bims-mitdis 2022-01-02 papers

bims-mitdis

Biomed News

on Mitochondrial disorders

Issue of 2022‒01‒02
twenty-six papers selected by
Catalina Vasilescu
University of Helsinki

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.
Induction and Detection of Mitophagy.
NAD kinase sustains lipogenesis and mitochondrial metabolismthrough fatty acid synthesis.
Mitochondrial Quality Surveillance: Mitophagy in cardiovascular health and disease.
A Leigh syndrome caused by compound heterozygous mutations on NDUFAF5 induce early infant death: A case report.
Mitochondrial Function and Parkinson's Disease: From the Perspective of the Electron Transport Chain.
Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.
Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease.
Computational modeling of mitochondrial K+- and H+-driven ATP synthesis.
Association Between Epilepsy and Leigh Syndrome With MT-ND3 Mutation, Particularly the m.10191T>C Point Mutation.
Variants in mitochondrial ATP synthase cause variable neurologic phenotypes.
Episodic weakness and axonal sensorimotor neuropathy caused by a mitochondrial MT-ATP6 mutation.
PNC2 (SLC25A36) deficiency associated with the hyperinsulinism/hyperammonemia syndrome.
p53 regulates skeletal muscle mitophagy and mitochondrial quality control following denervation-induced muscle disuse.
SOD2 V16A Amplifies Vascular Dysfunction in Sickle Cell Patients by Curtailing Mitochondria Complex IV Activity.
Mitochondriotropic antioxidant based on caffeic acid AntiOxCIN4 activates Nrf2-dependent antioxidant defenses and quality control mechanisms to antagonize oxidative stress-induced cell damage.
The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.
The role of FUNDC1 in mitophagy, mitochondrial dynamics and human diseases.
MITOL regulates phosphatidic acid-binding activity of RMDN3/PTPIP51.
Genome-wide association study of mitochondrial copy number.
Mitochondrial-derived peptides: New markers for cardiometabolic dysfunction.
Feasible and clinical relevant outcome measures for adults with mitochondrial disease.
Mitochondrial phenotypes in iPSC AD models.
VDAC1 oligomerization may enhance DDP-induced hepatocyte apoptosis by exacerbating oxidative stress and mitochondrial DNA damage.
Biology of Activating Transcription Factor 4 (ATF4) and Its Role in Skeletal Muscle Atrophy.
Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness.

Elife. 2021 Dec 31. pii: e68213. [Epub ahead of print]10

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

Sven Dennerlein, Sabine Poerschke, Silke Oeljeklaus, Cong Wang, Ricarda Richter-Dennerlein, Johannes Sattmann, Diana Bauermeister, Elisa Hanitsch, Stefan Stoldt, Thomas Langer, Stefan Jakobs, Bettina Warscheid, Peter Rehling.

  Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Keywords:  assembly; biochemistry; cell biology; chemical biology; mitochondria; oxidative phosphorylation; ribosome; translation

DOI:  https://doi.org/10.7554/eLife.68213
Methods Mol Biol. 2022 ;2445 227-239

Induction and Detection of Mitophagy.

Maria A Yapryntseva, Boris Zhivotovsky, Vladimir Gogvadze.

  Mitophagy, a process of selective elimination of mitochondria by autophagy, is a mechanism of mitochondrial quality control that maintains mitochondrial network functionality. The elimination of damaged mitochondria through autophagy requires two steps: induction of general autophagy and priming of damaged mitochondria for selective autophagic recognition. Mitophagy impairment is linked to various pathologies; thus, removal of malfunctioning or even harmful mitochondria is vital to cellular physiology. Here, we describe methods that can be applied to the investigation of mitophagy.

Keywords:  Autophagy; Confocal microscopy; Flow cytometry; Mitochondria; Mitophagy; Respiration

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_14
Cell Rep. 2021 Dec 28. pii: S2211-1247(21)01653-3. [Epub ahead of print]37(13): 110157

NAD kinase sustains lipogenesis and mitochondrial metabolismthrough fatty acid synthesis.

Mengyao Xu, Long Ding, Jingjing Liang, Xiao Yang, Yuan Liu, Yingchun Wang, Mei Ding, Xun Huang.

  Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, we found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function.

Keywords:  Drosophila; FASN; NADK; lipogenesis; mitochondrial metabolism

DOI:  https://doi.org/10.1016/j.celrep.2021.110157
Am J Physiol Cell Physiol. 2021 12 29.

Mitochondrial Quality Surveillance: Mitophagy in cardiovascular health and disease.

Rachel Y Diao, Asa B Gustafsson.

  Selective autophagy of the mitochondria, known as mitophagy, is a major mitochondrial quality control pathway in the heart that is involved in removing unwanted or dysfunctional mitochondria from the cell. Baseline mitophagy is critical for maintaining the fitness of the mitochondrial population by continuous turnover of aged and less functional mitochondria. Mitophagy is also critical in adapting to stress associated with mitochondrial damage or dysfunction. The removal of damaged mitochondria prevents ROS-mediated damaged to proteins and DNA and suppresses activation of inflammation and cell death. Impairments in mitophagy are associated with the pathogenesis of many diseases, including cancers, inflammatory diseases, neurodegeneration, and cardiovascular disease. Mitophagy is a highly regulated and complex process that requires the coordination of labeling dysfunctional mitochondria for degradation while simultaneously promoting de novo autophagosome biogenesis adjacent to the cargo. In this review, we provide an update on our current understanding of these steps in mitophagy induction and discuss the physiological and pathophysiological consequences of altered mitophagy in the heart.

