MT-TN mutations lead to progressive mitochondrial encephalopathy and promotes mitophagy.

Mitochondrial encephalopathy is a neurological disorder caused by impaired mitochondrial function and energy production. One of the genetic causes of this condition is the mutation of MT-TN, a gene that encodes the mitochondrial transfer RNA (tRNA) for asparagine. MT-TN mutations affect the stability and structure of the tRNA, resulting in reduced protein synthesis and complex enzymatic deficiency of the mitochondrial respiratory chain. Our patient cohort manifests with epileptic encephalopathy, ataxia, hypotonia, and bilateral basal ganglia calcification, which differs from previously reported cases. MT-TN mutation deficiency leads to decreased basal and maximal oxygen consumption rates, disrupted spare respiratory capacity, declined mitochondrial membrane potential, and impaired ATP production. Moreover, MT-TN mutations promote mitophagy, a process of selective degradation of damaged mitochondria by autophagy. Excessive mitophagy further leads to mitochondrial biogensis as a compensatory mechanism. In this study, we provided evidence of pathogenicity for two MT-TN mutations, m.5688 T > C and m.G5691A, explored the molecular mechanisms, and summarized the clinical manifestations of MT-TN mutations. Our study expanded the genotype and phenotypic spectrum and provided new insight into mt-tRNA (Asn)-associated mitochondrial encephalopathy.

Large heterozygous deletion and uniparental disomy masquerading as homozygosity in CHKB gene.

BACKGROUND: CHKB mutations have been described in 49 patients with megaconial congenital muscular dystrophy, which is a rare autosomal recessive disorder, of which 40 patients showed homozygosity. METHODS: Peripheral blood genomic DNA samples were extracted from patients and their parents and were tested by whole exome sequencing. Quantitative PCR was performed to detect deletion. Single nucleotide polymorphism analysis was performed to identify uniparental disomy. Quantitative PCR and western blot were used to measure the expression level of CHKB in patient 1-derived immortalized lymphocytes. Mitochondria were observed in lymphocytes by electron microscopy. RESULTS: Two unrelated cases born to non-consanguineous parents were diagnosed with megaconial congenital muscular dystrophy due to apparently homozygous mutations (patient 1: c.225-2A>T; patient 2: c.701C>T) in the CHKB gene using whole exome sequencing. Quantitative PCR revealed that patient 1 had a large deletion encompassing the CHKB gene, inherited from the mother. Single nucleotide polymorphism analysis revealed patient 2 had paternal uniparental isodisomy containing the CHKB gene. In the immortalized lymphocytes from patient 1, decreased expression of CHKB was revealed by quantitative PCR and western blot, and giant mitochondria were observed using electron microscopy. CONCLUSION: We provide a possibility to detect giant mitochondria in other cells when muscle was not available. Moreover, clinicians should be aware that homozygous variants can be masqueraded by uniparental disomy or large deletions in offspring of non-consanguineous parents, and excessive homozygosity may be misdiagnosed.

Use of dual genomic sequencing to screen mitochondrial diseases in pediatrics: a retrospective analysis.

Mitochondrial diseases (MDs) were a large group multisystem disorders, attributable in part to the dual genomic control. The advent of massively sequencing has improved diagnostic rates and speed, and was increasingly being used as a first-line diagnostic test. Paediatric patients (aged < 18 years) who underwent dual genomic sequencing were enrolled in this retrospective multicentre study. We evaluated the mitochondrial disease criteria (MDC) and molecular diagnostic yield of dual genomic sequencing. Causative variants were identified in 177 out of 503 (35.2%) patients using dual genomic sequencing. Forty-six patients (9.1%) had mitochondria-related variants, including 25 patients with nuclear DNA (nDNA) variants, 15 with mitochondrial DNA (mtDNA) variants, and six with dual genomic variants (MT-ND6 and POLG; MT-ND5 and RARS2; MT-TL1 and NARS2; MT-CO2 and NDUFS1; MT-CYB and SMARCA2; and CHRNA4 and MT-CO3). Based on the MDC, 15.2% of the patients with mitochondria-related variants were classified as "unlikely to have mitochondrial disorder". Moreover, 4.5% of the patients with non-mitochondria-related variants and 1.43% with negative genetic tests, were classified as "probably having mitochondrial disorder". Dual genomic sequencing in suspected MDs provided a more comprehensive and accurate diagnosis for pediatric patients, especially for patients with dual genomic variants.

Functional Investigation of TUBB4A Variants Associated with Different Clinical Phenotypes.

