Megalencephalic leukoencephalopathy with subcortical cysts: a variant update and review of the literature.

The leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC) is characterized by infantile-onset macrocephaly and chronic edema of the brain white matter. With delayed onset, patients typically experience motor problems, epilepsy and slow cognitive decline. No treatment is available. Classic MLC is caused by bi-allelic recessive pathogenic variants in MLC1 or GLIALCAM (also called HEPACAM). Heterozygous dominant pathogenic variants in GLIALCAM lead to remitting MLC, where patients show a similar phenotype in early life, followed by normalization of white matter edema and no clinical regression. Rare patients with heterozygous dominant variants in GPRC5B and classic MLC were recently described. In addition, two siblings with bi-allelic recessive variants in AQP4 and remitting MLC have been identified. The last systematic overview of variants linked to MLC dates back to 2006. We provide an updated overview of published and novel variants. We report on genetic variants from 508 patients with MLC as confirmed by MRI diagnosis (258 from our database and 250 extracted from 64 published reports). We describe 151 unique MLC1 variants, 29 GLIALCAM variants, 2 GPRC5B variants and 1 AQP4 variant observed in these MLC patients. We include experiments confirming pathogenicity for some variants, discuss particularly notable variants, and provide an overview of recent scientific and clinical insight in the pathophysiology of MLC.

Intragenic homozygous duplication in HEPACAM is associated with megalencephalic leukoencephalopathy with subcortical cysts type 2A.

Disease-causing variants in HEPACAM are associated with megalencephalic leukoencephalopathy with subcortical cysts 2A (MLC2A, MIM# 613,925, autosomal recessive), and megalencephalic leukoencephalopathy with subcortical cysts 2B, remitting, with or without impaired intellectual development (MLC2B, MIM# 613,926, autosomal dominant). These disorders are characterised by macrocephaly, seizures, motor delay, cognitive impairment, ataxia, and spasticity. Brain magnetic resonance imaging (MRI) in these individuals shows swollen cerebral hemispheric white matter and subcortical cysts, mainly in the frontal and temporal regions. To date, 45 individuals from 39 families are reported with biallelic and heterozygous variants in HEPACAM, causing MLC2A and MLC2B, respectively. A 9-year-old male presented with developmental delay, gait abnormalities, seizures, macrocephaly, dysarthria, spasticity, and hyperreflexia. MRI revealed subcortical cysts with diffuse cerebral white matter involvement. Whole-exome sequencing (WES) in the proband did not reveal any clinically relevant single nucleotide variants. However, copy number variation analysis from the WES data of the proband revealed a copy number of 4 for exons 3 and 4 of HEPACAM. Validation and segregation were done by quantitative PCR which confirmed the homozygous duplication of these exons in the proband and carrier status in both parents. To the best of our knowledge, this is the first report of an intragenic duplication in HEPACAM causing MLC2A.

Glial Cell Adhesion Molecule (GlialCAM) Determines Proliferative versus Invasive Cell States in Glioblastoma.

The malignant brain cancer glioblastoma (GBM) contains groups of highly invasive cells that drive tumor progression as well as recurrence after surgery and chemotherapy. The molecular mechanisms that enable these GBM cells to exit the primary mass and disperse throughout the brain remain largely unknown. Here we report using human tumor specimens and primary spheroids from male and female patients that glial cell adhesion molecule (GlialCAM), which has normal roles in brain astrocytes and is mutated in the developmental brain disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC), is differentially expressed in subpopulations of GBM cells. High levels of GlialCAM promote cell-cell adhesion and a proliferative GBM cell state in the tumor core. In contrast, GBM cells with low levels of GlialCAM display diminished proliferation and enhanced invasion into the surrounding brain parenchyma. RNAi-mediated inhibition of GlialCAM expression leads to activation of proinvasive extracellular matrix adhesion and signaling pathways. Profiling GlialCAM-regulated genes combined with cross-referencing to single-cell transcriptomic datasets validates functional links among GlialCAM, Mlc1, and aquaporin-4 in the invasive cell state. Collectively, these results reveal an important adhesion and signaling axis comprised of GlialCAM and associated proteins including Mlc1 and aquaporin-4 that is critical for control of GBM cell proliferation and invasion status in the brain cancer microenvironment.SIGNIFICANCE STATEMENT Glioblastoma (GBM) contains heterogeneous populations of cells that coordinately drive proliferation and invasion. We have discovered that glial cell adhesion molecule (GlialCAM)/hepatocyte cell adhesion molecule (HepaCAM) is highly expressed in proliferative GBM cells within the tumor core. In contrast, GBM cells with low levels of GlialCAM robustly invade into surrounding brain tissue along blood vessels and white matter. Quantitative RNA sequencing identifies various GlialCAM-regulated genes with functions in cell-cell adhesion and signaling. These data reveal that GlialCAM and associated signaling partners, including Mlc1 and aquaporin-4, are key factors that determine proliferative and invasive cell states in GBM.