Keywords:  Parkin; autophagy; heart; mitochondria; mitophagy

DOI:  https://doi.org/10.1152/ajpcell.00360.2021
Mol Genet Genomic Med. 2021 Dec 28. e1852

A Leigh syndrome caused by compound heterozygous mutations on NDUFAF5 induce early infant death: A case report.

Yan Wen, Guoyan Lu, Lina Qiao, Yifei Li.

  BACKGROUND: The NADH:ubiquinone oxidoreductase complex assembly factor gene (NDUFAF5) has been linked to the occurrence of Leigh syndrome, but few causative mutations have been identified. Here we report a rare case of Leigh syndrome in an infant who died in the early postnatal period.METHODS: We performed whole-exome sequencing (WES) and mutation analysis of NDUFAF5 to obtain genetic data on the patient and describe the clinical and genetic characteristics.
RESULTS: The proband was a 2-month-old male infant who suffered from recurrent vomiting and persistent seizure and died at 2 months of age after early medical support and treatment. His parents reported the unexplained death of the infant's older brother at 1 year of age. WES of the patient's DNA revealed c.357C>G and c.611C>T compound heterozygous mutations in NDUFAF5; analysis with the MutationTaster application indicated that both were pathogenic (p = 0.99). Significant structural changes in the transport domain of the protein were predicted using SWISS-MODEL. We estimated the stability of the mutant protein using a mutation cutoff scanning matrix and found reductions in Gibbs free energy (-0.623 kcal/mol for p.D119E and -0.813 kcal/mol for p.A204V), indicating that the mutations led to an unstable protein structure. We speculated that the patient died as a result of impaired mitochondrial function caused by the NDUFAF5 mutations, and made a diagnosis of Leigh syndrome.
CONCLUSION: Our results demonstrate that molecular genetic screening is useful for the diagnosis of mitochondrial diseases, especially in children with a positive family history. Leigh syndrome should be considered in the diagnosis of patients presenting with severe recurrent vomiting and feeding difficulties with persistent seizure. Our findings expand the mutation spectrum of the NDUFAF5 gene and contribute to the genotype-phenotype map of mitochondrial respiratory chain complex I deficiency.

Keywords:   NDUFAF5 ; Leigh syndrome; case report; genomic sequence; mitochondrial deficiency

DOI:  https://doi.org/10.1002/mgg3.1852
Front Mol Neurosci. 2021 ;14 797833

Mitochondrial Function and Parkinson's Disease: From the Perspective of the Electron Transport Chain.

Jeng-Lin Li, Tai-Yi Lin, Po-Lin Chen, Ting-Ni Guo, Shu-Yi Huang, Chun-Hong Chen, Chin-Hsien Lin, Chih-Chiang Chan.

  Parkinson's disease (PD) is known as a mitochondrial disease. Some even regarded it specifically as a disorder of the complex I of the electron transport chain (ETC). The ETC is fundamental for mitochondrial energy production which is essential for neuronal health. In the past two decades, more than 20 PD-associated genes have been identified. Some are directly involved in mitochondrial functions, such as PRKN, PINK1, and DJ-1. While other PD-associate genes, such as LRRK2, SNCA, and GBA1, regulate lysosomal functions, lipid metabolism, or protein aggregation, some have been shown to indirectly affect the electron transport chain. The recent identification of CHCHD2 and UQCRC1 that are critical for functions of complex IV and complex III, respectively, provide direct evidence that PD is more than just a complex I disorder. Like UQCRC1 in preventing cytochrome c from release, functions of ETC proteins beyond oxidative phosphorylation might also contribute to the pathogenesis of PD.

Keywords:  Parkinson’s disease; apoptosis; electron transport chain; mitochondria quality control; mitophagy

DOI:  https://doi.org/10.3389/fnmol.2021.797833
Methods Mol Biol. 2022 ;2445 207-226

Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy.

Benjamin S Padman, Michael Lazarou.

  Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.

Keywords:  ImageJ/FIJI; Object-based image analysis (OBIA); PINK1/Parkin mitophagy; Phagophore biogenesis; Regions of interest (ROI)

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_13
Mol Genet Metab. 2021 Dec 18. pii: S1096-7192(21)01191-4. [Epub ahead of print]

Advanced approach for comprehensive mtDNA genome testing in mitochondrial disease.

Jing Wang, Jorune Balciuniene, Maria Alejandra Diaz-Miranda, Elizabeth M McCormick, Erfan Aref-Eshghi, Alison M Muir, Kajia Cao, Juliana Troiani, Alicia Moseley, Zhiqian Fan, Zarazuela Zolkipli-Cunningham, Amy Goldstein, Rebecca D Ganetzky, Colleen C Muraresku, James T Peterson, Nancy B Spinner, Douglas C Wallace, Matthew C Dulik, Marni J Falk.

  Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations.

Keywords:  Heteroplasmy; Mitochondrial genome; Multiple deletions; Single large-scale deletion; mtDNA mutation

DOI:  https://doi.org/10.1016/j.ymgme.2021.12.006
J Mol Cell Cardiol. 2021 Dec 23. pii: S0022-2828(21)00232-7. [Epub ahead of print]165 9-18

Computational modeling of mitochondrial K+- and H+-driven ATP synthesis.

