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

Abstract

Purpose

Sengers-syndrome (S.S) is a genetic disorder characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. All reported cases were genetically caused by biallelic mutations in the AGK gene. We herein report a pathogenic variant in TIMM29 gene, encoding Tim29 protein, as a novel cause of S.S. Notably, AGK and Tim29 proteins are components of the TIM22 complex, which is responsible for importing carrier proteins into the inner mitochondrial membrane.

Method

Clinical data of 17 consanguineous patients featuring S.S was obtained. Linkage analysis, and sequencing were used to map and identify the disease-causing gene. Tissues derived from the study participants and a Drosophila melanogaster model were used to evaluate the effects of TIMM29 variant on S.S.

Results

The patients presented with a severe phenotype of S.S, markedly elevated serum creatine-phosphokinase, combined mitochondrial-respiratory-chain-complexes deficiency, reduced pyruvate-dehydrogenase complex activity, and reduced adenine nucleotide translocator 1 protein.

Histopathological studies showed accumulation of abnormal mitochondria. Homozygosity mapping and gene sequencing revealed a biallelic variant in TIMM29 NM_138358.4:c.514T > C NP_612367.1:p.(Trp172Arg). The knockdown of the Drosophila TIMM29 orthologous gene (CG14270) recapitulated the phenotype and pathology observed in the studied cohort. We expand the clinical phenotype of S.S and provide substantial evidence supporting TIMM29 as the second causal gene of a severe type of S.S, designated as S.S- TIMM29.

Conclusion

The present study uncovers several biochemical differences between the two S.S types, including the hyperCPKemia being almost unique for S.S-TIMM29 cohort, the different frequency of MMRCC and PDHc deficiencies among the two S.S types. We propose to designate the S.S associated with TIMM29 homozygous variant as S.S-TIMM29.

Introduction

Sengers syndrome (S.S) is an autosomal recessive mitochondrial disorder, characterized by hypertrophic cardiomyopathy, skeletal myopathy, congenital cataracts and lactic acidosis [1,2,3,4,5]. The clinical course of the disease can vary widely, ranging from a severe fatal form- leading to death within the third year of age in around 86% of cases- [6] to a more benign form that allows survival into adulthood [5, 7]. There is also an isolated form of congenital cataract [8].

A significant step in delineating the genetic defect underlying S.S was the identification of variants in the Acyl-glycerol kinase (AGK) by three research groups [4, 9, 10]. Since then, bi-allelic variants in the AGK have been found in most reported S.S cases (~ 40) [5, 6, 11, 12]. A decade ago, AGK was known as a mitochondrial transmembrane kinase that catalyzed the phosphorylation of monoacylglycerol and diacylglycerol into lysophosphatidic and phosphatidic acid, which play important roles as secondary messengers and regulators of mitochondrial ultrastructure [4]. However, the abnormal AGK's kinase activity could not explain the pathophysiology underlying the discovery of adenine nucleotide translocator 1 (ANT1) protein deficiency reported in many S.S patients [13, 14], including those in our cohort.

Mitochondria require more than 1000 nuclear-encoded proteins to function properly [15]. Evolutionary conserved translocation machineries are responsible for their specific mitochondrial sub-compartment delivery and integration into the outer membrane, intermembrane space, inner membrane, and matrix [15,16,17]. Mitochondrial inner membrane contains two translocase machineries: TIM23 and TIM22. TIM23 transports proteins that possess matrix-targeting N-terminal presequence, [18, 19], whereas TIM22 complex mediates the import of hydrophobic proteins, which are targeted by internal targeting signals into the inner membrane. Variants in genes encoding various subunits of these mitochondrial translocation machineries cause a growing list of mitochondrial diseases [20].

