Genetics and mitochondrial abnormalities in autism spectrum disorders: a review

We review the current status of the role and function of the mitochondrial DNA (mtDNA) in the etiology of autism spectrum disorders (ASD) and the interaction of nuclear and mitochondrial genes. High lactate levels reported in about one in five children with ASD may indicate involvement of the mitochondria in energy metabolism and brain development. Mitochondrial disturbances include depletion, decreased quantity or mutations of mtDNA producing defects in biochemical reactions within the mitochondria. A subset of individuals with ASD manifests copy number variation or small DNA deletions/duplications, but fewer than 20 percent are diagnosed with a single gene condition such as fragile X syndrome. The remaining individuals with ASD have chromosomal abnormalities (e.g., 15q11-q13 duplications), other genetic or multigenic causes or epigenetic defects. Next generation DNA sequencing techniques will enable better characterization of genetic and molecular anomalies in ASD, including defects in the mitochondrial genome particularly in younger children.     

Quantification of random mutations in the mitochondrial genome

Mitochondrial DNA (mtDNA) mutations contribute to the pathology of a number of age-related disorders, including Parkinson disease [A. Bender et al., Nat. Genet. 38 (2006) 515,Y. Kraytsberg et al., Nat. Genet. 38 (2006) 518], muscle-wasting [J. Wanagat, Z. Cao, P. Pathare, J.M. Aiken, FASEB J. 15 (2001) 322], and the metastatic potential of cancers [K. Ishikawa et al., Science 320 (2008) 661]. The impact of mitochondrial DNA mutations on a wide variety of human diseases has made it increasingly important to understand the mechanisms that drive mitochondrial mutagenesis. In order to provide new insight into the etiology and natural history of mtDNA mutations, we have developed an assay that can detect mitochondrial mutations in a variety of tissues and experimental settings [M. Vermulst et al., Nat. Genet. 40 (2008) 4, M. Vermulst et al., Nat. Genet. 39 (2007) 540]. This methodology, termed the Random Mutation Capture assay, relies on single-molecule amplification to detect rare mutations among millions of wild-type bases [J.H. Bielas, L.A. Loeb, Nat. Methods 2 (2005) 285], and can be used to analyze mitochondrial mutagenesis to a single base pair level in mammals.     

Diagnosis and molecular basis of mitochondrial respiratory chain disorders: Exome sequencing for disease gene identification

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Hippocampal RNA sequencing in mice selectively bred for high and low activity

High and Low Activity strains of mice were bidirectionally selected for differences in open-field activity (DeFries et al., 1978, Behavior Genetics, 8: 3–13) and subsequently inbred to use as a genetic model for studying anxiety-like behaviors (Booher et al., 2021, Genes, Brain and Behavior, 20: e12730). Hippocampal RNA-sequencing of the High and Low Activity mice identified 3901 differentially expressed protein-coding genes, with both sex-dependent and sex-independent effects. Functional enrichment analysis (PANTHER) highlighted 15 gene ontology terms, which allowed us to create a narrow list of 264 top candidate genes. Of the top candidate genes, 46 encoded four Complexes (I, II, IV and V) and two electron carriers (cytochrome c and ubiquinone) of the mitochondrial oxidative phosphorylation process. The most striking results were in the female high anxiety, Low Activity mice, where 39/46 genes relating to oxidative phosphorylation were upregulated. In addition, comparison of our top candidate genes with two previously curated High and Low Activity gene lists highlight 24 overlapping genes, where Ndufa13, which encodes the supernumerary subunit A13 of complex I, was the only gene to be included in all three lists. Mitochondrial dysfunction has recently been implicated as both a cause and effect of anxiety-related disorders and thus should be further explored as a possible novel pharmaceutical treatment for anxiety disorders.     

Mutations in cytochrome c oxidase subunit VIa cause neurodegeneration and motor dysfunction in Drosophila

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy(+) gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.     

Mitochondriale Erkrankungen

Mitochondrial disorders are clinically, biochemically, and genetically heterogeneous and surprisingly diverse. The clinical spectrum ranges from severe multisystem disorders of early infancy to monosymptomatic mild disorders in late adulthood. Causative for mitochondrial dysfunction are mutations in nuclear genes or/and the proper mitochondrial genome, the mitochondrial DNA. The last years have seen quite remarkable progress in identification of the molecular basis of mitochondrial diseases, which helps in the often difficult process of diagnosis. However, the clinical variability and complexity continue to increase and remain unexplained in many cases.     