Dominant TUBB4A variants result in different phenotypes, including hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC), dystonia type 4 (DYT4), and isolated hypomyelination. Here, we report four new patients with a novel TUBB4A variant (p.K324T) and three new patients with previously reported variants (p.Q292K, p.V255I, p.E410K). The individual carrying the novel p.K324T variant exhibits epilepsy of infancy with migrating focal seizures (EIMFS), while the other three have isolated hypomyelination phenotype. We also present a study of the cellular effects of TUBB4A variants responsible for H-ABC (p.D249N), DYT4 (p.R2G), a severe combined phenotype with combination of hypomyelination and EIMFS (p.K324T), and isolated hypomyelination (p.Q292K and p.E410K) on microtubule stability and dynamics, neurite outgrowth, dendritic spine development, and kinesin binding. Cellular-based assays reveal that all variants except p.R2G increase microtubule stability, decrease microtubule polymerization rates, reduce axonal outgrowth, and alter the density and shape of dendritic spines. We also find that the p.K324T and p.E410K variants perturb the binding of TUBB4A to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors. Taken together, our data suggest that impaired microtubule stability and dynamics, defected axonal growth, and dendritic spine development form the common molecular basis of TUBB4A-related leukodystrophy. Impairment of TUBB4A binding to KIF1A is more likely to be involved in the isolated hypomyelination phenotype, which suggests that alterations in kinesin binding may cause different phenotypes. In conclusion, our study extends the spectrum of TUBB4A mutations and related phenotypes and provides insight into why different TUBB4A variants cause distinct clinical phenotypes.

A novel homozygous missense mutation in the FASTKD2 gene leads to Lennox-Gastaut syndrome.

FASTKD2 encodes an RNA-binding protein, which is a key post-transcriptional regulator of mitochondrial gene expression. Mutations in FASTKD2 have recently been found in mitochondrial encephalomyopathy, which is characterized by a deficiency in mitochondrial function. To date, seven patients have been reported. Six patients were identified with nonsense or frameshift mutations in the FASTKD2 gene, and only one patient harbored a missense mutation and a nonsense mutation. Here, we identified a novel FASTKD2 homozygous mutation, c.911 T > C, in a patient diagnosed with Lennox-Gastaut syndrome. We observed that the expression of FASTKD2 and the levels of mitochondrial 16 S rRNA were lower in the patient than in the unaffected controls. In conclusion, the missense mutation c.911 T > C caused loss of function in FASTKD2, which was associated with a new phenotype, Lennox-Gastaut syndrome.

Phenotype-Genotype Analysis Based on Molecular Classification in 135 Children With Mitochondrial Disease.

OBJECTIVES: Over the past decades, mitochondrial disease classification has been mainly based on molecular defects. We aim to analyze phenotype-genotype correlation of mitochondrial disorders according to molecular classification. METHODS: In this cohort study, we identified 135 individuals diagnosed with mitochondrial disorders, and all patients were divided into four subgroups based on molecular functions: the Respiratory Chain group (including subunits and assembly proteins in the respiratory chain), the Protein Synthesis group (including mitochondrial RNA metabolism, mitochondrial translation), the mitcohindrial DNA (mtDNA) Replication group, and the Others group (including cofactors, homeostasis, substrates, and inhibitors). RESULTS: We found that in China, patients with the mtDNA variant constituted a large percentage of mitochondrial disease and were associated with a male preponderance in the Respiratory Chain group, whereas those in the Protein Synthesis group showed a relatively later onset and higher serum lactate level. In contrast, patients with nuclear DNA variants were younger at onset, with no specific lactate or cranial imaging features, especially in the Others group, which contained several mitochondrial diseases with corresponding treatment. CONCLUSION: The mtDNA was recommended to detect first in patients with typical lactate and cranial imaging features. A broader consideration and detection are necessary for a better prognosis in an atypical patient.

West Syndrome Caused By a Chloride/Proton Exchange-Uncoupling CLCN6 Mutation Related to Autophagic-Lysosomal Dysfunction.