Compared to yeast, the human TIM22 machinery has been less characterized. In humans, TIM22 is a 440-kDa complex comprising at least six components: the hypothetical channel-forming protein Tim22, three small Tim proteins (Tim9, Tim10a and Tim10b), Tim 29 and acylglycerol kinase (AGK) [19]. In 2017, two research groups demonstrated that, apart from the AGK having a first primary function of being a kinase, it also has a second major function, which is it being an essential component of the TIM22 protein translocase complex in human mitochondria. It facilitates the import and assembly of mitochondrial inner membrane proteins, including ANT1 [19, 21, 22].This discovery explained ANT1 deficiency existing in S.S individuals harboring biallelic variants in the AGK [4, 5, 11, 13, 23]. Recently, biallelic variants in the TIM22 pore forming subunit were identified and associated with hypotonia, gastroesophageal reflux and elevated lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity, altered mitochondrial morphology and ANT1 deficiency [24]. Variants in the TIMM8A gene also called DDP1 cause deafness dystonia syndrome [25].

This study focused on characterizing the clinical, biochemical and molecular mechanism underlying the pathogenicity of a homozygous missense variant identified in TIMM29 (C19orf52) encoding Tim29 protein. Initially, the function of the gene was unclear, but it was later identified as a subunit of TIM22 [19, 21, 26, 27]. We demonstrated that the variant segregated in a cohort of 17 consanguineous individuals, who displayed the severe infantile form of S.S, but had no variant in the AGK gene. RNA interference (RNAi) was used to knock down or reduce the expression of the orthologue of TIMM29 (CG14270) in Drosophila. The affected flies displayed characteristics similar to the human S.S infantile phenotype. This work has provided substantial evidence that TIMM29 constitutes the second causal gene of S.S, which is herein designated as Sengers-syndrome type2 (S.S-TIMM29). This designation makes it different from the S.S caused by mutations in the AKG gene (S.S-AGK).

Research participants and methods

Participants

Over the past three decades, we have been conducting investigations involving 17 individuals from a large consanguineous Arab family (Table 1, Fig. 1). The clinical and biochemical phenotype of these participants resembled those seen in patients with fatal infantile S.S. We conducted a comprehensive study, including clinical, biochemical and genetic analyses in all the available individuals. Detailed description and methods can be found in the Human Genomics manuscript. It includes information on clinical phenotype, biochemical analysis, metabolic, and human molecular evaluations as well the methodology used for Drosophila Knock down experiments. Also included aredetailed description of mitochondrial isolation and respiratory chain complexes analyses in human and fly samples [28,29,30,31], linkage analysis, DNA sequencing (Sanger and Next Generation Sequencing-NGS), mitochondrial DNA analysis, Western Blot analysis, RNA expression analysis and transmission electron microscopy (TEM).

Table 1 Clinical and biochemical characteristics of patients with Sengers syndrome-TIMM29

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 be found in the supplementary section

Results

Clinical and laboratory evaluation

The family disease of the study participants was compatible with an autosomal recessive disease (Fig. 1).

All affected participants displayed a fatal mitochondrial disorder which strikingly resembled the infantile form of S.S. The clinical and biochemical features of the 17 individuals are depicted in Table 1. Typical clinical findings which are not depicted in the column of clinical remarks in Table 1 are provided in the case reports (Human Genomics). 16 out of the 17 studied participants displayed progressive hypotonia, while 8 out of them had hypotonia during the first two days of their life. Hypotonia was not found in participant VII-11, who suddenly died at the age of 4 months. Her death was described as”crib death”. On follow-up, most of the participants had motor developmental delay and muscle weakness to various extents attributed to the progressive mitochondrial myopathy.

