Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)–encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)–binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation

Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analyzed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes), followed by the heart (375 genes), kidney (224 genes), colon (218 genes), and thyroid (163 genes). More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs, and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.Benjamin T. Mayne, Tina Bianco-Miotto, Sam Buckberry, James Breen, Vicki Clifton, Cheryl Shoubridge and Claire T. Robert

Incorrect dosage of IQSEC2, a known intellectual disability and epilepsy gene, disrupts dendritic spine morphogenesis

There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene (IQSEC2) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippocampal cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2, which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in the morphology of developing neurons.SJ Hinze, MR Jackson, S Lie, L Jolly, M Field, SC Barry, RJ Harvey and C Shoubridg

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding.

International audienceSco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients

Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer

Review of the articleMitochondria are the organelles responsible for producing the majority of a cell's ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically

Unraveling the pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approach

The Aristaless-related homeobox (ARX) gene is implicated in intellectual disability with the most frequent pathogenic mutations leading to expansions of the first two polyalanine tracts. Here, we describe analysis of the ARX gene outlining the approaches in the Australian and Portuguese setting, using an integrated clinical and molecular strategy. We report variants in the ARX gene detected in 19 patients belonging to 17 families. Seven pathogenic variants, being expansion mutations in both polyalanine tract 1 and tract 2, were identifyed, including a novel mutation in polyalanine tract 1 that expands the first tract to 20 alanines. This precise number of alanines is sufficient to cause pathogenicity when expanded in polyalanine tract 2. Five cases presented a probably non-pathogenic variant, including the novel HGVS: c.441_455del, classified as unlikely disease causing, consistent with reports that suggest that in frame deletions in polyalanine stretches of ARX rarely cause intellectual disability. In addition, we identified five cases with a variant of unclear pathogenic significance. Owing to the inconsistent ARX variants description, publications were reviewed and ARX variant classifications were standardized and detailed unambiguously according to recommendations of the Human Genome Variation Society. In the absence of a pathognomonic clinical feature, we propose that molecular analysis of the ARX gene should be included in routine diagnostic practice in individuals with either nonsyndromic or syndromic intellectual disability. A definitive diagnosis of ARX-related disorders is crucial for an adequate clinical follow-up and accurate genetic counseling of at-risk family members.Unit for Multidisciplinary Research in Biomedicine, UMIB, ICBAS-UP, Porto, Portugal was funded by FEDER funds of the Operational Program for Competitiveness Factors – COMPETE through FCT – Foundation for Science and Technology under the project: Fcomp-01-0124-FEDER-015896. The Neurogenetics research program in the Department of Paediatrics, University of Adelaide, Australia was funded by the Australian National Health and Medical Research Council (Grant No. 1063025). C. S. is supported Australian Research Council (Future Fellowship FT120100086

³¹P magnetization transfer magnetic resonance spectroscopy: assessing the activation induced change in cerebral ATP metabolic rates at 3 T

Purpose: In vivo ³¹P MRS magnetization transfer (MT) provides a direct measure of neuronal activity at the metabolic level. This work aims to use functional ³¹P MRS-MT to investigate the change in cerebral ATP metabolic rates in healthy adults upon repeated visual stimuli. Methods: A magnetization saturation transfer sequence with narrowband selective saturation of γ-ATP was developed for ³¹P MT experiments at 3 T. Results: Using progressive saturation of γ-ATP, the intrinsic T1 relaxation times of phosphocreatine (PCr) and inorganic phosphate (Pi) at 3 T were measured to be 5.1±0.8 s and 3.0±1.4 s, respectively. Using steady-state saturation of γ-ATP, a significant 24±14% and 11±7% increase in the forward creatine kinase (CK) pseudo-first-order reaction rate constant, k₁, was observed upon visual stimulation in the first and second cycles respectively of a paradigm consisting of 10min-rest followed by 10min-stimulation, with the measured baseline k₁ being 0.35±0.04 s⁻¹. No significant changes in forward ATP synthase (ATPase) reaction rate, PCr/γ- ATP, Pi/γ-ATP, and NAD(H)/γ-ATP ratios, or intracellular pH were detected upon stimulation. Conclusion: This work demonstrates the potential of studying cerebral bioenergetics using functional ³¹P MRS-MT to determine the change in the forward CK reaction rate at 3 T

Mechanistic insight into the pathology of polyalanine expansion disorders revealed by a mouse model for x linked hypopituitarism

Extent: 9 p.Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.James Hughes Sandra Piltz, Nicholas Rogers, Dale McAninch, Lynn Rowley and Paul Thoma

Novel Missense Mutation A789V in IQSEC2 underlies X-Linked intellectual disability in the MRX78 family

