The m.3243A > G mutation has become known as the MELAS mutation. However, many other clinical phenotypes associated with this mutation have been described, most frequently being maternally inherited ...diabetes and deafness (MIDD). The m.3243A > G mutation, can be detected in virtually all tissues, however heteroplasmy differs between samples. Recent reports indicate, a preference to perform mutation analysis in urinary epithelial cells (UEC). To test this, and to study a correlation between the mutational load in different tissues with two mitochondrial scoring systems (NMDAS and NPMDS) we investigated 34 families carrying the m.3243A > G mutation. Heteroplasmy was determined in three non-invasively collected samples, namely leucocytes, UEC and buccal mucosa. We included 127 patients, of which 82 carried the m.3243A > G mutation. None of the children (n = 11) had specific complaints. In adults (n = 71), a median NMDAS score of 15 (IQR 10-24) was found. The most prevalent symptoms were hearing loss(48%), gastro-intestinal problems(42%), exercise intolerance(38%) and glucose intolerance(37%). Ten patients had neurologic involvement. Buccal mucosa had the best correlation with the NMDAS in all adults (r = 0.437,p < 0.001), whereas UEC had the strongest correlation with the NMDAS in severely affected patients (r = 0.593,p = 0.002). Heteroplasmy declined significantly with increasing age in all three samples (leucocytes r = -0.705 (p < 0.001), UEC r = -0.374(p = 0.001), buccal mucosa r = -0.460(p < 0.001). In our cohort of 82 patients, the m.3243A > G mutation causes a wide variety of signs and symptoms, MIDD being far more prevalent than MELAS. Looking at the characteristics of the three non-invasively available tissues for testing heteroplasmy we confirm that UEC are the preferred sample to test.
Mitochondrial disorders are characterized by a broad clinical spectrum. Identical clinical signs and symptoms can be caused by mutations in different mitochondrial or nuclear genes. Vice versa, the ...same mutation can lead to different phenotypes. Genetic syndromes and neuromuscular disorders mimicking mitochondrial disorders further complicate the diagnostic process. Whole exome sequencing (WES) is the state of the art next generation sequencing technique to identify genetic defects in mitochondrial disorders. Until recently it has mainly been used as a research tool. In this study, the use of WES in routine diagnostics is described. The WES data of 109 patients, referred under the suspicion of a mitochondrial disorder, were examined in two steps. First, the data were filtered using a virtual gene panel of genes known to be associated with mitochondrial disease. If negative, the entire exome was examined. A molecular diagnosis was achieved in 39 % of the heterogeneous cohort, and in 57 % of the subgroup of 42 patients with the highest suspicion for a mitochondrial disease. In addition to mutations in genes known to be associated with mitochondrial disorders (e.g.
TUFM
,
MTFMT
,
FBXL4
), in the subgroup of patients with the lowest suspicion for a mitochondrial disorder we found mutations in several genes associated with neuromuscular disorders (
e.g. SEPN1, ACTA1
) and genetic syndrome (
e.g. SETBP1, ARID1B
). Our results show that WES technology has been successfully implemented as a state-of-the-art, molecular diagnostic test for mitochondrial disorders as well as for the mimicking disorders in daily clinical practice. It also illustrates that clinical and biochemical phenotyping is essential for successful application of WES to diagnose individual patients.
ABSTRACT
COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and ...isolated complex IV deficiency. For the first time, clinical details about a COA6‐deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady‐state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse‐chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2/CIVn‐supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6‐deficient patients is provided.
Cytochrome c oxidase (complex IV) levels are strongly reduced in cultured fibroblasts of a patient with a homozygous pathogenic variant in COA6 and severe hypertrophic neonatal cardiomyopathy. Copper supplementation partially recues complex IV deficiency in these cells providing a potential therapeutic lead for future patients.
Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in ...CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.
Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of ...the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.
Chronic kidney disease (CKD) is reported in 20%-30% of children after liver transplantation (LT). One of the proposed underlying causes is the long-term exposure to tacrolimus, a calcineurin ...inhibitor (CNI), which is the main immunosuppressive drug used after LT. Variation in tacrolimus absolute exposure and relative dose requirements are believed to be important risk factors for developing CNI-associated nephrotoxicity.
To describe the long-term renal outcome of pediatric LT recipients and determine the effects of tacrolimus exposure on renal outcome parameters.