No dysmorphic features were apparent in the studied group. However, two patients (VII −3 and VII-5) had facial features that were attributed to prematurity. 10 participants displayed cataract. Among these 10, two were diagnosed with cataract at birth, while cataract was diagnosed in the remaining 8 before they reached the age of three months. We assume that the other 7 died before they developed cataract. Hypertrophic cardiomyopathy was diagnosed in 14 individuals among the studied group. Out of these14 individuals, 2 were diagnosed with hypertrophic cardiomyopathy during the first week of their life, while the remaining 12 were diagnosed with hypertrophic cardiomyopathy between the ages of 2–12 months. We speculate that the other 3 participants died before they developed cardiac abnormalities which could be depicted by echocardiography (EC). This hypothesis is illustrated in the case of participant VII-4, where an EC performed five days prior to her death was reported as "normal". However, four hours before her death, upon arrival at the emergency room (ER), she featured cyanosis and bradycardia. Subsequent EC performed revealed for the first time moderate cardiac hypertrophy with an almost motionless left ventricle. Surprisingly, the electron microscopy (E.M) of her cardiac muscle revealed only a few left isolated disrupted myofibrils and most of the heart tissue was replaced by massive proliferation of abnormal shaped mitochondria. Most of these mitochondria are without cristae (Fig. 2a,b and c).

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)

Participant VII-11 did not show signs of cardiomyopathy on Echocardiography (EC) in at the age of 4 months. She died at home two weeks later, during sleep at night. We assume that her “sudden death" may have been caused by cardiac arrhythmia. This is similar to the case of a two-year-old patient that died suddenly of cardiorespiratory arrest, even though he had stable cardiac function 1 month prior to his death [11]. 15 of the studied participants died before reaching the age of one year. 4 of them died in the neonatal period; only 2 of them, who seemed to have a somewhat milder phenotype, survived until the age of 21 months (Fig. 1 and Table 1-VII-2).

All participants had elevated plasma lactate level which fluctuated between 2.9 and 17.5 (normal up to 2 mmol/l). Lactate level was elevated in the cerebrospinal fluid (CSF) of two out of three participants (VI-10 and VII-5) who underwent lumbar puncture, which is suggestive of the involvement of the central nervous system (CNS). Serum alanine, which is known as a marker of inborn errors of energy metabolism [14], was elevated in most of the studied participants.

Most of the routine and metabolic laboratory studies (see in the methods section) were found to be within normal ranges. However, remarkably, 13 out of 14 studied participants had elevated serum creatine phosphokinase (CK) levels greater than 1000 U/L (hyperCKemia), in the range compatible with rhabdomyolysis [32]. HyperCKemia has not been reported nor considered as a common marker in previously reported S.S-AGK cases. Recently, only one S.S-AGK reported case featured elevated CK and myoglobinuria [12].

Urinary organic acids of eight patients were studied. The results showed that the patients exhibited different urinary organic acids profiles, ranging from normal to intermittently increased secretion of various metabolites commonly secreted by patients with mitochondrial disorders. They included lactate, succinic acid, fumarate and 3-hydroxyglutaric, suggesting the involvement of the Kreb's cycle, 3- hydroxybutyrate (ketosis) and occasionally 3-methylglutaconic acid, known to constitute a non-specific marker for various mitochondrial disorders [33]. It is worth noting that we did not identify a constant and pathognomonic abnormal profile, for the SS-TIM29 mitochondrial disorder.

Histology and histopathology of skeletal and cardiac muscle specimens

Seven of the studied participants were subjected to histopathological analyses. Skeletal muscle biopsy specimens from 4 of them were subjected to histopathological analysis using light microscopy. While, histopathological analysis was performed on the cardiac muscle of the remaining three, using standard techniques. Light microscopy revealed minimal to significant variation in fiber size and increased glycogen and lipid droplets to various extents. We used specific stains for COX and succinate dehydrogenase (SDH). They revealed some of the participants had COX deficient fibers, increased SDH staining and ragged red fibers commonly reported in patients with mitochondrial myopathy.