Sonia Cortassa, Miguel A Aon, Magdalena Juhaszova, Evgeny Kobrinsky, Dmitry B Zorov, Steven J Sollott.

  ATP synthase (F1Fo) is a rotary molecular engine that harnesses energy from electrochemical-gradients across the inner mitochondrial membrane for ATP synthesis. Despite the accepted tenet that F1Fo transports exclusively H+, our laboratory has demonstrated that, in addition to H+, F1Fo ATP synthase transports a significant fraction of ΔΨm-driven charge as K+ to synthesize ATP. Herein, we utilize a computational modeling approach as a proof of principle of the feasibility of the core mechanism underlying the enhanced ATP synthesis, and to explore its bioenergetic consequences. A minimal model comprising the 'core' mechanism constituted by ATP synthase, driven by both proton (PMF) and potassium motive force (KMF), respiratory chain, adenine nucleotide translocator, Pi carrier, and K+/H+ exchanger (KHEmito) was able to simulate enhanced ATP synthesis and respiratory fluxes determined experimentally with isolated heart mitochondria. This capacity of F1Fo ATP synthase confers mitochondria with a significant energetic advantage compared to K+ transport through a channel not linked to oxidative phosphorylation (OxPhos). The K+-cycling mechanism requires a KHEmito that exchanges matrix K+ for intermembrane space H+, leaving PMF as the overall driving energy of OxPhos, in full agreement with the standard chemiosmotic mechanism. Experimental data of state 4➔3 energetic transitions, mimicking low to high energy demand, could be reproduced by an integrated computational model of mitochondrial function that incorporates the 'core' mechanism. Model simulations display similar behavior compared to the experimentally observed changes in ΔΨm, mitochondrial K+ uptake, matrix volume, respiration, and ATP synthesis during the energetic transitions at physiological pH and K+ concentration. The model also explores the role played by KHEmito in modulating the energetic performance of mitochondria. The results obtained support the available experimental evidence on ATP synthesis driven by K+ and H+ transport through the F1Fo ATP synthase.

Keywords:  Energy supply-demand matching; F(1)F(o) ATP synthase; Mitochondrial K(+) uptake; Mitochondrial K(+)/H(+) exchanger

DOI:  https://doi.org/10.1016/j.yjmcc.2021.12.005
Front Neurol. 2021 ;12 752467

Association Between Epilepsy and Leigh Syndrome With MT-ND3 Mutation, Particularly the m.10191T>C Point Mutation.

Ji-Hoon Na, Min Jung Lee, Chul Ho Lee, Young-Mock Lee.

  Background and Purpose: Recent advances in molecular genetic testing have led to a rapid increase in the understanding of the genetics of Leigh syndrome. Several studies have suggested that Leigh syndrome with MT-ND3 mutation is strongly associated with epilepsy. This study focused on the epilepsy-related characteristics of Leigh syndrome with MT-ND3 mutation identified in a single tertiary hospital in South Korea. Methods: We selected 31 patients with mitochondrial DNA (mtDNA) mutations who were genetically diagnosed with mtDNA-associated Leigh syndrome. Among them, seven patients with MT-ND3 mutations were detected. We reviewed various clinical findings such as laboratory findings, brain images, electroencephalography data, seizure types, seizure frequency, antiepileptic drug use history, and current seizure status. Results: The nucleotide changes in the seven patients with the Leigh syndrome with MT-ND3 mutation were divided into two groups: m.10191T>C and m.10158T>C. Six of the seven patients were found to have the m.10191T>C mutations. The median value of the mutant load was 82.5%, ranging from 57.9 to 93.6%. No particular tendency was observed for the first symptom or seizure onset or mutant load. The six patients with the m.10191T>C mutation were diagnosed with epilepsy. Three of these patients were diagnosed with Lennox-Gastaut syndrome (LGS). Conclusion: We reported a very strong association between epilepsy and MT-ND3 mutation in Leigh syndrome, particularly the m.10191T>C mutation. The possibility of an association between the epilepsy phenotype of the m.10191T>C mutation and LGS was noted.

Keywords:  Lennox-Gastaut syndrome; MT-ND3; epilepsy; m10191T>C; mitochondrial DNA-associated Leigh syndrome

DOI:  https://doi.org/10.3389/fneur.2021.752467
Ann Neurol. 2021 Dec 26.

Variants in mitochondrial ATP synthase cause variable neurologic phenotypes.

Michael Zech, Robert Kopajtich, Katja Steinbrücker, Céline Bris, Naig Gueguen, René G Feichtinger, Melanie T Achleitner, Neslihan Duzkale, Maximilien Périvier, Johannes Koch, Harald Engelhardt, Peter Freisinger, Matias Wagner, Theresa Brunet, Riccardo Berutti, Dmitrii Smirnov, Tharsini Navaratnarajah, Richard J T Rodenburg, Lynn S Pais, Christina Austin-Tse, Melanie O'Leary, Sylvia Boesch, Robert Jech, Somayeh Bakhtiari, Sheng Chih Jin, Friederike Wilbert, Michael C Kruer, Saskia B Wortmann, Matthias Eckenweiler, Johannes A Mayr, Felix Distelmaier, Robert Steinfeld, Juliane Winkelmann, Holger Prokisch.

