Unclassified white matter disorders: A diagnostic journey requiring close collaboration between clinical and laboratory services.

BACKGROUND: Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended. AIM: This study aimed to identify the genetic causes for a group of individuals with unclassified white matter disorders with suspected genetic aetiology and highlight the investigations required when the initial testing is non-diagnostic. METHODS: Twenty-six individuals from 22 families with unclassified white matter disorders underwent deep phenotyping and genome sequencing performed on trio, or larger, family groups. Functional studies and transcriptomics were used to resolve variants of uncertain significance with potential clinical relevance. RESULTS: Causative or candidate variants were identified in 15/22 (68.2%) families. Six of the 15 implicated genes had been previously associated with white matter disease (COL4A1, NDUFV1, SLC17A5, TUBB4A, BOLA3, DARS2). Patients with variants in the latter two presented with an atypical phenotype. The other nine genes had not been specifically associated with white matter disease at the time of diagnosis and included genes associated with monogenic syndromes, developmental disorders, and developmental and epileptic encephalopathies (STAG2, LSS, FIG4, GLS, PMPCA, SPTBN1, AGO2, SCN2A, SCN8A). Consequently, only 46% of the diagnoses would have been made via a current leukodystrophy gene panel test. DISCUSSION: These results confirm the importance of broad genomic testing for patients with white matter disorders. The high diagnostic yield reflects the integration of deep phenotyping, whole genome sequencing, trio analysis, functional studies, and transcriptomic analyses. CONCLUSIONS: Genetic white matter disorders are genetically and phenotypically heterogeneous. Deep phenotyping together with a range of genomic technologies underpin the identification of causes of unclassified white matter disease. A molecular diagnosis is essential for prognostication, appropriate management, and accurate reproductive counseling.

Brain Abnormalities in Patients with Germline Variants in H3F3: Novel Imaging Findings and Neurologic Symptoms Beyond Somatic Variants and Brain Tumors.

BACKGROUND AND PURPOSE: Pathogenic somatic variants affecting the genes Histone 3 Family 3A and 3B (H3F3) are extensively linked to the process of oncogenesis, in particular related to central nervous system tumors in children. Recently, H3F3 germline missense variants were described as the cause of a novel pediatric neurodevelopmental disorder. We aimed to investigate patterns of brain MR imaging of individuals carrying H3F3 germline variants. MATERIALS AND METHODS: In this retrospective study, we included individuals with proved H3F3 causative genetic variants and available brain MR imaging scans. Clinical and demographic data were retrieved from available medical records. Molecular genetic testing results were classified using the American College of Medical Genetics criteria for variant curation. Brain MR imaging abnormalities were analyzed according to their location, signal intensity, and associated clinical symptoms. Numeric variables were described according to their distribution, with median and interquartile range. RESULTS: Eighteen individuals (10 males, 56%) with H3F3 germline variants were included. Thirteen of 18 individuals (72%) presented with a small posterior fossa. Six individuals (33%) presented with reduced size and an internal rotational appearance of the heads of the caudate nuclei along with an enlarged and squared appearance of the frontal horns of the lateral ventricles. Five individuals (28%) presented with dysgenesis of the splenium of the corpus callosum. Cortical developmental abnormalities were noted in 8 individuals (44%), with dysgyria and hypoplastic temporal poles being the most frequent presentation. CONCLUSIONS: Imaging phenotypes in germline H3F3-affected individuals are related to brain features, including a small posterior fossa as well as dysgenesis of the corpus callosum, cortical developmental abnormalities, and deformity of lateral ventricles.

Imaging Patterns Characterizing Mitochondrial Leukodystrophies.

