Interferon signaling gene expression as a diagnostic biomarker for monogenic interferonopathies.

Interferon signaling gene (ISG) expression scores are potential markers of inflammation with significance from cancer to genetic syndromes. In Aicardi Goutières Syndrome (AGS), a disorder of abnormal DNA and RNA metabolism, this score has potential as a diagnostic biomarker, although the approach to ISG calculation has not been standardized or validated. To optimize ISG calculation and validate ISG as a diagnostic biomarker, mRNA levels of 36 type I interferon response genes were quantified from 997 samples (including 334 AGS), and samples were randomized into training and test datasets. An independent validation cohort (n = 122) was also collected. ISGs were calculated using all potential combinations up to 6 genes. A 4-gene approach (IFI44L, IFI27, USP18, IFI6) was the best-performing model [area under the curve (AUC) of 0.8872 (training dataset), 0.9245 (test dataset)]. The majority of top performing gene combinations included IFI44L. Performance of IFI44L-alone was 0.8762 (training dataset) and 0.9580 (test dataset) by AUC. The top approaches were able to discriminate cases of genetic interferonopathy from control samples. This study validates the context of use for the ISG score as a diagnostic biomarker and underscores the importance of IFI44L in diagnosis of genetic interferonopathies.

Consensus guidelines for the monitoring and management of metachromatic leukodystrophy in the United States.

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care.

Effective Gene Therapy for Metachromatic Leukodystrophy Achieved with Minimal Lentiviral Genomic Integrations.

Metachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-old Arsa -KO MLD mice transplanted with Arsa -KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Systematic analysis of genotype-phenotype variability in siblings with Aicardi Goutières Syndrome (AGS).

OBJECTIVE: Aicardi Goutières Syndrome (AGS) is a genetic interferonopathy associated with multisystemic heterogeneous disease and neurologic dysfunction. AGS includes a broad phenotypic spectrum which is only partially explained by genotype. To better characterize this variability, we will perform a systematic analysis of phenotypic variability in familial cases of AGS. METHODS: Among thirteen families, twenty-six siblings diagnosed with AGS were identified from the Myelin Disorders and Biorepository Project (MDBP) at the Children's Hospital of Philadelphia. Data were collected on the age of onset, genotype, neurologic impairment, and systemic complications. Neurologic impairment was assessed by a disease-specific scale (AGS Severity Scale) at the last available clinical encounter (range: 0-11 representing severe - attenuated phenotypes). The concordance of clinical severity within sibling pairs was categorized based on the difference in AGS Scale (discordant defined as >2-unit difference). The severity classifications were compared between sibling sets and by genotype. RESULTS: Five genotypes were represented: TREX1 (n = 4 subjects), RNASEH2B (n = 8), SAMHD1 (n = 8) ADAR1 (n = 4), and IFIH1 (n = 2). The older sibling was diagnosed later relative to the younger affected sibling (median age 7.32 years [IQR = 14.1] compared to 1.54 years [IQR = 10.3]). Common presenting neurologic symptoms were tone abnormalities (n = 10/26) and gross motor dysfunction (n = 9/26). Common early systemic complications included dysphagia and chilblains. The overall cohort median AGS severity score at the last encounter was 8, while subjects presenting with symptoms before one year had a median score of 5. The TREX1 cohort presented at the youngest age and with the most severe phenotype on average. AGS scores were discordant for 5 of 13 sibling pairs, most commonly in the SAMHD1 pairs. Microcephaly, feeding tube placement, seizures and earlier onset sibling were associated with lower AGS scores (respectively, Wilcoxon rank sum: p = 0.0001, p < 0.0001, p = 0.0426, and Wilcoxon signed rank: p = 0.0239). CONCLUSIONS: In this systematic analysis of phenotypic variability in familial cases, we found discordance between siblings affected by AGS. Our results underscore the heterogeneity of AGS and suggest factors beyond AGS genotype may affect phenotype. Understanding the critical variables associated with disease onset and severity can guide future therapeutic interventions and clinical monitoring. This report reinforces the need for further studies to uncover potential factors to better understand this phenotypic variability, and consequently identify potential targets for interventions in attempt to change the natural history of the disease.

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.

Dyschromatosis symmetrica hereditaria: A clue to early diagnosis of Aicardi-Goutières syndrome.

A 6-year-old female with a history of Aicardi-Goutières syndrome (AGS) presented to dermatology clinic with hypopigmented and hyperpigmented macules and patches consistent with dyschromatosis symmetrica hereditaria (DSH). Previous genetic workup demonstrated a de novo, heterozygous mutation in the adenosine deaminase acting on RNA 1 (ADAR) gene. While the co-occurrence of AGS and DSH has previously been described in mutations of the ADAR gene, our case highlights the potential association between these disorders that may aid in earlier future diagnosis of AGS.