The electron microscopy (EM) study of skeletal muscle and myocardial-biopsy specimens, performed within 1–2 h after the biopsy or post mortem, revealed interrupted myofibrils with numerous electron lucent mitochondria. Higher magnification shows isolated disrupted myofibril and replacement of heart muscle by marked proliferation namely, oncocytic transformation of abnormal looking mitochondria of various sizes and shapes (Fig. 2a,b and c). Such mitochondrial ultrastructural changes are not pathognomonic for S.S-AGK nor for S.S-TIMM29 since it has been found in other mitochondrial disorders [3, 34,35,36].

Mitochondrial respiratory-chain complexes activities

Over the years, muscle biopsies have been performed in most of the S.S-TIMM29 participants, in which the activities of respiratory chain complexes were assessed. Table 2 provides the results obtained from the study of respiratory chain complexes activities, citrate synthase (CS) and pyruvate dehydrogenase complex (PDHc) activities examined in mitochondria extracted from 4 fresh skeletal muscles and one frozen cardiac muscle. We demonstrate various combinations and extents of decreased mitochondrial respiratory-chain-complex activities in the participants. A similar pattern was demonstrated in a frozen cardiac muscle specimen. Fibroblast culture retrieved from one participant (VII-3) revealed only a mild defect in the mitochondrial function with an increased CS activity. This finding might denote that cultured fibroblast does not constitute the ideal tissue for studying the effect of Timm29 deficiency. Generally, complex II activity (composed solely of nuclear encoded proteins, without mitochondrial DNA (mtDNA) encoded proteins) ranged between low or slightly below normal to increased levels. Pyruvate dehydrogenase complex (PDHc) deficiency was also demonstrated in 4 of the studied participants (Table 2).

Table 2 enzymatic activities of the mitochondrial respiratory chain and mtDNA quantitation in mitochondria from fresh muscle, frozen myocard and fibroblasts

Linkage analysis and positional cloning

In our previous study on homozygosity mapping and positional cloning on participants VI-20, VII-4, VII-10 and VII-11 (Fig. 1) utilizing the DNA pooling strategy [37, 38], we localized the disease gene locus to 19p13.2–3 chromosomal region of 2.3 Mb interval between the markers D19S586 and D19S906 (LOD score of 7.89 for two-point linkage analysis for marker D19S906 (Fig. 1) and of 12.38, Θ = 0 for multipoint linkage analyses for the markers D19S584, D19S906 and D19S817). For LOD score calculation, we used the Superlink software (http://cbl-hap.cs.technion.ac.il/superlink-snp) [39].

TIMM29 mutated in S.S type2 individuals

We performed direct Sanger sequencing of 14 out of 84 genes located between the D19S586 and D19S906 markers (19p13.2–3; UCSC Genome Browser on Human Mar. 2006(NCBI36/hg18)), selected by their high scores as mitochondrial encoding proteins according to predicting tools (e.g.MitoPROT2, Mitopred, Predotar and TargetP). The sequences of all the genes were normal except one missense point variant (NM_138358.4:c.514T > C NP_612367.1:p.(Trp172Arg) in C19ORF52 (LOC90580) gene which was renamed as TIMM29 [19]. In genomic level, this variant is known as NC_000019.9:g.11040109T > C NM_138358.4:c.514T > C p.(Trp172Arg) (Fig. 3a). This variant was tested in 4 of our studied affected participants and in 51 healthy parents and siblings. The variant segregated with the disease and matched the haplotype carrier status, as described in Fig. 1. Thus, all affected individuals were homozygous for the c.514T > C variant. While the parents were heterozygous, the siblings and additional healthy family members were either heterozygous or homozygous for the wild allele (c.514T). Also, this variant was not present in 200 chromosomes from 100 unaffected, unrelated healthy individuals from the same ethnic background. A new SS-TIMM29 affected neonates were born in 2009 and 2024 (Table 1). The parents of both patients were 3rd degree cousins and belong to the expanded affected family. The patient 2009 was homozygous for the disease haplotype (data not shown) and her phenotype was extremely severe and she died at age 13 days of life. While, the patient HZ 2024 was 5 months old in his last examination. Both were homozygous and the parents were heterozygous for the TIMM29 NM_138358.4:c.514T > C.

