Sengers syndrome caused by biallelic TIMM29 variants and RNAi silencing in Drosophila orthologue recapitulates the human phenotype

Figures

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  Fig. 1. The pedigree of the autosomal recessive TIMM29-Sengers syndrome family and localization of the disease-associated gene. Haplotypes of tested members of the 9 nuclear subfamilies. The originally linked region was further refined to a 2.3 Mb region with the use of recombination events detected in the nuclear subfamily [V-12&V13]. The pedigree includes the allele sizes of the microsatellite markers in the critical region on chromosome 19. Individuals whose DNA was available for analysis are annotated. The region of shared homozygosity is marked. The markers of the haplotype are listed in the column on the left side of the pedigree. The lower inset demonstrates the two-point linkage analysis as described by the Superlink (http://cbl-hap.cs.technion.ac.il/superlink-snp). The Lod score results (Y axis) of 8 microsatellite markers (depicted on the X axis) and their physical position are indicated according to the UCSC Genome Browser on Human Dec. 2013 (GRCh38/hg38) Assembly). Superlink was used to generate these results: Number of markers = 8; Mode of inheritance (MOI) = Recessive 0 0 0.99; Disease mutant gene frequency = 0.01. The family data file as well as the multipoint analysis can…

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  Fig. 2. a Low magnification. X3000, of ultrathin sections of muscle showing interrupted myofibrils with numerous, small and large abnormal looking electron lucent mitochondria (“mitochondriosis” or oncotic transformation of mitochondria). Most of the mitochondria lost their cristae; only few irregular and degenerated cristae are seen. b Higher magnification, X9000, showing isolated and disrupted myofibrils and replacement of heart muscle by proliferation of mitochondria mass. The mitochondria themselves show fluffy bodies and abnormal cristae which have been reported in other mitochondrial disorders. The massive proliferation of the mitochondria could be termed: “oncocytic” transformation or “mitochondriosis”. c High magnification X15,000, showing a mass of abnormal mitochondria between isolate myofibrils. Most of the mitochondria lost their cristae; only few irregular and partially degenerated cristae are shown (arrows). In a few mitochondria paracrystalline inclusions are seen (arrow head)

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  Fig. 3. a Chromatograms showing the TIMM29 homozygous T > C transition found in patient sequences (VII-4 and VII-5), resulting in a missense variant in codon 172 where tryptophan was substituted for arginine (Trp172Arg). b The Tim29 Trp172 and neighboring amino residue orthologues ClustalW2 results. The Tim29 Trp172 is a highly conserved residue among species. c ClustalW2 aligment results of Tim29 human pW172 (Tryptophan) with 16 Drosophila species Tim29 Trp172 amino residue orthologues is replaced by a Tyrosine (Y) in most Drosophila species, including Drosophila melanogaster, and by a Phenylalanine (F) in certain species such as Drosophila persimilis. d Western blot was performed with mitochondrial(lanes a1 and b1)and intermyofibrillar homogenate (lanes a2 and b2), extracted proteins from two musculoskeletal samples of two healthy controls a and b. The blotted membrane was then exposed to anti Tim29 Ab, 1:400 dilution. The Tim29 protein was purely transported to the mitochondria and was not detected in the intermyofibrillar homogenate. e Mitochondria were isolated from quadriceps 1–3 and heart 4–6 from controls 1 and 4 and patients 2,3,5,6 (patients: VII-4 and VII-5, respectively). The…

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  Fig. 4. representative musculoskeletal nuclear and mitochondrial gene expression. We used musculoskeletal samples from four controls and three S.S TIMM29 patients for mRNA extraction. In order to compare the gene expression in the healthy and affected groups we used independent sample t-test. The expression data in S-Table 2 are the mean results of raw data. Apart from the TIMM29 gene, there were no significant differences in the gene expression level between healthy and affected groups in the tested genes (NDI, ND2, ATP6 & SLC25A4). The TIMM29 gene expression was more than five folds higher in the affected than in the healthy group (p value 0.005)

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  Fig. 5. Knocking down the TIMM29 orthologue CG14270 in mesodermal tissues leads to S.S-TIMM−29-like phenotypes. A A 107710kk/ + ;24B-gal4/ + (CG14270-KD) fly unable to eclose from the pupal case. B 107710kk/ + ;24B-gal4/ + flies that do eclose show extremely reduced motor activity and they fail to climb up the vial wall. The vial on the left contains 107710kk/ + ;24B-gal4/ + flies, and the vial on the right, control flies (arrows point to the position of flies). The flies in both vials were knocked down to the vial bottom and the picture was taken 30 s later (C–D). Heliyon video shows the locomotion activity of these flies. Surviving 107710kk/ + ;24B-gal4/ + flies do not eat as evident by their shrunken abdomen (arrow) (c) compared to control flies (D). E QFPCR amplification plot analysis in Drosophila melanogaster larva. Gene expression of two housekeeping genes, GAPDH and SDHA, was used as control. In both genes the expression results were similar and here we present the GAPDH data. For GC14270 gene expression, we used two sets of primers (EX_PR_1 and EX_PR_2) located within the GC14270 exon sequence. Here, we present the GC14270 expression in GC14270- RNAi expressing larvae (107710kk/…

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  Fig. 6. Transmission electron microscopy (TEM) analysis of indirect flight muscles of CG14270 knockdown versus normal flies. Representative TEM images of indirect flight muscles (IFM) from wild type (WT) and knockdown 107710kk/ + ;24B-gal4/ + flies (KDS). A Longitudinal and B transversal sections of IFM of WT showing normal muscle structure and orientation with normal mitochondrial morphology and cristae ultrastructure (insert in B). C–F IFM of KDS demonstrating swollen mitochondria with extensive vacuolization of the inner membrane (white arrows, D and F). Note the increase in mitochondrial number (C and E). Progressing mitochondrial pathology is accompanied by a disturbance in the orientation of the muscle fibers (E and F); Mt, mitochondria; Myf, myofibril; Scale bars: A–F, 1 µm, insert 0.5 µm

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  Fig. 7. A significant decrease in the respiratory chain enzymatic activities in the RNAi treated flies compared to the control group. There is a significant decrease of the respiratory chain enzymatic activities in the RNAi treated flies compared to the control group. We calculated the respiratory chain enzymatic activities assayed by spectrophotometry in muscle homogenates from flies grown at different temperatures (22 °C and 29 °C) and ratios to glycerol 3-phosophate dehydrogenase (GCCR) (*p < 0.05)

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  Fig. 8. a. Sequence conservation of Tim29 proteins. Sequence logo of the Tim29 protein family (pfam PF10171) uniprot sequence multiple-sequence alignment, with 1010 proteins, spanning chordates, arthropods, nematodes, mollusks, plathyhelminthes, rotifers, and cnidaria (https://www.ebi.ac.uk/interpro/entry/pfam/PF10171/). The height of each column relates to its conservation. The sequences were position-base weighted [https://doi.org/10.1016/0022–2836(94)90032-9]. The total conservation in each column is its relative entropy (Kullback–Leibler distance) using the amino-acids background frequencies in the SwissProt database, with small-sample correction [https://doi.org/10.1038/nbt0406-423]. The amino acid fractions in each column are relative to their weighted odds ratios, as previously described [https://doi.org/10.1016/0378–1119(95)00486-p]. Each column is labeled beneath by its position and residue in human Tim29 protein, with gaps indicating insertion in the alignment relative to the human protein, and the protein transit-peptide and trans-membrane regions, and Trp172 residue are marked. b Position of Tim29 W172 in the cryo-structure of human TIM22 complex…