Neurodegenerative disease after hematopoietic stem cell transplantation in metachromatic leukodystrophy.

OBJECTIVE: Metachromatic leukodystrophy is a lysosomal storage disease caused by deficient arylsulfatase A. It is characterized by progressive demyelination and thus mainly affects the white matter. Hematopoietic stem cell transplantation may stabilize and improve white matter damage, yet some patients deteriorate despite successfully treated leukodystrophy. We hypothesized that post-treatment decline in metachromatic leukodystrophy might be caused by gray matter pathology. METHODS: Three metachromatic leukodystrophy patients treated with hematopoietic stem cell transplantation with a progressive clinical course despite stable white matter pathology were clinically and radiologically analyzed. Longitudinal volumetric MRI was used to quantify atrophy. We also examined histopathology in three other patients deceased after treatment and compared them with six untreated patients. RESULTS: The three clinically progressive patients developed cognitive and motor deterioration after transplantation, despite stable mild white matter abnormalities on MRI. Volumetric MRI identified cerebral and thalamus atrophy in these patients, and cerebellar atrophy in two. Histopathology showed that in brain tissue of transplanted patients, arylsulfatase A expressing macrophages were clearly present in the white matter, but absent in the cortex. Arylsulfatase A expression within patient thalamic neurons was lower than in controls, the same was found in transplanted patients. INTERPRETATION: Neurological deterioration may occur after hematopoietic stem cell transplantation in metachromatic leukodystrophy despite successfully treated leukodystrophy. MRI shows gray matter atrophy, and histological data demonstrate absence of donor cells in gray matter structures. These findings point to a clinically relevant gray matter component of metachromatic leukodystrophy, which does not seem sufficiently affected by transplantation.

Novel biallelic variants in NRROS associated with a lethal microgliopathy, brain calcifications, and neurodegeneration.

Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat protein expressed by microglia and perivascular macrophages. To date, 9 individuals have been reported with biallelic NRROS variants. Here, we report one individual with a severe neurodegenerative phenotype in which exome sequencing identified 2 novel variants in NRROS, a missense variant (c.185T>C, p.Leu62Pro) and a premature stop codon (c.310C>T, p.Gln104Ter). Pathological examination revealed both extensive grey and white matter involvement, dystrophic calcifications, and infiltration of foamy macrophages. This is the first reported case of NRROS variants with a mitochondrial ultrastructure abnormality noted on electron microscopy analysis of post-mortem tissue.

Early-Onset Vascular Leukoencephalopathy Caused by Bi-Allelic NOTCH3 Variants.

OBJECTIVE: Heterozygous NOTCH3 variants are known to cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), with patients typically presenting in adulthood. We describe three patients presenting at an early age with a vascular leukoencephalopathy. Genome sequencing revealed bi-allelic variants in the NOTCH3 gene. METHODS: Clinical records and available MRI and CT scans of three patients from two unrelated families were retrospectively reviewed. RESULTS: The patients presented at 9 to 14 months of age with developmental delay, seizures, or both. The disease course was characterized by cognitive impairment and variably recurrent strokes, migraine attacks, and seizures. MRI findings pointed at a small vessel disease, with extensive cerebral white matter abnormalities, atrophy, lacunes in the basal ganglia, microbleeds, and microcalcifications. The anterior temporal lobes were spared. Bi-allelic cysteine-sparing NOTCH3 variants in exons 1, 32, and 33 were found. INTERPRETATION: This study indicates that bi-allelic loss-of-function NOTCH3 variants may cause a vascular leukoencephalopathy, distinct from CADASIL.

MRI surveillance of boys with X-linked adrenoleukodystrophy identified by newborn screening: Meta-analysis and consensus guidelines.