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 proteins were separated by SDS-PAGE (CII, AIF, TIM29) or urea-PAGE (CIV Subunit 2) and subjected to western blot analysis. Compared to control group, the TIM29 was decreased (muscle) or barely detectable (heart) in the patient group. ANT1 was undetectable in patient VII-4 (Western blot performed in a separate gel). f There is a decrease in two constituents of cytochrome C oxidase (complex IV) in both COX II, which is a mitochondrial encoded protein and in subunit IV, which is encoded by the nuclear genome. There is severe deficiency of ANT1. Regretfully, we only had a very limited amount of patient material and nothing was left after analysis, so we cannot repeat the blots. However the lanes were loaded with the same amount of the citrate synthase units, equivalent to 2.5 mU per lane. Thus, we relate to citrate synthase as a proper loading control that is not predicted to be affected by the TIMM29 variant. P = patient (represent all tested patients (14); C = Control

With the emergence of Next Generation Sequencing (NGS) technology, in order to cover all genes including those located in 19p13.2–3 mapped region (Human Genomics), we ran Exome Sequence (ES) analysis in the different participants using two different platforms six years apart and reanlyzed the last ES few months ago. In the first round, ES was performed in one nuclear family (patients VII-3 and VII-4 and parents VI-5, VI-6, Fig. 1) and the analysis did not reveal any pathogenic or likely pathogenic variant. NC_000019.9:g.11040109T > C NM_138358.4:c.514T > C p.(Trp172Arg) variant was not detected. To explain this, we focused on the 944 exons located within 19p13.2–3 disease refined mapped region. In conclusion, the coverage was not optimal since, only 605 exons (0.6408) were completely sequenced, while for the 339 remaining exons, the sequence coverage was incomplete. In 71 exons (0.075), over 80% of the exons’ length were covered and sequenced, in 73 exons (0.077), 50–80% of the exons’ length were covered and sequenced, in 92 exons (0.097) less than 50% of the exons’ length were covered and sequenced and 103 exons (0.109) were entirely uncovered. Moreover, we zoomed in TIMM29 gene by the Integrative Genome Viewer (IGV) and found a small region within exon 2 of TIMM29 that was entirely uncovered, including the NC_000019.9:g.11040109T and flanking sequence.

Six years later, we ran ES in patient VII-10 (Fig. 1) by using Nextera DNA Flex Enrichment reagents and NovaSeq 6000 sequencer (Illumina, San Diego, CA). The coverage and depth were obviously much higher (pptx VII-10 Human Genomics) and the analysis identified the TIMM29, NM_138358.4:c.514T > C NP_612367.1:p.(Trp172Arg) variant in homozygous state. Importantly, no other pathogenic or likely pathogenic variants were found. Of note, there was no evidence for nuclear copy number variation (CNV), mitochondrial genome point variants and deletions. Reanalysis of ES raw data performed 4 years later for patient VII-10 did not change our primary finding (Human Genomics).