OBJECTIVE: ATP synthase (ATPase) is responsible for the majority of ATP production. Nevertheless, disease phenotypes associated with mutations in ATPase subunits are extremely rare. We aimed at expanding the spectrum of ATPase-related diseases.METHODS: Whole-exome sequencing in cohorts with 2,962 mitochondrial-disease- and/or dystonia-diagnosed individuals and international collaboration were used to identify deleterious variants in ATPase-encoding genes. Findings were complemented by transcriptional and proteomic profiling of patient fibroblasts. ATPase integrity and activity were assayed using cells and tissues from five patients.
RESULTS: We present ten total individuals with biallelic or de-novo monoallelic variants in nuclear ATPase subunit genes. Three unrelated patients showed the same homozygous missense ATP5F1E mutation (including one published case). An intronic splice-disrupting alteration in compound heterozygosity with a nonsense variant in ATP5PO was found in one patient. Three patients had de-novo heterozygous missense variants in ATP5F1A, whereas another three were heterozygous for ATP5MC3 de-novo missense changes. Bioinformatics methods and populational data supported the variants` pathogenicity. Immunohistochemistry, proteomics, and/or immunoblotting revealed significantly reduced ATPase amounts in association to ATP5F1E and ATP5PO mutations. Diminished activity and/or defective assembly of ATPase was demonstrated by enzymatic assays and/or immunoblotting in cells bearing ATP5F1A-p.Arg207His, ATP5MC3-p.Gly79Val, and ATP5MC3-p.Asn106Lys. The associated clinical profiles were heterogeneous, ranging from hypotonia with spontaneous resolution (1/10) to epilepsy with early death (1/10) or variable persistent abnormalities including movement disorders, developmental delay, intellectual disability, hyperlactatemia, and other neurologic and systemic features. Although potentially reflecting an ascertainment bias, dystonia was common (7/10).
INTERPRETATION: Our results establish evidence for a previously unrecognized role of ATPase nuclear-gene defects in phenotypes characterized by neurodevelopmental and neurodegenerative features. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/ana.26293
J Formos Med Assoc. 2021 Dec 22. pii: S0929-6646(21)00536-2. [Epub ahead of print]

Episodic weakness and axonal sensorimotor neuropathy caused by a mitochondrial MT-ATP6 mutation.

Tzu-Hsuan Su, Ni-Chung Lee, Chao-Szu Wu, Steven Shinn-Forng Peng, Pi-Chuan Fan.

  Episodic weakness is typically associated with a group of disorders so called periodic paralyses. Their major causes are mutation of ion channels, and have rarely been linked to mitochondrial disorders. We report a 20-year-old man with episodic weakness and axonal sensorimotor neuropathy since the age of 10 years. Analysis of the next generation sequencing data of the entire mitochondrial genome extracted from the blood revealed a homoplasmic m.9185T > C variant in MT-ATP6. Acetazolamide may be responsive for episodic weakness, and supplements with l-carnitine with coenzyme-Q10 seem to be beneficial as well. To the best of our knowledge, this is the first report in Taiwan which reveals episodic weakness and sensorimotor polyneuropathy as a unique phenotype of MT-ATP6 mutations.

Keywords:  Episodic weakness; MT-ATP6 mutations; Mitochondrial disorders

DOI:  https://doi.org/10.1016/j.jfma.2021.12.003
J Clin Endocrinol Metab. 2021 Dec 31. pii: dgab932. [Epub ahead of print]

PNC2 (SLC25A36) deficiency associated with the hyperinsulinism/hyperammonemia syndrome.

Maher A Shahrour, Francesco Massimo Lasorsa, Vito Porcelli, Imad Dweikat, Maria Antonietta Di Noia, Michal Gur, Giulia Agostino, Avraham Shaag, Teresa Rinaldi, Giuseppe Gasparre, Flora Guerra, Alessandra Castegna, Simona Todisco, Bassam Abu-Libdeh, Orly Elpeleg, Luigi Palmieri.

  CONTEXT: The hyperinsulinism/hyperammonemia (HI/HA) syndrome, the second most common form of congenital hyperinsulinism, has been associated to dominant mutations in GLUD1, coding for the mitochondrial enzyme glutamate dehydrogenase, that increase enzyme activity by reducing its sensitivity to allosteric inhibition by GTP.OBJECTIVE: To identify the underlying genetic aetiology in two siblings who presented with the biochemical features of HI/HA syndrome but did not carry pathogenic variants in GLUD1, and to determine the functional impact of the newly identified mutation.
MAIN OUTCOME MEASURES: The patients were investigated by whole exome sequencing. Yeast complementation studies and biochemical assays on the recombinant mutated protein were performed. The consequences of stable slc25a36 silencing in HeLa cells were also investigated.
RESULTS: A homozygous splice site variant was identified in solute carrier family 25, member 36 (SLC25A36), encoding the pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine as well as guanine nucleotides across the inner mitochondrial membrane. The mutation leads to a 26 aa in-frame deletion in the first repeat domain of the protein which abolished transport activity. Furthermore, knockdown of slc25a36 expression in HeLa cells caused a marked reduction in the mitochondrial GTP content which likely leads to an hyperactivation of glutamate dehydrogenase in our patients.
CONCLUSIONS: We report for the first time a mutation in PNC2/SLC25A36 leading to HI/HA and provide functional evidence of the molecular mechanism responsible for this phenotype. Our findings underscore the importance of mitochondrial nucleotide metabolism and expand the role of mitochondrial transporters in insulin secretion.