Disease gene discovery in neurodevelopmental disorders, including X-linked intellectual disability (XLID) has recently been accelerated by next-generation DNA sequencing approaches. To date, more than 100 human X chromosome genes involved in neuronal signaling pathways and networks implicated in cognitive function have been identified. Despite these advances, the mutations underlying disease in a large number of XLID families remained unresolved. We report the resolution of MRX78, a large family with six affected males and seven affected females, showing X-linked inheritance. Although a previous linkage study had mapped the locus to the short arm of chromosome X (Xp11.4-p11.23), this region contained too many candidate genes to be analyzed using conventional approaches. However, our X-chromosome exome resequencing, bioinformatics analysis and inheritance testing revealed a missense mutation (c.C2366T, p.A789V) in IQSEC2, encoding a neuronal GDP-GTP exchange factor for Arf family GTPases (ArfGEF) previously implicated in XLID. Molecular modeling of IQSEC2 revealed that the A789V substitution results in the insertion of a larger side-chain into a hydrophobic pocket in the catalytic Sec7 domain of IQSEC2. The A789V change is predicted to result in numerous clashes with adjacent amino acids and disruption of local folding of the Sec7 domain. Consistent with this finding, functional assays revealed that recombinant IQSEC2A789V was not able to catalyze GDP-GTP exchange on Arf6 as efficiently as wild-type IQSEC2. Taken together, these results strongly suggest that the A789V mutation in IQSEC2 is the underlying cause of XLID in the MRX78 family

A new role for peroxidases in bone repair

When bone undergoes trauma or the architecture deteriorates, due to disease and is neglected or misdiagnosed, non-unions can occur, whereby bone does not heal correctly. As a consequence, patients experience pain, stiffness, loss of mobility and disability. In many cases this can result in an inability to perform normal duties in employment, which causes significant financial burden to the patient and economy. The repair of these large bone defects remains a significant challenge for orthopaedic surgeons. Bone grafting strategies have been developed to repair and restore bone function, however the demand for functional bone grafts is extremely high, with an estimated 2.2 million patients worldwide undergoing bone grafting procedures annually. Due to an aging population these numbers are expected to double by 2020, which will put further burden on health care costs worldwide. Autologous bone grafting remains the current standard to repair bone defects and fractures, however, this method of treatment has numerous surgical-associated morbidities and complication rates of up to 30%. Therefore, researchers are attempting to identify substitute grafting materials which possess the critical bone reparative characteristics required for successful healing. To date, a bone graft material which is comparable to autologous bone, with fewer associated morbidities is yet to be identified, thus, continued research is required to identify and develop new agents to promote and accelerate bone repair. Agents which have been thoroughly investigated to enhance the bone repair process in combination with bone graft substitutes include the use of BMP-2. BMP-2 has proven to be successful due to its pro-osteogenic role whereby it promotes osteoblast functionality through the regulation of genes necessary for collagen biosynthesis and mineralisation of the extracellular matrix (ECM). Osteoblasts are one of the main cell types responsible for bone formation and bone repair. These cells are derived from the mesenchymal progenitor cell population, along with endothelial cells and fibroblasts. Work published by our laboratory provides evidence that a group of enzymes with peroxidase activity, namely mammalian-derived myeloperoxidase (MPO) and eosinophil peroxidase (EPO) as well as plant-derived soybean peroxidase (SBP) stimulate the migration of fibroblastic cells and promote their ability to generate a functional ECM. In addition, we have presented evidence demonstrating the ability of these peroxidases, in promoting endothelial cell function and inhibiting osteoclastogenesis, suggesting a potential role for these enzymes in bone repair. The work described in this thesis aims to provide evidence that mammalian and plant derived peroxidase enzymes including, MPO, EPO and SBP possess pro-osteogenic activities by influencing osteoblast functionality. Using physiologically relevant concentrations of peroxidases, this study showed that the enzymatic catalytic activities and substrate specificities of each of these enzymes which were shown to be different, resulted in differential responses in the context of osteoblast function. EPO and SBP demonstrated a well-conserved pro-osteogenic capacity to stimulate the biosynthesis of collagen I by primary human osteoblasts and promote mineralisation of the deposited ECM. In contrast, MPO, while it was able to promote ECM deposition, it failed to promote mineralisation and therefore unlikely to contribute to bone formation. The ability of EPO and SBP to stimulate mineralisation by osteoblasts suggests that these enzymes may possess key properties for promoting bone repair. Of the two tested peroxidases however, SBP is more readily available and significantly cheaper than EPO, making it an attractive and realistic candidate for further pre-clinical assessment. Data presented in this thesis demonstrate for the first time the pro-osteogenic ability of SBP, in combination with a commercially available scaffold to significantly accelerate bone repair in an ovine critical-sized defect model. This was confirmed by quantitative micro-CT analysis. Histological assessment showed evidence of intramembranous bone formation and viable osteoblast and osteocyte cell populations, indicative of bone repair and maturation. These results suggest that SBP may be beneficial as a therapeutic agent to accelerate localised repair of damaged bone. The use of rodents over larger animals for different models of bone repair allows for high throughput analyses of multiple variables, such as dose and time. Using wildtype mice, we established a critical size defect model to validate SBP in this species, prior to investigating other models of bone repair. The doses of SBP investigated in this study demonstrated significant inhibition of bone formation with increased fibrous tissue present and an absence of bone remodelling indicators. The results presented in this thesis highlight the importance of further mechanistic investigation, to determine how SBP regulates the remodelling process and the necessity for optimisation before assessing the role of SBP in fracture healing. In conclusion, our findings demonstrate for the first time that peroxidase enzymes likely regulate multiple cellular processes involved in new bone formation, including collagen I biosynthesis, bone matrix mineralisation and osteogenic regulation. Specifically, the plant derived peroxidase, SBP, displays significant pro-osteogenic potential by promoting intramembranous ossification. The studies presented in this thesis provide the first in vivo evidence for peroxidase enzymes as therapeutic agents with the potential to enhance bone repair and, identifies peroxidase inhibitors as a preventative target of pathological ossificationThesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201