Retrospective single center study of renal function (GFR, proteinuria) and pharmacokinetic parameters (C
, AUC
) obtained during annual follow-up in children after liver transplantation, between 1998 and 2019. Relevant pharmacogenetic variants for tacrolimus disposition (CYP3A5 and ABCB1) were determined in recipients and donors. The evolution of individual renal function and tacrolimus exposure was evaluated using linear mixed models for repeated measurements.
Twenty-six children were included (mean follow-up: 10.4 years (range 2-18.9)). Mean estimated GFR was 109.3 (SE: 7.4), vs. measured: 91.3 mL/min/1.73 m
(SE: 6.3), which remained stable during follow-up. CKD stage ≥2 was observed in 32.8% of the visits based on eGFR versus 50.0% on mGFR. CKD stage ≥3 was uncommon (4.1% and 6.2% resp.). Mean tacrolimus C
was 5.3 ng/mL (SE: 2.5) with a AUC
of 72.7 ng*h/mL (SE: 30.3), which demonstrated a small decrease during follow-up. There was a negative correlation between C
and mGFR (r
= -0.3; p < .001). We found no correlation between GFR and tacrolimus dose requirements ((ng/mL)/(mg/kg)) or pharmacogenetic background.
Renal function during long-term follow-up after pediatric LT remained stable for the majority of our cohort. However, mild CKD was relatively common, warranting follow-up into adulthood. Although absolute tacrolimus exposure has a small depressing effect on concurrent GFR, there is no progressive deterioration of GFR due to long-term exposure, dose requirements or genetic background under the current target levels. These findings should be confirmed in a larger sample set, ideally including data from multiple centers.
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The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is ...the formation of a “living membrane”, consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2mgml−1 3,4-dihydroxy-l-phenylalanine (4min coating, 1h dissolution) and 25μgml−1 Coll IV (4min coating). The successful transport of 14C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney.
Unilateral nephrectomy is a relatively common procedure in children which results in a solitary functioning kidney (SFK). Living with an SFK predisposes to kidney injury, but it remains unknown which ...children are most at risk. We aimed to investigate kidney injury rates in patients who underwent unilateral nephrectomy in childhood and to investigate differences among nephrectomies performed for a congenital anomaly, malignancy or other condition.
MEDLINE and EMBASE were searched for studies reporting kidney injury rates i.e. proteinuria, hypertension and/or a decreased glomerular filtration rate (GFR) of patients who underwent unilateral nephrectomy during childhood. Studies including five or more patients with at least 12 months of follow-up were eligible. Analyses were performed using random effects models and stratified by indication for nephrectomy. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines were used for reporting.
Over 5000 unique articles were screened, of which 53 studies reporting on >4000 patients were included in the analyses. Proteinuria, hypertension and a decreased GFR were present in 15.3, 14.5 and 11.9% of patients, respectively. Heterogeneity among the studies was large in several subgroups, impairing quantitative meta-analyses. However, none of our analyses indicated differences in injury rates between a congenital anomaly or malignancy as an indication for nephrectomy.
Unilateral nephrectomy during childhood results in signs of kidney injury in >10% of patients, with no clear difference between the indications for nephrectomy. Therefore, structured follow-up is necessary in all children who underwent nephrectomy, regardless of the indication.
Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a ...set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs.
We have developed an iterative orthology prediction method, Ortho-Profile, that uses reciprocal best hits at the level of sequence profiles to infer orthology. It increases ortholog detection by 20% compared to sequence-to-sequence comparisons. Ortho-Profile predicts 598 human orthologs of mitochondrial proteins from Saccharomyces cerevisiae and Schizosaccharomyces pombe with 94% accuracy. Of these, 181 were not known to localize to mitochondria in mammals. Among the predictions of the Ortho-Profile method are 11 human cytochrome c oxidase (COX) assembly proteins that are implicated in mitochondrial function and disease. Their co-expression patterns, experimentally verified subcellular localization, and co-purification with human COX-associated proteins support these predictions. For the human gene C12orf62, the ortholog of S. cerevisiae COX14, we specifically confirm its role in negative regulation of the translation of cytochrome c oxidase.
Divergent homologs can often only be detected by comparing sequence profiles and profile-based hidden Markov models. The Ortho-Profile method takes advantage of these techniques in the quest for orthologs.
Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. ...Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.
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