The Effect of Interleukin-1 Antagonists on Brain Volume and Cognitive Function in Two Patients With Megalencephalic Leukoencephalopathy With Subcortical Cysts.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation. The MLC1 protein plays a role in astrocyte activation during neuroinflammation and regulates volume decrease following astrocyte osmotic swelling. Loss of MLC1 function activates interleukin (IL)-1ß-induced inflammatory signals. Theoretically, IL-1 antagonists (such as anakinra and canakinumab) can slow the progression of MLC. Herein, we present two boys from different families who had MLC due to biallelic MLC1 gene mutations and were treated with the anti-IL-1 drug anakinra. METHODS: Two boys from different families presented with megalencephaly and psychomotor retardation. Brain magnetic resonance imaging findings in both patients were compatible with the diagnosis of MLC. The diagnosis of MLC was confirmed via Sanger analysis of the MLC1 gene. Anakinra was administered to both patients. Volumetric brain studies and psychometric evaluations were performed before and after anakinra treatment. RESULTS: After anakinra therapy, brain volume in both patients decreased significantly and cognitive functions and social interactions improved. No adverse effects were observed during anakinra therapy. CONCLUSIONS: Anakinra or other IL-1 antagonists can be used to suppress disease activity in patients with MLC; however, the present findings need to be confirmed via additional research.

The alpha7 integrin subunit in astrocytes promotes endothelial blood-brain barrier integrity.

The blood-brain barrier (BBB) is a vascular endothelial cell boundary that partitions the circulation from the central nervous system to promote normal brain health. We have a limited understanding of how the BBB is formed during development and maintained in adulthood. We used quantitative transcriptional profiling to investigate whether specific adhesion molecules are involved in BBB functions, with an emphasis on understanding how astrocytes interact with endothelial cells. Our results reveal a striking enrichment of multiple genes encoding laminin subunits as well as the laminin receptor gene Itga7, which encodes the alpha7 integrin subunit, in astrocytes. Genetic ablation of Itga7 in mice led to aberrant BBB permeability and progressive neurological pathologies. Itga7-/- mice also showed a reduction in laminin protein expression in parenchymal basement membranes. Blood vessels in the Itga7-/- brain showed separation from surrounding astrocytes and had reduced expression of the tight junction proteins claudin 5 and ZO-1. We propose that the alpha7 integrin subunit in astrocytes via adhesion to laminins promotes endothelial cell junction integrity, all of which is required to properly form and maintain a functional BBB.

GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.

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.

Mlc1-Expressing Perivascular Astrocytes Promote Blood-Brain Barrier Integrity.

In the mammalian brain, perivascular astrocytes (PAs) closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). Deciphering specific functions for PAs in BBB biology, however, has been limited by the ability to distinguish these cells from other astrocyte populations. In order to characterize selective roles for PAs in vivo, a new mouse model has been generated in which the endogenous megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) gene drives expression of Cre fused to a mutated estrogen ligand-binding domain (Mlc1-T2A-CreERT2). This knock-in mouse model, which we term MLCT, allows for selective identification and tracking of PAs in the postnatal brain. We also demonstrate that MLCT-mediated ablation of PAs causes severe defects in BBB integrity, resulting in premature death. PA loss results in aberrant localization of Claudin 5 and -VE-Cadherin in endothelial cell junctions as well as robust microgliosis. Collectively, these data reveal essential functions for Mlc1-expressing PAs in regulating endothelial barrier integrity in mice and indicate that primary defects in astrocytes that cause BBB breakdown may contribute to human neurologic disorders.SIGNIFICANCE STATEMENT Interlaced among the billions of neurons and glia in the mammalian brain is an elaborate network of blood vessels. Signals from the brain parenchyma control the unique permeability properties of cerebral blood vessels known as the blood-brain barrier (BBB). However, we understand very little about the relative contributions of different neural cell types in the regulation of BBB functions. Here, we show that a specific subpopulation of astrocyte is essential for control of BBB integrity, with ablation of these cells leading to defects in endothelial cell junctions, BBB breakdown, and resulting neurologic deficits.