BACKGROUND AND PURPOSE: Achieving a specific diagnosis in leukodystrophies is often difficult due to clinical and genetic heterogeneity. Mitochondrial defects cause 5%-10% of leukodystrophies. Our objective was to define MR imaging features commonly shared by mitochondrial leukodystrophies and to distinguish MR imaging patterns related to specific genetic defects. MATERIALS AND METHODS: One hundred thirty-two patients with a mitochondrial leukodystrophy with known genetic defects were identified in the data base of the Amsterdam Leukodystrophy Center. Numerous anatomic structures were systematically assessed on brain MR imaging. Additionally, lesion characteristics were scored. Statistical group analysis was performed for 57 MR imaging features by hierarchic testing on clustered genetic subgroups. RESULTS: MR imaging features indicative of mitochondrial disease that were frequently found included white matter rarefaction (n = 50 patients), well-delineated cysts (n = 20 patients), T2 hyperintensity of the middle blade of the corpus callosum (n = 85 patients), and symmetric abnormalities in deep gray matter structures (n = 42 patients). Several disorders or clusters of disorders had characteristic features. The combination of T2 hyperintensity in the brain stem, middle cerebellar peduncles, and thalami was associated with complex 2 deficiency. Predominantly periventricular localization of T2 hyperintensities and cystic lesions with a distinct border was associated with defects in complexes 3 and 4. T2-hyperintense signal of the cerebellar cortex was specifically associated with variants in the gene NUBPL. T2 hyperintensities predominantly affecting the directly subcortical cerebral white matter, globus pallidus, and substantia nigra were associated with Kearns-Sayre syndrome. CONCLUSIONS: In a large group of patients with a mitochondrial leukodystrophy, general MR imaging features suggestive of mitochondrial disease were found. Additionally, we identified several MR imaging patterns correlating with specific genotypes. Recognition of these patterns facilitates the diagnosis in future patients.

Severe Leukoencephalopathy with Clinical Recovery Caused by Recessive BOLA3 Mutations.

AIM: To identify the genetic aetiology of a distinct leukoencephalopathy causing acute neurological regression in infancy with apparently complete clinical recovery. METHODS: We performed trio whole genome sequencing (WGS) to determine the genetic basis of the disorder. Mitochondrial function analysis in cultured patient fibroblasts was undertaken to confirm the pathogenicity of candidate variants. RESULTS: The patient presented at 18 months with acute hemiplegia and cognitive regression without obvious trigger. This was followed by clinical recovery over 4 years. MRI at disease onset revealed bilateral T2 hyperintensity involving the periventricular and deep white matter and MR spectroscopy of frontal white matter demonstrated a lactate doublet. Lactate levels and mitochondrial respiratory chain enzyme activity in muscle, liver and fibroblasts were normal. Plasma glycine was elevated. The MRI abnormalities improved. WGS identified compound heterozygous variants in BOLA3: one previously reported (c.136C>T, p.Arg46*) and one novel variant (c.176G>A, p.Cys59Tyr). Analysis of cultured patient fibroblasts demonstrated deficient pyruvate dehydrogenase (PDH) activity and reduced quantity of protein subunits of mitochondrial complexes I and II, consistent with BOLA3 dysfunction. Previously reported cases of multiple mitochondrial dysfunctions syndrome 2 (MMDS2) with hyperglycinaemia caused by BOLA3 mutations have leukodystrophy with severe, progressive neurological and multisystem disease. CONCLUSIONS: We report a novel phenotype for MMDS2 associated with apparently complete clinical recovery and partial resolution of MRI abnormalities. We have identified a novel disease-causing variant in BOLA3 validated by functional cellular studies. Our patient's clinical course broadens the phenotypic spectrum of MMDS2 and highlights the potential for some genetic leukoencephalopathies to spontaneously improve.

Recessive mutations in NDUFA2 cause mitochondrial leukoencephalopathy.

