Mitochondrial DNA maintenance defects El-Hattab, Ayman W; Craigen, William J; Scaglia, Fernando
Biochimica et biophysica acta. Molecular basis of disease
1863, Številka:
6
Journal Article
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The maintenance of mitochondrial DNA (mtDNA) depends on a number of nuclear gene-encoded proteins including a battery of enzymes forming the replisome needed to synthesize mtDNA. These enzymes need ...to be in balanced quantities to function properly that is in part achieved by exchanging intramitochondrial contents through mitochondrial fusion. In addition, mtDNA synthesis requires a balanced supply of nucleotides that is achieved by nucleotide recycling inside the mitochondria and import from the cytosol. Mitochondrial DNA maintenance defects (MDMDs) are a group of diseases caused by pathogenic variants in the nuclear genes involved in mtDNA maintenance resulting in impaired mtDNA synthesis leading to quantitative (mtDNA depletion) and qualitative (multiple mtDNA deletions) defects in mtDNA. Defective mtDNA leads to organ dysfunction due to insufficient mtDNA-encoded protein synthesis, resulting in an inadequate energy production to meet the needs of affected organs. MDMDs are inherited as autosomal recessive or dominant traits, and are associated with a broad phenotypic spectrum ranging from mild adult-onset ophthalmoplegia to severe infantile fatal hepatic failure. To date, pathogenic variants in 20 nuclear genes known to be crucial for mtDNA maintenance have been linked to MDMDs, including genes encoding enzymes of mtDNA replication machinery (POLG, POLG2, TWNK, TFAM, RNASEH1, MGME1, and DNA2), genes encoding proteins that function in maintaining a balanced mitochondrial nucleotide pool (TK2, DGUOK, SUCLG1, SUCLA2, ABAT, RRM2B, TYMP, SLC25A4, AGK, and MPV17), and genes encoding proteins involved in mitochondrial fusion (OPA1, MFN2, and FBXL4).
Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders.
To perform clinical whole-exome sequencing and report (1) the rate of ...molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome.
Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient's physician. The patients were primarily pediatric (1756 88%; mean age, 6 years; 888 females 44%, 1101 males 55%, and 11 fetuses 1% gender unknown), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay.
Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings.
A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%-31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%-27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%-47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%-25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics.
Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.
BACKGROUNDTranscriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate ...DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq-guided method to diagnose individuals across a wide range of ages and clinical phenotypes.METHODSOne hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and skin fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network clinical site from 2014 to 2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis.RESULTSThe transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Skin fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts.CONCLUSIONSFor our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.TRIAL REGISTRATIONNot applicable.FUNDINGNIH Common Fund, BCM Intellectual and Developmental Disabilities Research Center, Eunice Kennedy Shriver National Institute of Child Health & Human Development.
ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter γ-aminobutyric acid (GABA). We report an essential role for ABAT in a seemingly unrelated pathway, ...mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause an autosomal recessive neurometabolic disorder and mtDNA depletion syndrome (MDS). We describe a family with encephalomyopathic MDS caused by a homozygous missense mutation in ABAT that results in elevated GABA in subjects’ brains as well as decreased mtDNA levels in subjects’ fibroblasts. Nucleoside rescue and co-IP experiments pinpoint that ABAT functions in the mitochondrial nucleoside salvage pathway to facilitate conversion of dNDPs to dNTPs. Pharmacological inhibition of ABAT through the irreversible inhibitor Vigabatrin caused depletion of mtDNA in photoreceptor cells that was prevented through addition of dNTPs in cell culture media. This work reveals ABAT as a connection between GABA metabolism and nucleoside metabolism and defines a neurometabolic disorder that includes MDS.
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•ABAT converts dNDP to dNTP in the mitochondrial nucleoside salvage pathway•Inhibition of ABAT causes decreased copy number of mitochondrial genome•mtDNA depletion induced by ABAT inhibition is rescued by dNTP supplementation in vitro•ABAT causes human mitochondrial DNA depletion syndrome
ABAT is a key enzyme responsible for catabolism of principal inhibitory neurotransmitter γ-aminobutyric acid (GABA). Besse et al. report an essential role for ABAT in a seemingly unrelated pathway, mitochondrial nucleoside salvage, and demonstrate that mutations in this enzyme cause autosomal recessive mtDNA depletion syndrome.
Mitochondria are critically involved in necrotic cell death induced by Ca2+ overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore - a protein complex that spans ...both the outer and inner mitochondrial membranes - is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1-Vdac3-null mice exhibited a Ca2+- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca2+- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1-Vdac3 and Vdac1-Vdac2-Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.
Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the ...diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4-aminobutyrate aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in
and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (
-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in
. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (
= 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the
variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.
While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined.
To determine ...the diagnostic yield and use of clinical exome sequencing in critically ill infants.
Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants.
Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders.
The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling.
Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.
Voltage-dependant Anion Channels, also known as mitochondrial porins, are pore-forming proteins located in the mitochondrial outer membrane (MOM) that, in addition to forming complexes with other ...proteins that localize to the MOM, also function as the main conduit for transporting metabolites between the cytoplasm and mitochondria. VDACs are encoded by a multi-member gene family, and the number of isoforms and specific functions of VDACs varies between species. Translating the well-described in vitro characteristics of the VDAC isoforms into in vivo functions has been a challenge, with the generation of animal models of VDAC deficiency providing much of the available information about isoform-specific roles in biology. Here, we review the approaches used to create these insect and mammalian animal models, and the conclusions reached by studying the consequences of loss of function mutations on the genetic, physiologic, and biochemical properties of the resulting models. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.
► Animal models lacking VDAC isoforms have been created. ► Drosophila and mouse models share similar features. ► VDACs have roles in fertility, learning and memory, glucose homeostasis, and apoptosis. ► The models have been instrumental in identifying the function of each VDAC protein.
To develop criteria to interpret mitochondrial transfer RNA (mt-tRNA) variants based on unique characteristics of mitochondrial genetics and conserved structural/functional properties of tRNA.
We ...developed rules on a set of established pathogenic/benign variants by examining heteroplasmy correlations with phenotype, tissue distribution, family members, and among unrelated families from published literature. We validated these deduced rules using our new cases and applied them to classify novel variants.
Evaluation of previously reported pathogenic variants found that 80.6% had sufficient evidence to support phenotypic correlation with heteroplasmy levels among and within families. The remaining variants were downgraded due to the lack of similar evidence. Application of the verified criteria resulted in rescoring 80.8% of reported variants of uncertain significance (VUS) to benign and likely benign. Among 97 novel variants, none met pathogenic criteria. A large proportion of novel variants (84.5%) remained as VUS, while only 10.3% were likely pathogenic. Detection of these novel variants in additional individuals would facilitate their classification.
Proper interpretation of mt-tRNA variants is crucial for accurate clinical diagnosis and genetic counseling. Correlations with tissue distribution, heteroplasmy levels, predicted perturbations to tRNA structure, and phenotypes provide important evidence for determining the clinical significance of mt-tRNA variants.
Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP ...to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.
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