Alexander Disease.

Main teaching point: The main differential diagnosis of leukodystrophy associated with macrocephaly consists of Alexander disease, Canavan disease, and megalencephalic leukodystrophy with subcortical cysts. Distinguishing imaging characteristics of Alexander disease are an apicoposterior gradient of white matter involvement and a periventricular T2-hypointense rim.

Astrocyte-Oligodendrocyte-Microglia Crosstalk in Astrocytopathies.

Defective astrocyte function due to a genetic mutation can have major consequences for microglia and oligodendrocyte physiology, which in turn affects the white matter integrity of the brain. This review addresses the current knowledge on shared and unique pathophysiological mechanisms of astrocytopathies, including vanishing white matter, Alexander disease, megalencephalic leukoencephalopathy with subcortical cysts, Aicardi-Goutières syndrome, and oculodentodigital dysplasia. The mechanisms of disease include protein accumulation, unbalanced secretion of extracellular matrix proteins, pro- and anti-inflammatory molecules, cytokines and chemokines by astrocytes, as well as an altered gap junctional network and a changed ionic and nutrient homeostasis. Interestingly, the extent to which astrogliosis and microgliosis are present in these astrocytopathies is highly variable. An improved understanding of astrocyte-microglia-oligodendrocyte crosstalk might ultimately lead to the identification of druggable targets for these, currently untreatable, severe conditions.

Bipolar disorder in megalencephalic leukoencephalopathy with subcortical cysts: a case report.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC), or Van der Knaap disease, is a rare spongiform leukodystrophy that is characterized by macrocephaly, progressive motor dysfunction, and mild mental retardation. It is very rare for mental illness such as psychotic disorders, affective disorders and anxiety disorders to occur in MLC. CASE PRESENTATION: A 17-year-old boy was admitted to our hospital after he developed symptoms of depressive state with catatonia after being diagnosed as having MLC with confirmed MLC1 mutation. His catatonic symptoms were improved with administration of olanzapine and sertraline and he was discharged after 4 months. Several months later, he became hypomanic. He was diagnosed with bipolar II disorder. Mood swings were controlled with the administration of carbamazepine. CONCLUSIONS: This case is the first report of bipolar disorder during the clinical course of MLC. This case indicate the possibility that MLC influences the development of bipolar disorder in MLC, however, further studies involving more patients are required to clarify this.

White matter abnormality in Jacobsen syndrome assessed by serial MRI.

INTRODUCTION: Jacobsen syndrome (JS) is caused by a deletion at the terminus of the long arm of chromosome 11. There are few reports of JS associated with cerebral white matter abnormalities (WMA), and the etiology, pathophysiology, and time-dependent changes in WMA with JS still remain unclear. CASE REPORT: The patient was a 2-month-old female with several morphological anomalies, including trigonocephaly, ectropion, flat nasal bridge, low-set ears, and sparse eyebrows. Chromosome analysis (G-banding karyotyping) of 46,XX,del(11)(q23.3) led to the diagnosis of JS. Head MRI performed at age 9 months indicated diffuse WMA with hyperintense signals on T2-weighted imaging. MRI at age 2.5 years demonstrated a decrease in the WMA and progressive myelination. DISCUSSION: These findings suggested that the WMA in the present patient were due to chronic white matter edema associated with a deletion in the 11q terminal region of HEPACAM/GlialCAM, a causative gene for megalencephalic leukoencephalopathy with subcortical cysts type 2B (MLC2B). As with some of MLC2B patients, the WMA in the present patient improved over time. The present report is the first to document dramatic changes in WMA in JS visualized by serial MRI examinations from the neonatal period through early childhood. CONCLUSION: The findings of the present study suggested that WMA in JS are due to chronic white matter edema associated with HEPACAM/GlialCAM deletion and show gradual improvement over time, as seen in some MLC2B patients.

