether antibodies in AGS could be found in the central nervous system, IgGs were identified to target in vivo endothelial cells in vivo and astrocytes in brain sections of deceased patients with AGS. Using a proteomics approach, we were able to confirm that IgGs in serum samples from AGS patients bind epitopes present in the cerebral white matter. CONCLUSIONS: Patients with AGS produce a broad spectrum of autoantibodies unique from other autoimmune diseases. Some of these autoantibodies target endothelial cells and astrocytes in the brain of the affected patients, perhaps explaining the prominence of neurological disease in the AGS phenotype.

A de novo mutation in the ß-tubulin gene TUBB4A results in the leukoencephalopathy hypomyelination with atrophy of the basal ganglia and cerebellum.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and ß-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC's clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.