BACKGROUND: Among boys with X-Linked adrenoleukodystrophy, a subset will develop childhood cerebral adrenoleukodystrophy (CCALD). CCALD is typically lethal without hematopoietic stem cell transplant before or soon after symptom onset. We sought to establish evidence-based guidelines detailing the neuroimaging surveillance of boys with neurologically asymptomatic adrenoleukodystrophy. METHODS: To establish the most frequent age and diagnostic neuroimaging modality for CCALD, we completed a meta-analysis of relevant studies published between January 1, 1970 and September 10, 2019. We used the consensus development conference method to incorporate the resulting data into guidelines to inform the timing and techniques for neuroimaging surveillance. Final guideline agreement was defined as >80% consensus. RESULTS: One hundred twenty-three studies met inclusion criteria yielding 1285 patients. The overall mean age of CCALD diagnosis is 7.91 years old. The median age of CCALD diagnosis calculated from individual patient data is 7.0 years old (IQR: 6.0-9.5, n = 349). Ninety percent of patients were diagnosed between 3 and 12. Conventional MRI was most frequently reported, comprised most often of T2-weighted and contrast-enhanced T1-weighted MRI. The expert panel achieved 95.7% consensus on the following surveillance parameters: (a) Obtain an MRI between 12 and 18 months old. (b) Obtain a second MRI 1 year after baseline. (c) Between 3 and 12 years old, obtain a contrast-enhanced MRI every 6 months. (d) After 12 years, obtain an annual MRI. CONCLUSION: Boys with adrenoleukodystrophy identified early in life should be monitored with serial brain MRIs during the period of highest risk for conversion to CCALD.

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.

Metachromatic leukodystrophy and transplantation: remyelination, no cross-correction.

OBJECTIVE: In metachromatic leukodystrophy, a lysosomal storage disorder due to decreased arylsulfatase A activity, hematopoietic stem cell transplantation may stop brain demyelination and allow remyelination, thereby halting white matter degeneration. This is the first study to define the effects and therapeutic mechanisms of hematopoietic stem cell transplantation on brain tissue of transplanted metachromatic leukodystrophy patients. METHODS: Autopsy brain tissue was obtained from eight (two transplanted and six nontransplanted) metachromatic leukodystrophy patients, and two age-matched controls. We examined the presence of donor cells by immunohistochemistry and microscopy. In addition, we assessed myelin content, oligodendrocyte numbers, and macrophage phenotypes. An unpaired t-test, linear regression or the nonparametric Mann-Whitney U-test was performed to evaluate differences between the transplanted, nontransplanted, and control group. RESULTS: In brain tissue of transplanted patients, we found metabolically competent donor macrophages expressing arylsulfatase A distributed throughout the entire white matter. Compared to nontransplanted patients, these macrophages preferentially expressed markers of alternatively activated, anti-inflammatory cells that may support oligodendrocyte survival and differentiation. Additionally, transplanted patients showed higher numbers of oligodendrocytes and evidence for remyelination. Contrary to the current hypothesis on therapeutic mechanism of hematopoietic cell transplantation in metachromatic leukodystrophy, we detected no enzymatic cross-correction to resident astrocytes and oligodendrocytes. INTERPRETATION: In conclusion, donor macrophages are able to digest accumulated sulfatides and may play a neuroprotective role for resident oligodendrocytes, thereby enabling remyelination, albeit without evidence of cross-correction of oligo- and astroglia. These results emphasize the importance of immunomodulation in addition to the metabolic correction, which might be exploited for improved outcomes.

Neuropathological Findings in a Case of IFIH1-Related Aicardi-Goutières Syndrome.

Aicardi-Goutières syndrome (AGS) is a rare syndrome characterized by calcification, diffuse demyelination, and variable degree of brain atrophy. The syndrome is genetically heterogeneous with mutations in 7 genes, including TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, and IFIH1 (interferon-induced helicase c domain-containing protein 1) associated with the syndrome, so far. These mutations lead to the overproduction of α-interferon within the central nervous system. Mutations in IFIH1 have been recently described in a subset of AGS, with only 1 previous report of neuropathological findings. We report neuropathological findings in a second case of AGS with a known mutation in IFIH1 gene. The patient is a 16-year-old adolescent boy with early-onset symptoms that progressed to profound loss of cognitive and motor functions. The patient experienced sudden cardiopulmonary arrest at the age of 16 years. At autopsy, the cause of death was determined to be pulmonary thromboembolism. Neuropathological examination revealed microcephaly (brain weight: 916 g) with relatively mild brain atrophy on gross examination. Microscopic examination revealed multifocal calcifications limited to small to medium central nervous system arteries (no evidence of calcification in other organs), involving bilateral cerebral cortex, basal ganglia, thalamus, and cerebellum. Ultrastructural examination showed Calcospherules limited to the vessel walls and the perivasulcar area without evidence of neuronal ferrugination or tubuloreticular bodies. The extent of calcifications was variable across different brain regions, resembling findings in previously reported cases and correlated with the extent of IFIH1 protein expression (data derived from Allen Brain Institute). AGS is a rare cause of brain calcifications that can closely mimic congenital and neonatal infections such as Rubella and similar infections.

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.

Adulthood leukodystrophies.