Expression of various genes is controlled by DNA methylation during mammalian development

Despite thousands of articles about 5-methylcytosine (m(5)C) residues in vertebrate DNA, there is still controversy concerning the role of genomic m(5)C in normal vertebrate development. Inverse correlations between expression and methylation are seen for many gene regulatory regions [Heard et al., 1997; Attwood et al., 2002; Plass and Soloway, 2002] although much vertebrate DNA methylation is in repeated sequences [Ehrlich et al., 1982]. At the heart of this debate is whether vertebrate DNA methylation has mainly a protective role in limiting expression of foreign DNA elements and endogenous transposons [Walsh and Bestor, 1999] or also is important in the regulation of the expression of diverse vertebrate genes involved in differentiation [Attwood et al., 2002]. Enough thorough studies have now been reported to show that many tissue- or development-specific changes in methylation at vertebrate promoters, enhancers, or insulators regulate expression and are not simply inconsequential byproducts of expression differences. One line of evidence comes from mutants with inherited alterations in genes encoding DNA methyltransferases and from rodents or humans with somatically acquired changes in DNA methylation that illustrate the disease-producing effects of abnormal methylation. Another type of evidence derives from studies of in vivo correlations between tissue-specific changes in DNA methylation and gene expression coupled with experiments demonstrating cause-and-effect associations between DNA hyper- or hypomethylation and gene expression. In this review, I summarize some of the strong evidence from both types of studies. Taken together, these studies demonstrate that DNA methylation in mammals modulates expression of many genes during development, causing major changes in or important fine-tuning of expression. Also, I discuss previously established and newly hypothesized mechanisms for this epigenetic control.     

Mitochondrial DNA Mutations and Pathogenesis

Approximately three years ago, this journal published a review on the clinical and molecular analysis of mitochondrial encephalomyopathies, with emphasis on defects in mitochondrial DNA (mtDNA). At that time, approximately 30 point mutations associated with a variety of maternally-inherited (or rarely, sporadic) disorders had been described. Since that time, almost twenty new pathogenic mtDNA point mutations have been described, and the pace of discovery of such mutations shows no signs of abating. This accumulating body of data has begun to reveal some patterns that may be relevant to pathogenesis.     

Clue to a New Deafness Gene: A Large Chinese Nonsyndromic Hearing Loss Family Linked to DFNA4

Hereditary hearing loss is one of the most common neurosensory defects in humans.Approximately 70％ of cases are nonsyndromic and could be inherited in autosomal dominant,autosomal recessive,mitochondrial,X-linked,and Y-linked manners (Wang et al.,2004;Alford,2011).The autosomal dominant type,comprising 15％-20％ of nonsyndromic hearing loss,is monogenic and genetically heterogeneous.Since the first dominant deafness locus (DFNA1) was identified in 1992,a total of 64 DFNA loci have been mapped (DFNA1-DFNA64),and 27 corresponding genes have been identified (http://hereditaryhearingloss.org).Previous studies have revealed that one deafness locus can be linked to more than one gene (Bayazit and Yilmaz,2006),and the question "one locus,how many genes?" was first raised about a decade ago (Van-Hauwe et al.,1999).So far,several loci,including DFNA2 and DFNA3,have been shown to be related to one or more genes,showing high genetic heterogeneity in hereditary hearing loss (Grifa et al.,1999;Goldstein and Lalwani,2002;Yan et al.,2011).     

Systematic affinities of the lyrebirds (Passeriformes: Menura), with a novel classification of the major groups of passerine birds