Deficiencies of mitochondrial respiratory chain complex I frequently result in leukoencephalopathy in young patients, and different mutations in the genes encoding its subunits are still being uncovered. We report 2 patients with cystic leukoencephalopathy and complex I deficiency with recessive mutations in NDUFA2, an accessory subunit of complex I. The first patient was initially diagnosed with a primary systemic carnitine deficiency associated with a homozygous variant in SLC22A5, but also exhibited developmental regression and cystic leukoencephalopathy, and an additional diagnosis of complex I deficiency was suspected. Biochemical analysis confirmed a complex I deficiency, and whole-exome sequencing revealed a homozygous mutation in NDUFA2 (c.134A>C, p.Lys45Thr). Review of a biorepository of patients with unsolved genetic leukoencephalopathies who underwent whole-exome or genome sequencing allowed us to identify a second patient with compound heterozygous mutations in NDUFA2 (c.134A>C, p.Lys45Thr; c.225del, p.Asn76Metfs*4). Only 1 other patient with mutations in NDUFA2 and a different phenotype (Leigh syndrome) has previously been reported. This is the first report of cystic leukoencephalopathy caused by mutations in NDUFA2.

Therapies in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a genetically determined disorder, affecting most particularly the brain and the skin, characterized by the inappropriate induction of a type I interferon-mediated immune response. In most, but not all, cases the condition is severe, with a high associated morbidity and mortality. A number of important recent advances have helped to elucidate the biology of the AGS-related proteins, thus providing considerable insight into disease pathology. In this study, we outline the clinical phenotype of AGS, paying particular attention to factors relevant to therapeutic intervention. We then discuss the pathogenesis of AGS from a molecular and cell biology perspective. Finally, we suggest possible treatment strategies in light of these emerging insights.

GFAP mutations, age at onset, and clinical subtypes in Alexander disease.

OBJECTIVE: To characterize Alexander disease (AxD) phenotypes and determine correlations with age at onset (AAO) and genetic mutation. AxD is an astrogliopathy usually characterized on MRI by leukodystrophy and caused by glial fibrillary acidic protein (GFAP) mutations. METHODS: We present 30 new cases of AxD and reviewed 185 previously reported cases. We conducted Wilcoxon rank sum tests to identify variables scaling with AAO, survival analysis to identify predictors of mortality, and χ(2) tests to assess the effects of common GFAP mutations. Finally, we performed latent class analysis (LCA) to statistically define AxD subtypes. RESULTS: LCA identified 2 classes of AxD. Type I is characterized by early onset, seizures, macrocephaly, motor delay, encephalopathy, failure to thrive, paroxysmal deterioration, and typical MRI features. Type II is characterized by later onset, autonomic dysfunction, ocular movement abnormalities, bulbar symptoms, and atypical MRI features. Survival analysis predicted a nearly 2-fold increase in mortality among patients with type I AxD relative to those with type II. R79 and R239 GFAP mutations were most common (16.6% and 20.3% of all cases, respectively). These common mutations predicted distinct clinical outcomes, with R239 predicting the most aggressive course. CONCLUSIONS: AAO and the GFAP mutation site are important clinical predictors in AxD, with clear correlations to defined patterns of phenotypic expression. We propose revised AxD subtypes, type I and type II, based on analysis of statistically defined patient groups.

New-onset afebrile seizures in infants: role of neuroimaging.

OBJECTIVE: To investigate the presenting characteristics of new-onset afebrile seizures in infants (age 1-24 months) and the yield of neuroimaging. METHODS: Prospective data were obtained from a standardized evaluation and management plan mandated by a critical care pathway. A total of 317 infants presented with new-onset afebrile seizures between 2001 and 2007. EEG was performed on 90.3%, head CT was obtained on 94%, and MRI was obtained on 57.4%. RESULTS: We found half of the infants had partial features to their seizures, yet evidence for primary generalized seizures was rare. The majority had more than 1 seizure upon presentation. Seizures in this age group tended to be brief, with 44% lasting less than 1 minute. EEG abnormalities were found in half. One-third of CTs were abnormal, with 9% of all CTs requiring acute medical management. Over half of MRIs were abnormal, with cerebral dysgenesis being the most common abnormality (p < 0.05). One-third of normal CTs had a subsequent abnormal MRI-only 1 resulted in altered medical management. CONCLUSIONS: Infantile seizures are usually brief, but commonly recurrent, and strong consideration should be made for inpatient observation. Acute imaging with CT can alter management in a small but important number of infants. Due to the superior yield, strong consideration for MRI should be given for all infants, as primary generalized seizures are rare, and there is a high rate of cerebral dysgenesis.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases.

