Homozygous variant of MLC1 results in megalencephalic leukoencephalopathy with subcortical cysts.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare, inherited disorder that causes epilepsy, intellectual disorders, and early onset macrocephaly. MLC1 has been identified as a main pathogenic gene. METHODS: Clinical data such as magnetic resonance imaging (MRI), routine blood tests, and physical examinations were collected from proband. Trio whole-exome sequencing (WES) of the family was performed, and all variants with a minor allele frequency (<0.01) in the exon and canonical splicing sites were selected for further pathogenic evaluation. Candidate variants were validated using Sanger sequencing. RESULTS: Here, we report a new homozygous variant identified in two children from the same family in the MLC1 gene [NM_015166.4: c.838_843delinsATTTTA, (p.Ser280_Phe281delinsIleLeu)]. This variant is classified as variant of uncertain significance (VUS) according to the ACMG guidelines. Further experiments demonstrate that the newly identified variant causes a decrease of MLC1 protein levels when expressed in a heterologous expression system. CONCLUSION: Our case expands on this genetic variation and provides new evidence for the clinical diagnosis of MLC1-related MLC.

A novel variant of the POLR3A gene in a Chinese patient with POLR3-related leukodystrophy.

BACKGROUND: POLR3-related leukodystrophy is a group of rare neurodegenerative disorders characterized by degeneration of the white matter with different combinations of major clinical features. CASE: An 18-year-old lady was admitted for no menstruation since childhood. She gradually developed slight symptoms, such as choking after drinking water and unsteady walking in the last 2 years. Furthermore, her test scores and response capability were far lower than that of her peers. Physical examination revealed her to be of a slightly short stature, with stiff expressions and bilateral breast enlargement. She revealed clumsy movements when examined for ataxia, with an SARA score of 9. FINDINGS: The laboratory data revealed a decreased level of estradiol, FSH, and LH, with a MoCA score of 7. Conventional karyotype analysis revealed a 46 XX 9qh + karyotype. Ultrasound indicated primordial uterus (19 × 11 × 10 mm). Brain MRI showed bilateral cerebral hemisphere myelin dysplasia, brain atrophy, thin corpus callosum, and small pituitary gland with uneven reinforcement and enlarged ventricles. Exome sequencing exhibited two missense mutations in the POLR3A gene (c.3013C > T and c.1757C > T), which were inherited from her mother and father, respectively. CONCLUSION: Collectively, we identified novel compound heterozygous mutations of the POLR3A gene that caused POLR3A-related hypomyelinating leukodystrophy with hypogonadism in the patient combined with the clinical presentation, MRI brain pattern, and medical exome sequencing. TEACHING POINTS: The complexity of clinical phenotypes and heterogeneity of genotypes raise new challenges in genetic diagnoses. This study will further aid our understanding of POLR3A-related leukodystrophy and promote further analysis of phenotype-genotype correlations of related diseases.

Prevalent MLC1 mutation causing autosomal recessive megalencephalic leukoencephalopathy in consanguineous Palestinian families.

BACKGROUND: Recessive forms of megalencephalic leukoencephalopathy with subcortical cysts (MLC, OMIM 604004) is a rare early-onset leukodystrophy that presents with macrocephaly, seizures, slowly progressive gross motor deterioration, and MRI evidence of diffuse symmetric white matter swelling and subcortical cysts in the anterior temporal and frontoparietal regions. Later in the disease course, significant spasticity and ataxia develop, which may be accompanied by intellectual deterioration. This disease is caused mostly by biallelic pathogenic variants in the MLC1 gene. METHODS: In this study, we analysed the clinical and molecular architecture of 6 individuals, belonging to 4 unrelated consanguineous Palestinian families, presenting with consistent MLC features. We sequenced the entire coding and flanking intronic regions of the MLC1 gene. RESULTS: In all recruited individuals, we detected one recurrent homozygous splice donor mutation NM_015166.4: c.423 + 1G > A. All parents were heterozygous carriers. The mutation abolishes a highly conserved splice site in humans and other species. In silico splice predictors suggested the loss of a canonical splice donor site (CADD score 33.0. SpliceAI: 0.980). The c.423 + 1G > A variant is rare; it was detected in only 4 heterozygous carriers in gnomAD. CONCLUSION: In this study, we identified a recurrent MLC1 variant (c.423 + 1G > A) as the cause of MLC among a group of Palestinian patients originating from a particular region of the country. Cost-effective studies should be performed to evaluate the implementation of carrier screening in adults originating from this region. Our findings have the potential to contribute to improved genetic diagnosis and carrier testing for individuals within this population and the wider community.