Keywords:  GTP; Hyperinsulinism/hyperammonemia syndrome; Mitochondrial carrier; Nucleotide metabolism; PNC2; SLC25A36

DOI:  https://doi.org/10.1210/clinem/dgab932
J Biol Chem. 2021 Dec 24. pii: S0021-9258(21)01350-8. [Epub ahead of print] 101540

p53 regulates skeletal muscle mitophagy and mitochondrial quality control following denervation-induced muscle disuse.

Jonathan M Memme, Ashley N Oliveira, David A Hood.

  Persistent inactivity promotes skeletal muscle atrophy, marked by mitochondrial aberrations that affect strength, mobility, and metabolic health leading to the advancement of disease. Mitochondrial quality control (MQC) pathways include biogenesis (synthesis), mitophagy/lysosomal turnover, and the mitochondrial unfolded protein response (UPRmt) which serve to maintain an optimal organelle network. Tumor suppressor p53 has been implicated in regulating muscle mitochondria in response to cellular stress; however, its role in the context of muscle disuse has yet to be explored, and whether p53 is necessary for MQC remains unclear. To address this, we subjected p53 muscle-specific knockout (mKO) and wild-type (WT) mice to unilateral denervation. Transcriptomic and pathway analyses revealed dysregulation of pathways pertaining to mitochondrial function, and especially turnover, in mKO muscle following denervation. Protein and mRNA data of the MQC pathways indicated activation of the UPRmt and mitophagy-lysosome systems along with reductions in mitochondrial biogenesis and content in WT and mKO tissue following chronic denervation. However, p53 ablation also attenuated the expression of autophagy/mitophagy machinery, reduced autophagic flux, and enhanced lysosomal dysfunction. While similar reductions in mitochondrial biogenesis and content were observed between genotypes, MQC dysregulation exacerbated mitochondrial dysfunction in mKO fibers, evidenced by elevated reactive oxygen species (ROS). Moreover, acute experiments indicate that p53 mediates the expression of transcriptional regulators of MQC pathways as early as 1 day following denervation. Together, our data illustrate exacerbated mitochondrial dysregulation with denervation stress in p53 mKO tissue, thus indicating that p53 contributes to organellar maintenance via regulation of MQC pathways during muscle atrophy.

Keywords:  lysosome; mitochondria; mitochondrial biogenesis; mitochondrial quality control; mitophagy; muscle atrophy; p53; skeletal muscle; transcriptomics; unfolded protein response (UPR)

DOI:  https://doi.org/10.1016/j.jbc.2021.101540
Blood. 2021 Dec 27. pii: blood.2021013350. [Epub ahead of print]

SOD2 V16A Amplifies Vascular Dysfunction in Sickle Cell Patients by Curtailing Mitochondria Complex IV Activity.

Atinuke Dosunmu-Ogunbi, Shuai Yuan, Michael Reynolds, Luca Giordano, Subramaniam Sanker, Mara Sullivan, Donna B Stolz, Brett A Kaufman, Katherine C Wood, Yingzhe Zhang, Sruti Shiva, Seyed Mehdi Nouraie, Adam C Straub.

Superoxide dismutase 2 (SOD2) catalyzes the dismutation of superoxide to hydrogen peroxide in mitochondria limiting mitochondrial damage. The SOD2 amino acid valine-to-alanine substitution at position 16 (V16A) in the mitochondrial leader sequence is a common genetic variant among patients with sickle cell disease (SCD). However, little is known about the cardiovascular consequences of SOD2V16A in SCD patients or its impact on endothelial cell function. Here, we show SOD2V16A associates with increased tricuspid regurgitant velocity (TRV), systolic blood pressure, right ventricle area at systole and declined 6-minute walk distance in 410 SCD patients. Plasma lactate dehydrogenase, a marker of oxidative stress and hemolysis, significantly associated with higher TRV. To define the impact of SOD2V16A in the endothelium, we introduced the SOD2V16A variant into endothelial cells. SOD2V16A increases hydrogen peroxide and mitochondrial reactive oxygen species (ROS) production compared to controls. Unexpectedly, the increased ROS was not due to SOD2V16A mislocalization but was associated with mitochondrial Complex IV and a concomitant decrease in basal respiration and Complex IV activity. In sum, SOD2V16A is a novel clinical biomarker of cardiovascular dysfunction in SCD patients through its ability to decrease mitochondrial Complex IV activity and amplify ROS production in the endothelium.

DOI: https://doi.org/10.1182/blood.2021013350
Free Radic Biol Med. 2021 Dec 22. pii: S0891-5849(21)01156-4. [Epub ahead of print]179 119-132

Mitochondriotropic antioxidant based on caffeic acid AntiOxCIN4 activates Nrf2-dependent antioxidant defenses and quality control mechanisms to antagonize oxidative stress-induced cell damage.

Ricardo Amorim, Fernando Cagide, Ludgero C Tavares, Rui F Simões, Pedro Soares, Sofia Benfeito, Inês Baldeiras, John G Jones, Fernanda Borges, Paulo J Oliveira, José Teixeira.

  Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. We have previously shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 μM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 μM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the oxidative stress response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative stress by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). Here we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by a 1.5-fold increase in basal respiration and a 1.2-fold increase in extracellular acidification. AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. AntiOxCIN4 also up-regulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data open a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes.

Keywords:  Antioxidant defenses; Autophagy; Caffeic acid; Dietary antioxidants; Mitochondria; Mitochondria-targeted antioxidants; Nrf2

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.12.304
Front Physiol. 2021 ;12 777770

The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.