Identification in Chinese patients with GLIALCAM mutations of megalencephalic leukoencephalopathy with subcortical cysts and brain pathological study on Glialcam knock-in mouse models.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare neurological degenerative disorder caused by the mutations of MLC1 or GLIALCAM with autosomal recessive or autosomal dominant inheritance and a different prognosis, characterized by macrocephaly, delayed motor and cognitive development, and bilateral abnormal signals in cerebral white matter (WM) with or without cysts on magnetic resonance imaging (MRI). This study aimed to reveal the clinical and genetic features of MLC patients with GLIALCAM mutations and to explore the brain pathological characteristics and prognosis of mouse models with different modes of inheritance. METHODS: Clinical information and peripheral venous blood were collected from six families. Genetic analysis was performed by Sanger sequencing of GLIALCAM. GlialcamArg92Trp/+ and GlialcamLys68Met/Thr132Asn mouse models were generated based on mutations from patients (c.274C>T(p.Arg92Trp) (c.203A>T(p.Lys68Met), and c.395C>A (p.Thr132Asn))). Brain pathologies of the mouse models at different time points were analyzed. RESULTS: Six patients were clinically diagnosed with MLC. Of the six patients, five (Pt1-Pt5) presented with a heterozygous mutation in GLIALCAM (c.274C>T(p.Arg92Trp) or c.275G>C(p.Arg92Pro)) and were diagnosed with MLC2B; the remaining patient (Pt6) with two compound heterozygous mutations in GLIALCAM (c.203A>T (p.Lys68Met) and c.395C>A (p.Thr132Asn)) was diagnosed with MLC2A. The mutation c.275C>G (p.Arg92Pro) has not been reported before. Clinical manifestations of the patient with MLC2A (Pt6) progressed with regression, whereas the course of the five MLC2B patients remained stable or improved. The GlialcamArg92Trp/+ and GlialcamLys68Met/ Thr132Asn mouse models showed vacuolization in the anterior commissural WM at 1 month of age and vacuolization in the cerebellar WM at 3 and 6 months, respectively. At 9 months, the vacuolization of the GlialcamLys68Met/ Thr132Asn mouse model was heavier than that of the GlialcamArg92Trp/+ mouse model. Decreased expression of Glialcam in GlialcamArg92Trp/+ and GlialcamLys68Met/ Thr132Asn mice may contribute to the vacuolization. CONCLUSIONS: Clinical and genetic characterization of patients with MLC and GLIALCAM mutations revealed a novel mutation, expanding the spectrum of GLIALCAM mutations. The first Glialcam mouse model with autosomal recessive inheritance and a new Glialcam mouse model with autosomal dominant inheritance were generated. The two mouse models with different modes of inheritance showed different degrees of brain pathological features, which were consistent with the patients' phenotype and further confirmed the pathogenicity of the corresponding mutations.

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.

Analysis of clinical characteristics and gene mutations of megalencephalic leukoencephalopathy with subcortical cysts in a family / 中华实用儿科临床杂志

Objective To analyze the clinical characteristics and genetic variation of megalencephalic leu-koencephalopathy with subcortical cysts(MCL),then to explore the genetic characteristics so as to help families by pro-viding genetic counseling. Methods The clinical data of the children and their family members were collected,and the peripheral blood DNA of the children and family members were extracted. Then,the MLC1 gene mutation in the children was detected by using the target sequence capture high-throughput sequencing technology and Sanger sequencing tech-nology. Results (1)MCL often presented abnormal head circumference in infants as the first symptom. The main clini-cal manifestations were hypoevolutism in motor development,retrogression of early school age,then the movement disor-der progressed and finally paralyzed;epilepsy was common in early childhood;head magnetic resonance imaging showed white matter in bilateral cerebral hemisphere diffusing abnormal signal with temporal lobe cystic change in the early stage,and then showed brain atrophy. (2)The gene results showed that the 2 girls with MLC had both c. 368C >T (p. Thr123Ile)and c. 353C > T (p. Thr118Met)complex heterozygous variation,which existed in the MLC1 gene. The girls′ father and a sister carried c. 368C > T (p. Thr123Ile),while the mother carried c. 353C > T (p. Thr118Met) heterozygous variation,all of whom were normal phenotypes. Conclusions MCL is one cause of hypoevolutism in motor development in children and abnormal head circumference of infants is usually the first symptom. The MLC1 gene c. 368C> T(p. Thr123Ile)is a pathogenic mutation for MLC,and may be another new pa-thogenic mutation.