A homozygous missense variant in the MLC1 gene underlies megalencephalic leukoencephalopathy with subcortical cysts in large kindred: Heterozygous carriers show seizure and mild motor function deterioration.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by epileptic seizures, macrocephaly, and vacuolization of myelin and astrocyte. The magnetic resonance imaging of the brain of MLC patients shows diffuse white-matter anomalies and the occurrence of subcortical cysts. MLC features have been observed in individuals having mutations in the MLC1 or HEPACAM genes. In this study, we recruited a six generation large kindred with five affected individuals manifesting clinical features of epileptic seizures, macrocephaly, ataxia, and spasticity. In order to identify the underlying genetic cause of the clinical features, we performed whole-genome genotyping using Illumina microarray followed by detection of loss of heterozygosity (LOHs) regions. One affected individual was exome sequenced as well. Homozygosity mapping detected several LOH regions due to extensive consanguinity. An unbiased and hypothesis-free exome data analysis identified a homozygous missense variant (NM_015166.3:c.278C>T) in the exon 4 of the MLC1 gene. The variant is present in the LOH region on chromosome 22q (50 Mb) and segregates perfectly with the disorder within the family in an autosomal recessive manner. The variant is present in a highly conserved first cytoplasmic domain of the MLC1 protein (NM_015166.3:p.(Ser93Leu)). Interestingly, heterozygous individuals show seizure and mild motor function deterioration. We propose that the heterozygous variant in MLC1 might disrupt the functional interaction of MLC1 with GlialCAM resulting in mild clinical features in carriers of the variant.

Novel variants causing megalencephalic leukodystrophy in Sudanese families.

Mutations in MLC1 cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare form of leukodystrophy characterized by macrocephaly, epilepsy, spasticity, and slow mental deterioration. Genetic studies of MLC are lacking from many parts of the world, especially in Sub-Saharan Africa. Genomic DNA was extracted for 67 leukodystrophic patients from 43 Sudanese families. Mutations were screened using the NGS panel testing 139 leukodystrophies and leukoencephalopathies causing genes (NextSeq500 Illumina). Five homozygous MLC1 variants were discovered in seven patients from five distinct families, including three consanguineous families from the same region of Sudan. Three variants were missense (c.971 T > G, p.Ile324Ser; c.344 T > C, p.Phe115Ser; and c.881 C > T, p.Pro294Leu), one duplication (c.831_838dupATATCTGT, p.Ser280Tyrfs*8), and one synonymous/splicing-site mutation (c.762 C > T, p.Ser254). The segregation pattern was consistent with autosomal recessive inheritance. The clinical presentation and brain MRI of the seven affected patients were consistent with the diagnosis of MLC1. Due to the high frequency of distinct MLC1 mutations found in our leukodystrophic Sudanese families, we analyzed the coding sequence of MLC1 gene in 124 individuals from the Sudanese genome project in comparison with the 1000-genome project. We found that Sudan has the highest proportion of deleterious variants in MLC1 gene compared with other populations from the 1000-genome project.

Biallelic PI4KA variants cause a novel neurodevelopmental syndrome with hypomyelinating leukodystrophy.

Phosphoinositides are lipids that play a critical role in processes such as cellular signalling, ion channel activity and membrane trafficking. When mutated, several genes that encode proteins that participate in the metabolism of these lipids give rise to neurological or developmental phenotypes. PI4KA is a phosphoinositide kinase that is highly expressed in the brain and is essential for life. Here we used whole exome or genome sequencing to identify 10 unrelated patients harbouring biallelic variants in PI4KA that caused a spectrum of conditions ranging from severe global neurodevelopmental delay with hypomyelination and developmental brain abnormalities to pure spastic paraplegia. Some patients presented immunological deficits or genito-urinary abnormalities. Functional analyses by western blotting and immunofluorescence showed decreased PI4KA levels in the patients' fibroblasts. Immunofluorescence and targeted lipidomics indicated that PI4KA activity was diminished in fibroblasts and peripheral blood mononuclear cells. In conclusion, we report a novel severe metabolic disorder caused by PI4KA malfunction, highlighting the importance of phosphoinositide signalling in human brain development and the myelin sheath.

Hypomyelination and Congenital Cataract: Clinical, Imaging, and Genetic Findings in Three Tunisian Families and Literature Review.