Phylogenetic relationships of the lyrebirds are investigated using DNA sequence data. The aligned data matrix consists of 4027 bp obtained from three nuclear genes (c-myc, RAG-1 and myoglobin intron II) and two mitochondrial genes (cytochrome b and ND2). Both maximum-likelihood and parsimony analyses show that the lyrebirds unambiguously belong to the oscine radiation, and that they are the sister taxon to all other oscines. The results do not support the suggestion based on DNA-DNA hybridization data (Sibley and Ahlquist, 1990) that the treecreepers and bowerbirds are part of the lyrebird clade. Nevertheless, treecreepers and bowerbirds are sister taxa to all other oscines (except the lyrebirds) and may constitute a monophyletic group, although bootstrap support values for this clade are low. A major disagreement between the present analysis and that based on DNA-DNA hybridization data is that the Corvida (sensu Sibley and Ahlquist, 1990) and Passerida are not reciprocally monophyletic, as we find the latter group be nested within the Corvida. Also, the superfamilies Meliphagoidea and Corvoidea sensu, are not recovered as monophyletic in the present study. Within the oscine radiation, all taxa belonging to the earliest splits are confined to the Australo-Papuan region. This suggests strongly that the origins and early radiation of the oscines occurred in the southern supercontinent Gondwana. A new classification of the major groups of passerines is presented following from the results presented in the present study, as well as those published recently on analyses of sequence data from the nuclear c-myc and RAG-1 genes (Ericson et al., 2002; Irestedt et al., 2001).     

Measurement of oxidatively induced base lesions in liver from Wistar rats of different ages.

Mitochondrial and nuclear DNA were isolated from the livers of young (6-7 month) and old (23-24 month) Wistar rats and the levels of 10 different oxidatively induced lesions were analyzed by gas chromatography/mass spectrometry. This is the first study to measure several different oxidatively induced base lesions in both mitochondrial and nuclear DNA as a function of age. No significant age effects were observed for any lesion. Furthermore, contrary to expectations, we did not observe elevated levels of oxidatively induced base lesions in mitochondrial DNA. This contrasts with 50-fold differences reported for several lesions between mitochondrial and nuclear DNA from porcine liver (Zastawny et al., Free Radic. Biol. Med. 24:722-725, 1998). The fact that different lesion levels are observed even when similar techniques are employed emphasizes that the role of oxidative mitochondrial DNA damage and its repair in aging must continue to be the subject of intense investigation. Questions concerning endogenous levels of damage should be revisited as existing methods are improved and new methods become available.     

The potential of stem cells for the treatment of brain tumors and globoid cell leukodystrophy

Stem cells of different origin are under careful scrutiny as potential new tools for the treatment of several neurological diseases. The major focus of these reaserches have been neurodegenerative disorders, such as Huntington Chorea or Parkinson Disease (Shihabuddin et al., 1999). More recently attention has been devoted to their use for brain repair after stroke (Savitz et al., 2002). In this review we will focus on the potential of stem cell treatments for glioblastoma multiforme (Holland, 2000), the most aggressive primary brain tumor, and globoid cell leukodystrophy (Krabbe disease), a metabolic disorder of the white matter (Berger et al., 2001). These two diseases may offer a paradigm of what the stem cell approach may offer in term of treatment, alone or in combination with other therapeutic approaches. Two kinds of stem cells will be consideredhere: neural stem cells and hematopoietic stem cells, both obtained after birth. The review will focus on experimental models, with an eye on clinical perspectives. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of mitochondrial complex IV leads to secondary loss complex II-III activity: implications for the pathogenesis and treatment of mitochondrial encephalomyopathies.

Mitochondrial encephalomyopathies, arising from deficiencies of the electron transport chain (ETC) give rise to a wide clinical spectrum of presentation and are often progressive in nature. The aetiology of mitochondrial encephalomyopathies have yet to be fully elucidated, however, a successive loss of ETC function may contribute to the progressive nature of these disorders. The possibility arises that as a consequence of a primary impairment of ETC activity, secondary damage to the ETC may occur. In order to investigate this hypothesis, we established a model of cytochrome oxidase (Complex IV) deficiency in cultured human astrocytoma 1321N cells. Potassium cyanide (KCN, 1mM) resulted in a sustained 50% (p<0.01) loss of complex IV. At 24h activities of the other ETC complexes were unaffected. However, at 72h significant loss of succinate-cytochrome c reductase (complex II-III) activity expressed as a ratio to the mitochondrial marker, citrate synthase was observed. (KCN treated; 0.065+/-0.011 vs controls; 0.118+/-0.017 mean+/-SEM, n=8, p<0.05). These results provide a possible mechanism for the progressive nature of ETC defects and why in some patients multiple patterns of ETC deficiencies can be demonstrated.     