Selective expression of eGFP in mouse perivascular astrocytes by modification of the Mlc1 gene using T2A-based ribosome skipping.

Perivascular astrocyte end feet closely juxtapose cerebral blood vessels to regulate important developmental and physiological processes including endothelial cell proliferation and sprouting as well as the formation of the blood-brain barrier (BBB). The mechanisms underlying these events remain largely unknown due to a lack of experimental models for identifying perivascular astrocytes and distinguishing these cell types from other astroglial populations. Megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) is a transmembrane protein that is expressed in perivascular astrocyte end feet where it controls BBB development and homeostasis. On the basis of this knowledge, we used T2A peptide-skipping strategies to engineer a knock-in mouse model in which the endogenous Mlc1 gene drives expression of enhanced green fluorescent protein (eGFP), without impacting expression of Mlc1 protein. Analysis of fetal, neonatal and adult Mlc1-eGFP knock-in mice revealed a dynamic spatiotemporal expression pattern of eGFP in glial cells, including nestin-expressing neuroepithelial cells during development and glial fibrillary acidic protein (GFAP)-expressing perivascular astrocytes in the postnatal brain. EGFP was not expressed in neurons, microglia, oligodendroglia, or cerebral vascular cells. Analysis of angiogenesis in the neonatal retina also revealed enriched Mlc1-driven eGFP expression in perivascular astrocytes that contact sprouting blood vessels and regulate blood-retinal barrier permeability. A cortical injury model revealed that Mlc1-eGFP expression is progressively induced in reactive astrocytes that form a glial scar. Hence, Mlc1-eGFP knock-in mice are a new and powerful tool to identify perivascular astrocytes in the brain and retina and characterize how these cell types regulate cerebral blood vessel functions in health and disease.

A Unique Mutational Spectrum of MLC1 in Korean Patients With Megalencephalic Leukoencephalopathy With Subcortical Cysts: p.Ala275Asp Founder Mutation and Maternal Uniparental Disomy of Chromosome 22.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare inherited disorder characterized by infantile-onset macrocephaly, slow neurologic deterioration, and seizures. Mutations in the causative gene, MLC1, are found in approximately 75% of patients and are inherited in an autosomal recessive manner. We analyzed MLC1 mutations in five unrelated Korean patients with MLC. METHODS: Direct Sanger sequencing was used to identify MLC1 mutations. A founder effect of the p.Ala275Asp variant was demonstrated by haplotype analysis using single-nucleotide polymorphic (SNP) markers. Multiple ligation-dependent probe amplification (MLPA) and comparative genomic hybridization plus SNP array were used to detect exonic deletions or uniparental disomy (UPD). RESULTS: The most prevalent pathogenic variant was c.824C>A (p.Ala275Asp) found in 7/10 (70%) alleles. Two pathogenic frameshift variants were found: c.135delC (p.Cys46Alafs*12) and c.337_353delinsG (p.Ile113Glyfs*4). Haplotype analysis suggested that the Korean patients with MLC harbored a founder mutation in p.Ala275Asp. The p.(Ile113Glyfs*4) was identified in a homozygous state, and a family study revealed that only the mother was heterozygous for this variant. Further analysis of MLPA and SNP arrays for this patient demonstrated loss of heterozygosity of chromosome 22 without any deletion, indicating UPD. The maternal origin of both chromosomes 22 was demonstrated by haplotype analysis. CONCLUSIONS: This study is the first to describe the mutational spectrum of Korean patients with MLC, demonstrating a founder effect of the p.Ala275Asp variant. This study also broadens our understanding of the mutational spectrum of MLC1 by demonstrating a homozygous p.(Ile113Glyfs*4) variant resulting from UPD of chromosome 22.