The leukodystrophies are a group of inherited white matter disorders with a heterogeneous genetic background, considerable phenotypic variability and disease onset at all ages. This Review focuses on leukodystrophies with major prevalence or primary onset in adulthood. We summarize 20 leukodystrophies with adult presentations, providing information on the underlying genetic mutations and on biochemical assays that aid diagnosis, where available. Definitions, clinical characteristics, age of onset, MRI findings and treatment options are all described, providing a comprehensive overview of the current knowledge of the various adulthood leukodystrophies. We highlight the distinction between adult-onset leukodystrophies and other inherited disorders with white matter involvement, and we propose a diagnostic pathway for timely recognition of adulthood leukodystrophies in a routine clinical setting. In addition, we provide detailed clinical information on selected adult-onset leukodystrophies, including X-linked adrenoleukodystrophy, metachromatic leukodystrophy, cerebrotendinous xanthomatosis, hereditary diffuse leukoencephalopathy with axonal spheroids, autosomal dominant adult-onset demyelinating leukodystrophy, adult polyglucosan body disease, and leukoencephalopathy with vanishing white matter. Ultimately, this Review aims to provide helpful suggestions to identify treatable adulthood leukodystrophies at an early stage in the disease course.

UFM1 founder mutation in the Roma population causes recessive variant of H-ABC.

OBJECTIVE: To identify the gene defect in patients with hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) who are negative for TUBB4A mutations. METHODS: We performed homozygosity mapping and whole exome sequencing (WES) to detect the disease-causing variant. We used a Taqman assay for population screening. We developed a luciferase reporter construct to investigate the effect of the promoter mutation on expression. RESULTS: Sixteen patients from 14 families from different countries fulfilling the MRI criteria for H-ABC exhibited a similar, severe clinical phenotype, including lack of development and a severe epileptic encephalopathy. The majority of patients had a known Roma ethnic background. Single nucleotide polymorphism array analysis in 5 patients identified one large overlapping homozygous region on chromosome 13. WES in 2 patients revealed a homozygous deletion in the promoter region of UFM1. Sanger sequencing confirmed homozygosity for this variant in all 16 patients. All patients shared a common haplotype, indicative of a founder effect. Screening of 1,000 controls from different European Roma panels demonstrated an overall carrier rate of the mutation of 3%-25%. Transfection assays showed that the deletion significantly reduced expression in specific CNS cell lines. CONCLUSIONS: UFM1 encodes ubiquitin-fold modifier 1 (UFM1), a member of the ubiquitin-like family involved in posttranslational modification of proteins. Its exact biological role is unclear. This study associates a UFM1 gene defect with a disease and sheds new light on possible UFM1 functional networks.

X-linked hypomyelination with spondylometaphyseal dysplasia (H-SMD) associated with mutations in AIFM1.

An X-linked condition characterized by the combination of hypomyelinating leukodystrophy and spondylometaphyseal dysplasia (H-SMD) has been observed in only four families, with linkage to Xq25-27, and recent genetic characterization in two families with a common AIFM1 mutation. In our study, 12 patients (6 families) with H-SMD were identified and underwent comprehensive assessment accompanied by whole-exome sequencing (WES). Pedigree analysis in all families was consistent with X-linked recessive inheritance. Presentation typically occurred between 12 and 36 months. In addition to the two disease-defining features of spondylometaphyseal dysplasia and hypomyelination on MRI, common clinical signs and symptoms included motor deterioration, spasticity, tremor, ataxia, dysarthria, cognitive defects, pulmonary hypertension, nystagmus, and vision loss due to retinopathy. The course of the disease was slowly progressive. All patients had maternally inherited or de novo mutations in or near exon 7 of AIFM1, within a region of 70 bp, including synonymous and intronic changes. AIFM1 mutations have previously been associated with neurologic presentations as varied as intellectual disability, hearing loss, neuropathy, and striatal necrosis, while AIFM1 mutations in this small region present with a distinct phenotype implicating bone. Analysis of cell lines derived from four patients identified significant reductions in AIFM1 mRNA and protein levels in osteoblasts. We hypothesize that AIFM1 functions in bone metabolism and myelination and is responsible for the unique phenotype in this condition.

Functionally pathogenic EARS2 variants in vitro may not manifest a phenotype in vivo.

OBJECTIVE: To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC). METHODS: Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity. RESULTS: Whole-exome sequencing uncovered compound heterozygous mutations in EARS2, c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA (SNORD118) responsible for LCC. CONCLUSIONS: Here, we report seemingly pathogenic EARS2 mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant. CLINICALTRIALSGOV IDENTIFIER: NCT00001671.