At the genomic level, TIMM29 is composed of 1494 base pairs and has two exons separated by 83 base pairs intron. The 1394 nucleotide mRNA encodes for a 260 amino acids protein of 29.233 kDa molecular weight. TIMM29 has no splice isoforms. TIMM29 is a ubiquitously gene with abundant orthologues present across a wide range of species, including mammalian, amphibian, avian, fish, vertebrates, sea urchin (Strongylocentrotus purpuratus), phylum Placozoa (Trichoplax adhaerens), Drosophila and c. elegans. For sea urchin, phylum Placozoa, Drosophila and c. elegans, it has 40%, 29%, 28, and 25% conservation over the length of the protein, respectively. At the protein level, Tim29 Trp172 residue is highly conserved in evolution (Fig. 8a) [19, 26]. It is almost fully buried within the TIM22 complex structure facing the inner membrane, and only directly interacts with other Tim29 residues [22] (Fig. 8b). A blast search analysis revealed that Tim29 protein shares high amino acid homology that ranges from 35% up to 98% with proteins from various species. The analysis and a multiple sequence alignment also revealed a high protein similarity among Tim29 orthologues across diverse species. Of note, as the Trp172Arg variant is a change that is not observed in any orthologue (Fig. 3a, b and 8a), the p.Trp172Arg variant results in the substitution of the hydrophobic aromatic side chain tryptophan for the positively charged side chain arginine at position 172 of the Tim29 protein [19]. 14 prediction tools considered this variant as “pathogenic”, while five others considered it as “benign” (Human Genomics xls2) selectively. Here, we mention c. 514T > C variant scores in parentheses as they appear in different prediction tools, such as CADD (32.0); REVEL(0.646); phyloP(6.25) and PolyPhen(0.964). This supports the pathogenicity of this variant. Also, according to the gnomAD v4.10 (GRCh38) database, only three heterozygous variants in TIMM29 gene (ENSG00000142444.7) p.Trp172 codon were described. The NC_000019.9:10929433T > C (chr19-10929433-T-C) (c. 514T > C) variant found in this study was reported to be heterozygous in only one individual of Middle Eastern origin with an allele frequency of 0.0001650. Moreover, NC_000019.9: 10929434G > A chr19-10929434-G-A (p.Trp172Ter) variant (rs778791266) was reported heterozygous in two individuals of European non-Finnish origin. The NC_000019.9: 10929434G > T (chr19-10929434-G-T) variant was reported to be heterozygous in three individuals (one of South Asian origin and remaining two unspecified). Finally, NC_000019.9: 10929435G > A (chr19-10929435-G-A) variant was reported to be heterozygous in one individual of European non-Finnish origin. The termination variants were predicted to be pLoF with high-confidence. pTrp172Leu variant was predicted to be probably-damaging in Polyphen and damagaing in SIFT.

From the information obtained from the databases (gnomAD, ClinVar, ExAC), we can say that TIMM29 variants appear in low allele frequency. From gnomAD v4.10 (GRCh38), there are 734 variants (61 LoF, 454 missense and 219 synonymous) in TIMM29 gene. All of the LoF and missense variants were reported in heterozygous state only. Also, repeated GeneMatcher searches were negative.

Tim29, ANT1 and OXPHOS protein levels

We studied the impact of ^172RTim29 variant on the protein level of Tim29 itself and on several other mitochondrial proteins, using available skeletal-muscle biopsy specimens and primary fibroblasts cultures. It was done to show that TIMM29 variant is linked to S.S-TIMM29. Western blot analysis showed markedly reduced Tim29 levels along with reduced levels of ANT1 and other OXPHOS proteins (Fig. 3b, c, d, e ,f). These findings support the notion that the ^172RTim29 variant adversely affects the stability of the Tim29 protein, ANT1 and several OXPHOS proteins encoded by both the mitochondrial and the nuclear genomes. The decreased levels seen in the various OXPHOS proteins in tissues with Tim29 deficiency show that Tim29 plays an essential role in the TIM22 complex [19, 26].

Mitochondrial DNA content and transcription

In all the studied participants described above, the function of the respiratory complexes I, III, IV, and V was disrupted, while the function of complex II, solely composed of nuclear encoded proteins was elevated, within normal range or slightly reduced. Thus, we examined mtDNA content in the studied participants, which was found to be normal or elevated (results not shown). In addition, we sequenced the entire mtDNA (coding genes, mitochondrial tRNA and rRNA genes) excluding pathogenic mtDNA variants, depletion and mtDNA translation defects.