The R336Q mutation in human mitochondrial EFTu prevents the formation of an active mt-EFTu·GTP·aa-tRNA ternary complex

The mitochondrial translational machinery allows the genes encoded by mitochondrial DNA (mtDNA) to be translated in situ. Mitochondrial translation requires a number of nucleus-encoded protein factors, some of which have been found to carry mutations in patients affected by mitochondrial encephalomyopathies. We have previously described the first, and so far only, mutation in the mitochondrial elongation factor Tu, mt-EFTu, in a baby girl with polycystic encephalopathy, micropolygyria, and leukodystrophic changes. Despite that the mutant mt-EFTu was present in normal amount in the patient's tissues, mitochondrial translation was severely reduced, determining multiple defects in the amount and activity of mtDNA-dependent respiratory chain complexes. By an in-vitro reconstructed translational system, we here provide evidence that the mutant mt-EFTu variant fails to bind to aminoacylated mitochondrial tRNAs, thus explaining the observed impairment of mitochondrial translation. This is the first analysis on the molecular mechanism of a mtDNA translation defect due to a nuclear gene mutation.     

Identification of Mitochondrial Coenzyme A Transporters from Maize and Arabidopsis

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.CoA acts as an acyl carrier in many reactions of primary and secondary metabolism, and some 8% of the nearly 4,900 enzymes described in the Enzyme Commission database are CoA dependent (Bairoch, 2000). CoA occupies a central position in lipid metabolism, respiration, gluconeogenesis, and other pathways (Leonardi et al., 2005). It is present in all forms of life, but while all organisms can synthesize it from pantothenate (vitamin B₅), only prokaryotes, plants, and fungi are able to synthesize pantothenate; animals obtain pantothenate from the diet (Daugherty et al., 2002; Leonardi et al., 2005; Webb and Smith, 2011).In plants, the steps that convert pantothenate to CoA are almost certainly cytosolic (Webb and Smith, 2011; Gerdes et al., 2012). CoA, however, is required in mitochondria for the citric acid cycle, in chloroplasts for fatty acid synthesis, and in peroxisomes for β-oxidation. CoA, therefore, must be imported into these organelles from the cytosol, and indeed, early work demonstrated a CoA transport system in potato (Solanum tuberosum) mitochondria (Neuburger et al., 1984). Yeast (Saccharomyces cerevisiae) and mammalian mitochondria and peroxisomes likewise import CoA because they cannot make it (Fiermonte et al., 2009; Agrimi et al., 2012b). The compartmentation of CoA in all eukaryotes appears to be closely regulated, with cytosol and organelles maintaining separate CoA pools whose levels can modulate fluxes through CoA-dependent reactions (Hunt and Alexson, 2002; Leonardi et al., 2005; De Marcos Lousa et al., 2013).Mitochondrial CoA transporters belonging to the mitochondrial carrier family (MCF) have been identified in yeast (Leu-5p; Prohl et al., 2001) and human (SLC25A42; Fiermonte et al., 2009). Furthermore, peroxisomal CoA carriers from human (SLC25A17; Agrimi et al., 2012b) and Arabidopsis (Arabidopsis thaliana; peroxisomal CoA and NAD carrier [PXN]; Agrimi et al., 2012a) have also been identified. However, no transporters for CoA are known for plant mitochondria or chloroplasts (Palmieri et al., 2011; Gerdes et al., 2012).In this study, a comparative genomic analysis first identified close Arabidopsis and maize (Zea mays) homologs of the yeast and mammalian mitochondrial CoA carriers as candidates for the missing plant mitochondrial or chloroplast transporters. Experimental evidence then demonstrated that the candidate proteins transport CoA when expressed in yeast, that they are targeted to mitochondria in vitro and in planta, and that they are expressed throughout the plant.     

Mitochondrial encephalomyopathies: Clinical and molecular analysis

The classification of mitochondrial encephalomyopathies relied upon clinical, biochemical, and histological features until the discovery of mitochondrial DNA defects in 1988. Since then, an outburst of molecular genetic information has aided our understanding of the pathogenesis and the classification of these heterogeneous disorders. Novel concepts of maternal inheritance, mitochondrial DNA (mtDNA) heteroplasmy, tissue distribution, and threshold have explained many of the clinical characteristics. The discovery of point mutations, large-scale mtDNA deletions, duplications, and autosomally inherited disorders with multiple mtDNA deletions have revealed new genetic phenomena. Despite our rapidly expanding understanding of the molecular genetic defects, many questions remain to be explored to fill the gap in our knowledge of the relationship between genotype and clinical phenotype.