Prenatal diagnosis of two families with megalencephalic leukoencephalopathy with subcortical cysts / 中华围产医学杂志

Objectives To provide genetic counseling and prenatal molecular diagnosis for two families with megalencephalic leukoencephalopathy with subcortical cysts (MLC).Methods Two MLC patients (probands 1 and 2) were admitted to the Department of Pediatrics of Peking University First Hospital in June 2011 and June 2009,respectively.Peripheral blood was collected and DNA sequencing was performed for genetic analysis for the two MLC patients and their parents.Amniotic fluid and villus of two fetuses (fetus 1 and 2) were collected at 21+4 and 12+3 weeks of gestational age from their mothers when they were pregnant again.The genomic DNA of the two fetuses was extracted and corresponding sites of MLC1 gene were sequenced.Haplotype analysis using a combination of 3 microsatellite markers (AR,DXS6807 and DXS6797) on chromosome X and sex determining region of Y chromosome was performed to detect maternal cell contamination.Verification of the prenatal molecular diagnosis and follow up study after birth were conducted for both fetuses.Results Macrocephaly,motor development delay and typical findings on brain MRI were identified in the two probands,and were clinically diagnosed with MLC.Compound heterozygous mutations were detected in proband 1 [c.353C＞T (p.T118M) and c.803C＞G (p.T268R)] and proband 2 [c.353C＞T (p.T118M) and c.836T＞C(p.L279P)],respectively.MLC was genetically diagnosed.Heterozygous variation in c.353[c.353C＞T (p.T118M)] and wild c.803C were identified in fetus 1,and both wild c.353C and c.836T were found in fetus 2.No maternal cell contamination was detected in both fetuses.Sequencing the corresponding sites after birth confirmed the prenatal diagnosis,and the head circumference and motor development were normal in fetus 1 at 5 months old.No macrocephaly was found and no DNA sequencing was done in fetus 2 at one month old.Conclusions Genetic counseling and prenatal molecular diagnosis for MLC families combined with clinical and genetic diagnosis are important in preventing MLC.Haplotype analysis with a combination of three microsatellite markers on chromosome X and sex determining region of Y chromosome is useful in detecting maternal cell contamination and avoiding its influence on prenatal diagnosis,and confirming the reliability of prenatal diagnosis.

A Unique Mutational Spectrum of MLC1 in Korean Patients With Megalencephalic Leukoencephalopathy With Subcortical Cysts: p.Ala275Asp Founder Mutation and Maternal Uniparental Disomy of Chromosome 22

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare inherited disorder characterized by infantile-onset macrocephaly, slow neurologic deterioration, and seizures. Mutations in the causative gene, MLC1, are found in approximately 75% of patients and are inherited in an autosomal recessive manner. We analyzed MLC1 mutations in five unrelated Korean patients with MLC. METHODS: Direct Sanger sequencing was used to identify MLC1 mutations. A founder effect of the p.Ala275Asp variant was demonstrated by haplotype analysis using single-nucleotide polymorphic (SNP) markers. Multiple ligation-dependent probe amplification (MLPA) and comparative genomic hybridization plus SNP array were used to detect exonic deletions or uniparental disomy (UPD). RESULTS: The most prevalent pathogenic variant was c.824C>A (p.Ala275Asp) found in 7/10 (70%) alleles. Two pathogenic frameshift variants were found: c.135delC (p.Cys46Alafs*12) and c.337_353delinsG (p.Ile113Glyfs*4). Haplotype analysis suggested that the Korean patients with MLC harbored a founder mutation in p.Ala275Asp. The p.(Ile113Glyfs*4) was identified in a homozygous state, and a family study revealed that only the mother was heterozygous for this variant. Further analysis of MLPA and SNP arrays for this patient demonstrated loss of heterozygosity of chromosome 22 without any deletion, indicating UPD. The maternal origin of both chromosomes 22 was demonstrated by haplotype analysis. CONCLUSIONS: This study is the first to describe the mutational spectrum of Korean patients with MLC, demonstrating a founder effect of the p.Ala275Asp variant. This study also broadens our understanding of the mutational spectrum of MLC1 by demonstrating a homozygous p.(Ile113Glyfs*4) variant resulting from UPD of chromosome 22.