In order to assess the effect of mtDNA transcription on the studied participants, we tested mitochondrial respiratory chain complexes representative genes, such as ND1 and ND2 for complex-I and ATP6 for complex V and detected normal or elevated levels. In addition, we tested two nuclear genes, SLC25A4 (ANT1) and TIMM29. The expression analysis determined a normal mtDNA transcription level (Fig. 4 and, Table 2 and xls3 in Human Genomics). This demonstrates there is a normal SLC25A4 mRNA expression, but markedly increased level (by more than five folds) of TIMM29 mRNA expression in our S.S-Tim29 participants compared to the healthy control samples (Table 2 in Human Genomics, Fig. 4). Thus, according to our results, Tim29 deficiency does not affect transcription negatively.

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)

Animal model

The knock down of CG14270 the Drosophila melanogaster TIMM29 orthologue

To further confirm the role of TIMM29 as the causative agent of S.S type 2 phenotype, we examined the loss-of-function phenotype of the Drosophila melanogaster orthologue of TIMM29. The fly genome contains a single gene, CG14270, of the Tim29 protein family that shows a significant sequence similarity to Tim29 throughout its entire length [40]. To test for possible involvement of the CG14270 gene in mitochondrial biogenesis or function, we knocked down its expression using RNA interference (RNAi) and examined the effects on the fly’s locomotion and general behavior. We used the mesodermal driver 24B-Gal4 to drive the expression of two independent CG14270-specific RNAi transgenes (P{kk100018}v107710 and P{GD8280}v17459) in mesodermal tissues, both of these RNAi constructs have zero off targets [41]. Testing the effect of P{kk100018}v107710 on CG14270 epression by QRT-PCR, demonstrated a 6.6 fold decrease in expression in P{kk100018}v107710/ + ;24B-gal4/ + larvae as compared to control P{kk100018}v107710 larvae (Fig. 5E). Driving P{GD8280}v17459 expression caused embryonic or larval lethality (in all temperatures tested from 18° to 29°). The expression of P{kk100018}v107710 caused semi-lethality. Approximately 50% of P{kk100018}v107710/ + ;24B-gal4/ + flies died as pharate adults (113/223). Some of the flies had difficulties eclosing from their pupal case and they died while trying to eclose (Fig. 5A). The progeny that did emerge appeared very lethargic, they did not eat and displayed dramatic reduction in climbing activity from day 1 of life (Fig. 5B–D, and video MVI-1 in Human Genomics). Whereas 83.5 ± 15% of control flies climbed from the bottom of the vial to a 5 cm height within 10 s or less (n = 49) after being knocked down to the bottom of the vial, none of the knockdown flies reached the 5 cm line within 10 s (n = 87).

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/ + ;24B-gal4/ +) (lower plot) and control larvae (107710kk/ +) (upper plots) groups. The GC14270 average expression in the RNAi-expressing larvae was reduced 6.6 folds (0.15) compared to the gene’s expression in the control group (1)

The P{kk100018}v107710/ + ;24B-gal4/ + flies started to die within a day or two post eclosion and did not survive more than six days, compared to the control flies that survived for several weeks.

The dramatic behavioral phenotypes caused by reduction in CG14270 expression led us to examine directly the mitochondrial phenotype in the flight muscles of CG14270 knocked-down flies, using transmission electron microscopy. The EM analysis showed substantial mitochondrial phenotype (Fig. 6), resembling the phenotype seen in muscle biopsies from S.S patients (Fig. 2). The mitochondria of the indirect flight muscles appeared swollen and presented extensive vacuolization of the inner membrane (Fig. 6). Disorganized musculature led to the progression of mitochondrial pathology. We could see a largely increased number and reduced size of mitochondria conforming with mitochondrial oncocytic.