Jörg W Wegener, Ahmed Wagdi, Eva Wagner, Dörthe M Katschinski, Gerd Hasenfuss, Tobias Bruegmann, Stephan E Lehnart.

  Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E GSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E GSH increase. Importantly, β-adrenergic stimulation resulted in excessive E GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial E GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial E GSH oxidization in RyR2-R2474S cardiomyocytes.

Keywords:  RyR2 Ca2+ leak; dantrolene; glutathione redox potential; mitochondria; mitochondrial oxidation; reactive oxygen species (ROS); ryanodine receptor

DOI:  https://doi.org/10.3389/fphys.2021.777770
Biochem Pharmacol. 2021 Dec 27. pii: S0006-2952(21)00517-7. [Epub ahead of print] 114891

The role of FUNDC1 in mitophagy, mitochondrial dynamics and human diseases.

Hui Liu, Caixia Zang, Fangyu Yuan, Cheng Ju, Meiyu Shang, Jingwen Ning, Yang Yang, Jingwei Ma, Gen Li, Xiuqi Bao, Dan Zhang.

  Mitochondria are the principal sites of energy metabolism and provide most of the energy needed for normal cellular function. They are dynamic organelles that constantly undergo fission, fusion and mitophagy to maintain their homeostasis and function. However, dysregulated mitochondrial dynamics and mitophagy leads to reduced ATP generation and mutation of their DNA, which ultimately leads to cell death. Increasing evidence has shown that the FUN14 domain-containing protein 1 (FUNDC1), a novel mitophagy receptor, participates in the process of mitochondrial dynamics and mitophagy and plays a critical role in various human diseases. Herein, we review the role of FUNDC1 in mitophagy and mitochondrial dynamics, thus providing a better understanding of the relationship between the two processes. Moreover, we summarize the treatments targeting FUNDC1, and suggest that FUNDC1 may represent a promising therapeutic target for the treatment of several human diseases such as cardiovascular diseases, metabolic syndrome, cancer and chronic obstructive pulmonary disease (COPD).

Keywords:  FUNDC1; human diseases; mitochondrial dynamics; mitophagy

DOI:  https://doi.org/10.1016/j.bcp.2021.114891
J Biochem. 2021 Dec 29. pii: mvab153. [Epub ahead of print]

MITOL regulates phosphatidic acid-binding activity of RMDN3/PTPIP51.

Naoki Ito, Takara Takahashi, Isshin Shiiba, Shun Nagashima, Ryoko Inatome, Shigeru Yanagi.

  The transfer of phospholipids from the endoplasmic reticulum to mitochondria via the mitochondria-endoplasmic reticulum (ER) contact site (MERCS) is essential for maintaining mitochondrial function and integrity. Here, we identified RMDN3/PTPIP51, possessing phosphatidic acid (PA)-transfer activity, as a neighboring protein of the mitochondrial E3 ubiquitin ligase MITOL/MARCH5 by proximity-dependent biotin labeling using APEX2. We found that MITOL interacts with and ubiquitinates RMDN3. Mutational analysis identified lysine residue 89 in RMDN3 as a site of ubiquitination by MITOL. Loss of MITOL or the substitution of lysine 89 to arginine in RMDN3 significantly reduced the PA-binding activity of RMDN3, suggesting that MITOL regulates the transport of PA to mitochondria by activating RMDN3. Our findings imply that ubiquitin signaling regulates phospholipid transport at the MERCS.

Keywords:  E3 ubiquitin ligase; MITOL; RMDN3; mitochondria-ER contact site; phospholipid

DOI:  https://doi.org/10.1093/jb/mvab153
Hum Mol Genet. 2021 Nov 23. pii: ddab341. [Epub ahead of print]

Genome-wide association study of mitochondrial copy number.

Manuel Gentiluomo, Matteo Giaccherini, Xīn Gào, Feng Guo, Hannah Stocker, Ben Schöttker, Hermann Brenner, Federico Canzian, Daniele Campa.

Mitochondrial DNA copy number (mtDNAcn) variation has been associated with increased risk of several human diseases in epidemiological studies. The quantification of mtDNAcn performed with real-time PCR is currently considered the de facto standard among several techniques. However, the heterogeneity of the laboratory methods (DNA extraction, storage, processing) used could give rise to results that are difficult to compare and reproduce across different studies. Several lines of evidence suggest that mtDNAcn is influenced by nuclear and mitochondrial genetic variability, however this relation is largely unexplored. The aim of this work was to elucidate the genetic basis of mtDNAcn variation. We performed a genome-wide association study (GWAS) of mtDNAcn in 6836 subjects from the ESTHER prospective cohort, and included, as replication set, the summary statistics of a GWAS that used 295 150 participants from the UK Biobank. We observed two novel associations with mtDNAcn variation on chromosome 19 (rs117176661), and 12 (rs7136238) that reached statistical significance at the genome-wide level. A polygenic score that we called mitoscore including all known single nucleotide polymorphisms explained 1.11% of the variation of mtDNAcn (p = 5.93 × 10-7). In conclusion, we performed a GWAS on mtDNAcn, adding to the evidence of the genetic background of this trait.

DOI: https://doi.org/10.1093/hmg/ddab341
Arch Cardiovasc Dis. 2021 Dec 28. pii: S1875-2136(21)00222-9. [Epub ahead of print]

Mitochondrial-derived peptides: New markers for cardiometabolic dysfunction.