Hypomyelination and congenital cataract (HCC) is characterized by congenital cataract, progressive neurologic impairment, and diffuse myelin deficiency. This autosomal recessive disorder is caused by homozygous variant in the FAM126A gene. Five consanguineous Tunisian patients, belonging to three unrelated families, underwent routine blood tests, electroneuromyography, and magnetic resonance imaging of the brain. The direct sequencing of FAM126A exons was performed for the patients and their relatives. We summarized the 30 previously published HCC cases. All of our patients were carriers of a previously reported c.414 + 1G > T (IVS5 + 1G > T) variant, but the clinical spectrum was variable. Despite the absence of a phenotype-genotype correlation in HCC disease, screening of this splice site variant should be performed in family members at risk.

MRI characteristics of Japanese macaque encephalomyelitis: Comparison to human diseases.

BACKGROUND AND PURPOSE: To describe MRI findings in Japanese macaque encephalomyelitis (JME) with emphasis on lesion characteristics, lesion evolution, normal-appearing brain tissue, and similarities to human demyelinating disease. METHODS: MRI data were obtained from 114 Japanese macaques, 30 presenting neurological signs of JME. All animals were screened for presence of T2 -weighted white matter signal hyperintensities; animals with behavioral signs of JME were additionally screened for contrast-enhancing lesions. Whole-brain quantitative T1 maps were collected, and histogram analysis was performed with regression across age to evaluate microstructural changes in normal appearing brain tissue in JME and neurologically normal animals. Quantitative estimates of blood-brain-barrier (BBB) permeability to gadolinium-based-contrast agent (GBCA) were obtained in acute, GBCA-enhancing lesions. Longitudinal imaging data were acquired for 15 JME animals. RESULTS: One hundred and seventy-three focal GBCA-enhancing lesions were identified in 30 animals demonstrating behavioral signs of neurological dysfunction. JME GBCA-enhancing lesions were typically focal and ovoid, demonstrating highest BBB GBCA permeability in the lesion core, similar to acute, focal multiple sclerosis lesions. New GBCA-enhancing lesions arose rapidly from normal-appearing tissue, and BBB permeability remained elevated for weeks. T1 values in normal-appearing tissue were significantly associated with age, but not with sex or disease. CONCLUSIONS: Intense, focal neuroinflammation is a key MRI finding in JME. Several features of JME compare directly to human inflammatory demyelinating diseases. Investigation of JME combined with the development and validation of noninvasive imaging biomarkers offers substantial potential to improve diagnostic specificity and contribute to the understanding of human demyelinating diseases.

Lesion evolution and neurodegeneration in RVCL-S: A monogenic microvasculopathy.

OBJECTIVE: To characterize lesion evolution and neurodegeneration in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) using multimodal MRI. METHODS: We prospectively performed MRI and cognitive testing in RVCL-S and healthy control cohorts. Gray and white matter volume and disruption of white matter microstructure were quantified. Asymmetric spin echo acquisition permitted voxel-wise oxygen extraction fraction (OEF) calculation as an in vivo marker of microvascular ischemia. The RVCL-S cohort was included in a longitudinal analysis of lesion subtypes in which hyperintense lesions on fluid-attenuated inversion recovery (FLAIR), T1-postgadolinium, and diffusion-weighted imaging were delineated and quantified volumetrically. RESULTS: Twenty individuals with RVCL-S and 26 controls were enrolled. White matter volume and microstructure declined faster in those with RVCL-S compared to controls. White matter atrophy in RVCL-S was highly linear (ρ = -0.908, p < 0.0001). Normalized OEF was elevated in RVCL-S and increased with disease duration. Multiple cognitive domains, specifically those measuring working memory and processing speed, were impaired in RVCL-S. Lesion volumes, regardless of subtype, progressed/regressed with high variability as a function of age, while FLAIR lesion burden increased near time to death (p < 0.001). CONCLUSION: RVCL-S is a monogenic microvasculopathy affecting predominantly the white matter with regard to atrophy and cognitive impairment. White matter volumes in RVCL-S declined linearly, providing a potential metric against which to test the efficacy of future therapies. Progressive elevation of white matter OEF suggests that microvascular ischemia may underlie neurodegeneration in RVCL-S.

yippee like 3 (ypel3) is a novel gene required for myelinating and perineurial glia development.