OBJECTIVE: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS). METHODS: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. RESULTS: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L). CONCLUSION: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Cerebellar ataxia with elevated cerebrospinal free sialic acid (CAFSA).

In order to identify new metabolic abnormalities in patients with complex neurodegenerative disorders of unknown aetiology, we performed high resolution in vitro proton nuclear magnetic resonance spectroscopy on patient cerebrospinal fluid (CSF) samples. We identified five adult patients, including two sisters, with significantly elevated free sialic acid in the CSF compared to both the cohort of patients with diseases of unknown aetiology (n = 144; P < 0.001) and a control group of patients with well-defined diseases (n = 91; P < 0.001). All five patients displayed cerebellar ataxia, with peripheral neuropathy and cognitive decline or noteworthy behavioural changes. Cerebral MRI showed mild to moderate cerebellar atrophy (5/5) as well as white matter abnormalities in the cerebellum including the peridentate region (4/5), and at the periventricular level (3/5). Two-dimensional gel analyses revealed significant hyposialylation of transferrin in CSF of all patients compared to age-matched controls (P < 0.001)--a finding not present in the CSF of patients with Salla disease, the most common free sialic acid storage disorder. Free sialic acid content was normal in patients' urine and cultured fibroblasts as were plasma glycosylation patterns of transferrin. Analysis of the ganglioside profile in peripheral nerve biopsies of two out of five patients was also normal. Sequencing of four candidate genes in the free sialic acid biosynthetic pathway did not reveal any mutation. We therefore identified a new free sialic acid syndrome in which cerebellar ataxia is the leading symptom. The term CAFSA is suggested (cerebellar ataxia with free sialic acid).

Sensitivity and specificity of decreased CSF asialotransferrin for eIF2B-related disorder.

OBJECTIVE: This is a study estimating diagnostic accuracy of CSF asialotransferrin to transferrin ratio measurement in eIF2B related disorders by using clinical evaluation and EIF2B mutation analysis as the reference standard. eIF2B-related disorder is a relatively common leukodystrophy with broad phenotypic variation that is caused by mutations in any of the five EIF2B genes. There is a need for a simple and clinically valid screening tool for physicians evaluating patients with an unclassified leukodystrophy. METHODS: CSF two-dimensional gel (2DG) electrophoresis analyses to measure asialotransferrin to transferrin ratios were performed in 60 subjects including 6 patients with documented EIF2B gene mutations, patients with other types of leukodystrophy, and patients with no leukodystrophy. RESULTS: All six patients with mutation proven eIF2B-related disease showed low to nearly undetectable amounts of asialotransferrin in their CSF when compared to 54 unaffected controls by CSF 2DG analyses in this study. eIF2B-like patients, with clinically similar presentations but no mutations in EIF2B1-5, were distinguished from patients with mutations in EIF2B1-5 by this biomarker. Patients with mutations in EIF2B1-5 had asialotransferrin/transferrin ratio levels significantly different from the group as a whole (p < 0.001). Using 8% asialotransferrin/transferrin ratio as a cutoff, this biomarker has a 100% sensitivity (95% CI = 52-100%) and 94% specificity (95% CI = 84-99%). CONCLUSION: Decreased asialotransferrin/transferrin ratio in the CSF of patients with eIF2B-related disorder is highly sensitive and specific. This rapid (<48 hours) and inexpensive diagnostic tool for eIF2B-related disorders has the potential to identify patients with likely eIF2B-related disorder for mutation analysis.