Luc Rochette, Eve Rigal, Geoffrey Dogon, Gabriel Malka, Marianne Zeller, Catherine Vergely, Yves Cottin.

  Great attention is being paid to the evaluation of new markers in blood circulation for the estimation of tissue metabolism disturbance. This endogenous disturbance may contribute to the onset and progression of cardiometabolic disease. In addition to their role in energy production and metabolism, mitochondria play a main function in cellular mechanisms, including apoptosis, oxidative stress and calcium homeostasis. Mitochondria produce mitochondrial-derived peptides that mediate the transcriptional stress response by translocating into the nucleus and interacting with deoxyribonucleic acid. This class of peptides includes humanin, mitochondrial open reading frame of the 12S ribosomal ribonucleic acid type c (MOTS-c) and small humanin-like peptides. Mitochondrial-derived peptides are regulators of metabolism, exerting cytoprotective effects through antioxidative stress, anti-inflammatory responses and antiapoptosis; they are emerging biomarkers reflecting mitochondrial function, and the circulating concentration of these proteins can be used to diagnose cardiometabolic dysfunction. The aims of this review are: (1) to describe the emerging role for mitochondrial-derived peptides as biomarkers; and (2) to discuss the therapeutic application of these peptides.

Keywords:  Biomarkers; Biomarqueurs; Cardiometabolic; Cardiométabolique; Humanin; Humanine; Mitochondrial-derived-peptides; Oxidative stress; Peptides mitochondriaux; Stress oxydatif

DOI:  https://doi.org/10.1016/j.acvd.2021.10.013
Mol Genet Metab. 2021 Dec 22. pii: S1096-7192(21)01195-1. [Epub ahead of print]

Feasible and clinical relevant outcome measures for adults with mitochondrial disease.

Peggy M J Bergs, Daphne M Maas, Mirian C H Janssen, Jan T Groothuis.

  There is no consensus on clinical outcome measures that reflect function, activities and participation which are suitable for adults with mitochondrial diseases (MD). The aim of this study was to determine feasible and clinically relevant outcome measures for patients with MD . In 156 adult patients with MD, endurance, balance, strength and mobility tests were evaluated. All tests showed a negative deviation to healthy reference values. Balance tests were feasible and significantly correlated with clinical severity. The Åstrand cycle test was not feasible in 55%, whereas the feasibility of the 6 min walking test is unclear in patients with MD.

Keywords:  Adults; Mitochondrial disease; Outcome measures; Physical therapy

DOI:  https://doi.org/10.1016/j.ymgme.2021.12.010
Alzheimers Dement. 2021 Dec;17 Suppl 2 e058489

Mitochondrial phenotypes in iPSC AD models.

Kaitlin Flannagan, Julia A Stopperan, Benjamin M Troutwine, Colton R Lysaker, Taylor Strope, Julia Draper, Cynthia Shaddy-Gouvion, Jay Vivian, Mohammad Haeri, Heather M Wilkins.

BACKGROUND: Mitochondrial dysfunction is observed in Alzheimer's disease (AD). Altered mitochondrial respiration, cytochrome oxidase (COX) Vmax, and mitophagy are observed in human subjects and animal models of AD. Models derived from induced pluripotent stem cells (iPSCs) may not recapitulate these phenotypes after reprogramming from differentiated adult cells. We examined mitochondrial function across iPSC derived models including cerebral organoids, forebrain neurons, and astrocytes. Postmortem brain tissue was used as a comparison.METHOD: iPSCs were reprogrammed from fibroblasts either from the University of Kansas Alzheimer's Disease Research Center (KU ADRC) cohort or purchased from WiCell. Postmortem brain samples were from the KU ADRC cohort when available. A total of four non-demented and four sporadic AD iPSC lines were examined. Postmortem brain tissue was derived from 9 ND and 12 AD subjects. iPSCs were differentiated into neurons, astrocytes, or cerebral organoids using StemCell Technologies protocols and reagents. iPSC derived models and postmortem brain tissue were subjected to mitochondrial respiration analysis using Seahorse XF technology and spectrophotometric COX Vmax assays. iPSC derived neurons and astrocytes underwent fluorescent assays to determine mitochondrial mass, mitochondrial membrane potential, and mitophagy levels.
RESULT: iPSC derived neurons and cerebral organoids showed reduced COX Vmax in AD subjects. These results were not observed in astrocytes. Postmortem human brain samples showed reduced COX Vmax in AD subjects. iPSC derived neurons had reduced mitochondrial respiration parameters, mitochondrial mass, mitophagy, mitochondrial membrane potential, and mitochondrial superoxide production. iPSC derived astrocytes had reduced mitochondrial respiration parameters but increased mitochondrial membrane potential and no change in mitochondrial superoxide production.
CONCLUSION: iPSC derived models from AD subjects show mitochondrial dysfunction phenotypes like what is observed in postmortem brain. As iPSCs do not maintain their epigenetic signatures after reprogramming the observed phenotypes are likely due to other somatic factors.

DOI: https://doi.org/10.1002/alz.058489
FEBS Open Bio. 2021 Dec 30.

VDAC1 oligomerization may enhance DDP-induced hepatocyte apoptosis by exacerbating oxidative stress and mitochondrial DNA damage.

Xueqin Zhu, Lei Luo, Yanyan Xiong, Nan Jiang, Yurun Wang, Yuan Lv, Ying Xie.