Hypomyelination, a neurological condition characterized by decreased production of myelin sheets by glial cells, often has no known etiology. Elucidating the genetic causes of hypomyelination provides a better understanding of myelination, as well as means to diagnose, council, and treat patients. Here, we present evidence that YIPPEE LIKE 3 (YPEL3), a gene whose developmental role was previously unknown, is required for central and peripheral glial cell development. We identified a child with a constellation of clinical features including cerebral hypomyelination, abnormal peripheral nerve conduction, hypotonia, areflexia, and hypertrophic peripheral nerves. Exome and genome sequencing revealed a de novo mutation that creates a frameshift in the open reading frame of YPEL3, leading to an early stop codon. We used zebrafish as a model system to validate that YPEL3 mutations are causative of neuropathy. We found that ypel3 is expressed in the zebrafish central and peripheral nervous system. Using CRISPR/Cas9 technology, we created zebrafish mutants carrying a genomic lesion similar to that of the patient. Our analysis revealed that Ypel3 is required for development of oligodendrocyte precursor cells, timely exit of the perineurial glial precursors from the central nervous system (CNS), formation of the perineurium, and Schwann cell maturation. Consistent with these observations, zebrafish ypel3 mutants have metabolomic signatures characteristic of oligodendrocyte and Schwann cell differentiation defects, show decreased levels of Myelin basic protein in the central and peripheral nervous system, and develop defasciculated peripheral nerves. Locomotion defects were observed in adult zebrafish ypel3 mutants. These studies demonstrate that Ypel3 is a novel gene required for perineurial cell development and glial myelination.

Identification of a deep intronic POLR3A variant causing inclusion of a pseudoexon derived from an Alu element in Pol III-related leukodystrophy.

Pseudoexon inclusion caused by deep intronic variants is an important genetic cause for various disorders. Here, we present a case of a hypomyelinating leukodystrophy with developmental delay, intellectual disability, autism spectrum disorder, and hypodontia, which are consistent with autosomal recessive POLR3-related leukodystrophy. Whole-exome sequencing identified only a heterozygous missense variant (c.1451G>A) in POLR3A. To explore possible involvement of a deep intronic variant in another allele, we performed whole-genome sequencing of the patient with variant annotation by SpliceAI, a deep-learning-based splicing prediction tool. A deep intronic variant (c.645 + 312C>T) in POLR3A, which was predicted to cause inclusion of a pseudoexon derived from an Alu element, was identified and confirmed by mRNA analysis. These results clearly showed that whole-genome sequencing, in combination with deep-learning-based annotation tools such as SpliceAI, will bring us further benefits in detecting and evaluating possible pathogenic variants in deep intronic regions.

[Analysis of a child with megalencephalic leukoencephalopathy with subcortical cyst type 2B caused by HEPACAM variant].

OBJECTIVE: To explore the clinical features and genetic variant in a child featuring megalencephalic leukoencephalopathy with subcortical cyst (MLC) type 2B. METHODS: Clinical and imaging data of the child was collected. Potential variant of hepatocyte adhesion molecule (HEPACAM) gene was detected by Sanger sequencing. The growth and development of her mother and uncle was also reviewed. RESULTS: The patient, a 1-year-and-7-month female, presented with convulsion, mental retardation and abnormally increased head circumference. Cranial MRI revealed extensive long T1 long T2 signals in the white matter of bilateral cerebral hemisphere, right anterior sac cyst, cerebral gyrus widening, and shallow sulcus. Sanger sequencing identified a c.437C>T missense variant in exon 3 of the HEPACAM gene. The same variant was detected in her mother but not father. Her mother and maternal uncle both had a history of increased head circumference when they were young. In their adulthood, the head circumference was in the normal range but still greater than the average. CONCLUSION: The heterozygous variant of the HEPACAM gene probably underlies the MLC2B in this child. The variant has derived from her asymptomatic mother, which suggested incomplete penetrance of the MLC2B.

POLR3A variants in striatal involvement without diffuse hypomyelination.