Vesicular chloride/proton exchangers of the CLC family are critically involved in the function of the endosomal-lysosomal pathway. Their dysfunction leads to severe disorders including intellectual disability and epilepsy for ClC-4, Dent's disease for ClC-5, and lysosomal storage disease and osteopetrosis for ClC-7. Here, we report a de novo variant p.Glu200Ala (p.E200A; c.599A>C) of the late endosomal ClC-6, encoded by CLCN6, in a patient with West syndrome (WS), severe developmental delay, autism, movement disorder, microcephaly, facial dysmorphism, and visual impairment. Mutation of this conserved glutamate uncouples chloride transport from proton antiport by ClC-6. This affects organellar ion homeostasis and was shown to be deleterious for other CLCs. In this study, we found that upon heterologous expression, the ClC-6 E200A variant caused autophagosome accumulation and impaired the clearance of autophagosomes by blocking autophagosome-lysosome fusion. Our study provides clinical and functional support for an association between CLCN6 variants and WS. Our findings also provide novel insights into the molecular mechanisms underlying the pathogenesis of WS, suggesting an involvement of autophagic-lysosomal dysfunction.

[Mutations in aminoacyl-tRNA synthetase genes: an analysis of 10 cases].

OBJECTIVE: To study the clinical features of the diseases associated with aminoacyl-tRNA synthetases (ARS) deficiency. METHODS: A retrospective analysis was performed of the clinical and gene mutation data of 10 children who were diagnosed with ARS gene mutations, based on next-generation sequencing from January 2016 to October 2019. RESULTS: The age of onset ranged from 0 to 9 years among the 10 children. Convulsion was the most common initial symptom (7 children). Clinical manifestations included ataxia and normal or mildly retarded intellectual development (with or without epilepsy; n=4) and onset of epilepsy in childhood with developmental regression later (n=2). Some children experienced disease onset in the neonatal period and had severe epileptic encephalopathy, with myoclonus, generalized tonic-clonic seizure, and convulsive seizure (n=4); 3 had severe delayed development, 2 had feeding difficulty, and 1 had hearing impairment. Mutations were found in five genes: 3 had novel mutations in the AARS2 gene (c.331G>C, c.2682+5G>A, c.2164C>T, and c.761G>A), 2 had known mutations in the DARS2 gene (c.228-16C>A and c.536G>A), 1 had novel mutations in the CARS2 gene (c.1036C>T and c.323T>G), 1 had novel mutations in the RARS2 gene (c.1210A>G and c.622C>T), and 3 had novel mutations in the AARS gene (c.1901T>A, c.229C>T, c.244C>T, c.961G>C, c.2248C>T, and Chr16:70298860-70316687del). CONCLUSIONS: A high heterogeneity is observed in the clinical phenotypes of the diseases associated with the ARS deficiency. A total of 14 novel mutations in 5 genes are reported in this study, which enriches the clinical phenotypes and genotypes of the diseases associated with ARS deficiency.

The Glycosylphosphatidylinositol biosynthesis pathway in human diseases.

Glycosylphosphatidylinositol biosynthesis defects cause rare genetic disorders characterised by developmental delay/intellectual disability, seizures, dysmorphic features, and diverse congenital anomalies associated with a wide range of additional features (hypotonia, hearing loss, elevated alkaline phosphatase, and several other features). Glycosylphosphatidylinositol functions as an anchor to link cell membranes and protein. These proteins function as enzymes, adhesion molecules, complement regulators, or co-receptors in signal transduction pathways. Biallelic variants involved in the glycosylphosphatidylinositol anchored proteins biosynthetic pathway are responsible for a growing number of disorders, including multiple congenital anomalies-hypotonia-seizures syndrome; hyperphosphatasia with mental retardation syndrome/Mabry syndrome; coloboma, congenital heart disease, ichthyosiform dermatosis, mental retardation, and ear anomalies/epilepsy syndrome; and early infantile epileptic encephalopathy-55. This review focuses on the current understanding of Glycosylphosphatidylinositol biosynthesis defects and the associated genes to further understand its wide phenotype spectrum.

Homozygous PIGT Mutation Lead to Multiple Congenital Anomalies-Hypotonia Seizures Syndrome 3.

PIGT encodes a subunit of the glycosylphosphatidylinositol transamidase complex, which catalyzes the attachment of proteins to GPI-anchors. A homozygous PIGT variant c.550G>A (p. E184K) in a Chinese boy with multiple malformations, hypotonia, seizure and profound development delay was identified by panel sequencing. Pathogenicity of the variant was confirmed by flow cytometry. The expression of CD16 and CD24 of this proband reduced to 16.92 and 22.16% compare with normal control respectively while which of his parents and sister were normal. This mutation raised the mRNA level on the peripheral blood mono nuclear cells of this patient. This study expanded the variant spectrum of MCAHS3, and CD16 could be an effective marker to evaluate the pathogenicity of PIGT mutation.