  Cisplatin (DDP)-based chemotherapy is a preferred treatment for a broad spectrum of cancers, but the precise mechanisms of its hepatotoxicity are not yet clear. Recently, the role of voltage-dependent anion channel protein 1 (VDAC1) in mitochondrial activity and cell apoptosis has attracted much attention. Our aim was to investigate the effects of mitochondrial outer membrane protein VDAC1 oligomerization in DDP-induced hepatocyte apoptosis. L-02 hepatocytes were divided into 4 groups: (1) control group, (2) 4,4'diisothiocyanate-2,2'-disulfonic acid (DIDS; 40 μM) group, (3) DDP (5μM) group, (4) DDP and DIDS combination group. Cell apoptosis was tested by annexin V/fluorescein isothiocyanate (FITC) assay, protein expression of caspase-3, γH2AX and NDUFB6 were observed by western blot assay, reactive oxygen species (ROS) and mitochondrial superoxide anion radical (O2 •- ) were detected by DCFH-DA and MitoSOX probe, and DNA damage was assessed by comet assay. Moreover, the activity of mitochondrial respiratory chain complex I was determined by the colorimetry method. Compared with the control group, apoptosis rate and activated cleaved-caspase-3 protein, ROS and O2 •- generation, DNA damage marker comet tail length and γH2AX protein level increased in the DDP treatment group (P<0.05). Activity of mitochondrial COXI decreased after DDP treatment (P<0.05). DIDS, as a VDAC1 oligomerization inhibitor, antagonized DDP-induced apoptosis by diminishing oxidative stress and DNA damage, and protecting mitochondrial complex protein. These results show that VDAC1 oligomerization may play an important role in DDP-induced hepatocyte apoptosis by increasing ROS and mitochondrial DNA (mtDNA) leakage from VDAC1 pores, exacerbating oxidative stress and mtDNA damage.

Keywords:  4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; apoptosis; cisplatin; hepatocyte; oligomerization; voltage-dependent anion channel 1

DOI:  https://doi.org/10.1002/2211-5463.13359
J Nutr. 2021 Dec 27. pii: nxab440. [Epub ahead of print]

Biology of Activating Transcription Factor 4 (ATF4) and Its Role in Skeletal Muscle Atrophy.

Scott M Ebert, Blake B Rasmussen, Andrew R Judge, Sarah M Judge, Lars Larsson, Ronald C Wek, Tracy G Anthony, George R Marcotte, Matthew J Miller, Mark A Yorek, Adrian Vella, Elena Volpi, Jennifer I Stern, Matthew D Strub, Zachary Ryan, John J Talley, Christopher M Adams.

  Activating Transcription Factor 4 (ATF4) is a multifunctional transcription regulatory protein in the basic leucine zipper (bZIP) superfamily. ATF4 can be expressed in most if not all mammalian cell types, and it can participate in a variety of cellular responses to specific environmental stresses, intracellular derangements, or growth factors. Because ATF4 is involved in a wide range of biological processes, its roles in human health and disease are not yet fully understood. Much of our current knowledge about ATF4 comes from investigations in cultured cell models, where ATF4 was originally characterized and where further investigations continue to provide new insights. ATF4 is also an increasingly prominent topic of in vivo investigations in fully differentiated mammalian cell types, where our current understanding of ATF4 is less complete. Here, we review some important high-level concepts and questions concerning the basic biology of ATF4. We then discuss current knowledge and emerging questions about the in vivo role of ATF4 in one fully differentiated cell type, mammalian skeletal muscle fibers.

Keywords:  ATF4; sarcopenia; skeletal muscle atrophy; tomatidine; ursolic acid

DOI:  https://doi.org/10.1093/jn/nxab440
Front Genet. 2021 ;12 773922

Detection and Functional Verification of Noncanonical Splice Site Mutations in Hereditary Deafness.

Penghui Chen, Longhao Wang, Yongchuan Chai, Hao Wu, Tao Yang.

  Splice site mutations contribute to a significant portion of the genetic causes for mendelian disorders including deafness. By next-generation sequencing of 4 multiplex, autosomal dominant families and 2 simplex, autosomal recessive families with hereditary deafness, we identified a variety of candidate pathogenic variants in noncanonical splice sites of known deafness genes, which include c.1616+3A > T and c.580G > A in EYA4, c.322-57_322-8del in PAX3, c.991-15_991-13del in DFNA5, c.6087-3T > G in PTPRQ and c.164+5G > A in USH1G. All six variants were predicted to affect the RNA splicing by at least one of the computational tools Human Splicing Finder, NNSPLICE and NetGene2. Phenotypic segregation of the variants was confirmed in all families and is consistent with previously reported genotype-phenotype correlations of the corresponding genes. Minigene analysis showed that those splicing site variants likely have various negative impact including exon-skipping (c.1616+3A > T and c.580G > A in EYA4, c.991-15_991-13del in DFNA5), intron retention (c.322-57_322-8del in PAX3), exon skipping and intron retention (c.6087-3T > G in PTPRQ) and shortening of exon (c.164+5G > A in USH1G). Our study showed that the cryptic, noncanonical splice site mutations may play an important role in the molecular etiology of hereditary deafness, whose diagnosis can be facilitated by modified filtering criteria for the next-generation sequencing data, functional verification, as well as segregation, bioinformatics, and genotype-phenotype correlation analysis.

Keywords:  RNA splicing; hereditary deafness; minigene; next-generation sequencing; splice site mutation

DOI:  https://doi.org/10.3389/fgene.2021.773922