BACKGROUND: Biallelic variants in POLR3A encoding the largest subunit of RNA polymerase III cause POLR3-related (or 4H) leukodystrophy characterized by neurologic dysfunction, abnormal dentition, endocrine abnormalities and ocular abnormality. Recently, whole-exome sequencing enabled the discovery of POLR3A variants in cases lacking diffuse hypomyelination, the principal MRI phenotype of POLR3-related leukodystrophy. Homozygous c.1771-6C > G variants in POLR3A were recently suggested to cause striatal and red nucleus involvement without white matter involvement. CASE REPORT: Here, we report three cases in two families with biallelic POLR3A variants. We identified two sets of compound heterozygous variants in POLR3A, c.1771-6C > G and c.791C > T, p.(Pro264Leu) for family 1 and c.1771-6C > G and c.2671C > T, p.(Arg891*) for family 2. Both families had the c.1771-6C > G variant, which led to aberrant mRNA splicing. Neuropsychiatric regression and severe intellectual disability were identified in three patients. Two cases showed dystonia and oligodontia. Notably, characteristic bilateral symmetric atrophy and abnormal signal of the striatum without diffuse white matter signal change were observed in brain MRI of all three individuals. CONCLUSIONS: Striatum abnormalities may be another distinctive MRI finding associated with POLR3A variants, especially in cases including c.1771-6C > G variants and our cases can expand the phenotypic spectrum of POLR3A-related disorders.

RARS1-related hypomyelinating leukodystrophy: Expanding the spectrum.

OBJECTIVE: Biallelic variants in RARS1, encoding the cytoplasmic tRNA synthetase for arginine (ArgRS), cause a hypomyelinating leukodystrophy. This study aimed to investigate clinical, neuroradiological and genetic features of patients with RARS1-related disease, and to identify possible genotype-phenotype relationships. METHODS: We performed a multinational cross-sectional survey among 20 patients with biallelic RARS1 variants identified by next-generation sequencing techniques. Clinical data, brain MRI findings and genetic results were analyzed. Additionally, ArgRS activity was measured in fibroblasts of four patients, and translation of long and short ArgRS isoforms was quantified by western blot. RESULTS: Clinical presentation ranged from severe (onset in the first 3 months, usually with refractory epilepsy and early brain atrophy), to intermediate (onset in the first year with nystagmus and spasticity), and mild (onset around or after 12 months with minimal cognitive impairment and preserved independent walking). The most frequent RARS1 variant, c.5A>G, led to mild or intermediate phenotypes, whereas truncating variants and variants affecting amino acids close to the ArgRS active centre led to severe phenotypes. ArgRS activity was significantly reduced in three patients with intermediate and severe phenotypes; in a fourth patient with intermediate to severe presentation, we measured normal ArgRS activity, but found translation mainly of the short instead of the long ArgRS isoform. INTERPRETATION: Variants in RARS1 impair ArgRS activity and do not only lead to a classic hypomyelination presentation with nystagmus and spasticity, but to a wide spectrum, ranging from severe, early-onset epileptic encephalopathy with brain atrophy to mild disease with relatively preserved myelination.

A novel compound heterozygous mutation in AARS2 gene (c.965 G > A, p.R322H; c.334 G > C, p.G112R) identified in a Chinese patient with leukodystrophy involved in brain and spinal cord.

Leukodystrophies are genetic disorders leading to progressive white matter degeneration in the central nervous system. Mitochondrial aminoacyl tRNA synthase protein is encoded by the nuclear gene AARS2. An autosomal recessive mutation in this gene has been linked to AARS2 mutation-related adult-onset leukodystrophy (AARS2-L) or infantile mitochondrial cardiomyopathy. To date, only 16 AARS2-L cases have been reported in English literature. Thus, the clinical and genetic characteristics of this disease remain to be defined. Through whole-exome sequencing, we identified a Chinese patient with leukodystrophy related to two novel compounds heterozygous mutation in AARS2 (c.965 G > A, p.R322H; c.334 G > C, p.G112R). These two compounds heterozygous variants in AARS2 gene co-segregated with disease in his family. And pyramidal tracts in the spinal cord were involved. Our findings have important implications on genetic counseling for any case with leukodystrophy and extend the mutational spectrum in AARS2 gene.

Megalencephalic leukoencephalopathy with subcortical cysts without macrocephaly: A case study of comorbid Turner's syndrome.

We present a case of megalencephalic leukoencephalopathy with subcortical cysts without macrocephaly and who initially presented with severe psychiatric symptoms. The patient presented with presented with late-onset secondary generalized focal motor seizures, gait ataxia and mild spasticity with hyperreflexia. MRI showed diffuse white matter hyperintensities and bilateral anterotemporal cysts. Genetic analysis confirmed the causal MLC1 mutation and Turner's syndrome. Surprisingly, our patient had no macrocephaly, which is a typical finding in MCL1 mutations; we emphasize that comorbid unrelated Turner's syndrome could explain the absence of macrocephaly: although short stature is typical, microcephaly is not associated with Turner's syndrome. Our observation thus argues for detailed investigations in cases presenting with an atypical clinical picture.