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

Encouraged by the mitochondrial phenotype observed by EM in the indirect flight muscles of the CG14270 knock-down flies, we studied the respiratory chain enzymatic activities assayed by spectrophotometry in muscle homogenates from the CG14270 knockdown flies compared to normal flies. As presented in Fig. 7 and xls4 in Human Genomics, there was a significant decrease in the respiratory chain enzymatic activities in the RNAi treated flies compared to the control group. Altogether, the CG14270 knock-down flies recapitulated the clinical and biochemical phenotype of S.S-TIMM29, type 2, which strongly provides evidence of the central role that TIMM29 plays in causing S.S-TIMM29.

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)

Discussion

Till date, all reported cases of S.S have been shown to be associated with homozygous or compound heterozygous variants in the AGK gene encoding the AGK protein (S.S-AGK) [4,5,6, 12]. We herein describe the results of the clinical, biochemical, molecular and functional studies in a cohort of participants who featured S.S phenotype, but had normal AGK gene sequence. Homozygosity mapping in this cohort located the disease gene in a 2.3 Mb interval on chromosome 19p13.2–3. A missense variant, NM_138358.4:c.514T > C NP_612367.1:p.(Trp172Arg) in TIMM29 segregated completely with the disease (Fig. 1, xls1 in Human Genomics).

The various experimental validations, including in vitro analysis of tissues from the S.S- TIMM29 participants and the use of a Drosophila model that recapitulated the human S.S-TIMM29 phenotype provide strong substantiation for the pathogenicity of NM_138358.4:c.514T > C NP_612367.1:p.(Trp172Arg) variant in the TIMM29 as the causal gene of S.S-TIMM29 mitochondrial disorder. The protein level of Tim29 was significantly decreased in the participants' tissues (Fig. 3), even though TIMM29 mRNA expression increased by almost fivefold (Fig. 4 and Table 2 in Human Genomics). A similar finding was reported in a patient with compound heterozygous variants in TIMM22 [24]. The increased mRNA levels of both TIMM29 and TIMM22 in the affected participants could result from an up-regulation of gene expression, a plausible hint for a common positive feedback regulatory system for TIM22 complex. The evolutionary conservation of the mutated Trp172 (Figs. 3; 8a), its replacement by the radically different Arginine residue, and its position and interactions within the TIM22 complex [22] (Fig. 8b) strongly suggest that the variant is highly deleterious. Indeed, we found that theTIMM29 homozygous variant destabilizes Tim29 protein and leads to its degradation, as seen in the muscle specimens of S.S-TIMM29 patients (Fig. 3). Consequently, Tim29 diminution in TIM22 complex is likely responsible for the severe phenotype of S.S-TIMM29 mitochondrial disorder. Of note, Tim29 Trp172 is not conserved in CG14270 (the DrosophilaTIMM29 orthologue), and it was replaced either by Tyrosine (Y), in most Drosophila species, including Drosophila melanogaster, or by a Phenylalanine (F) in other species such as Drosophila persimilis (Fig. 3b, c). Since W, Y and F are all nonpolar aromatic amino acid residues that have hydrophobic side chain, we assume that this replacement keeps Tim29 activity intact. Although uncommon, there are known examples for a replacement of conserved tryptophan residues by phenylalanine in functional protein domains. For instance, in 33 rice Aux/IAA proteins, a conserved tryptophan residue, which is located in the central position of the functional protein domain IV was found in 31 proteins. It was replaced by phenylalanine in the 2 remaining proteins, without affecting protein activity [42].

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://doiorg.publicaciones.saludcastillayleon.es/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://doiorg.publicaciones.saludcastillayleon.es/10.1038/nbt0406-423]. The amino acid fractions in each column are relative to their weighted odds ratios, as previously described [https://doiorg.publicaciones.saludcastillayleon.es/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 [doi.org/10.1038/s41422-020–00400-w]. Subunit colors are: TIM22 in light green, AGK in light blue, Tim29 in red with Trp172 in cyan, Tim9 chains in orange, Tim10a chains in olive, and Tim10b in light yellow. The atoms of the proteins aa are shown as spheres, except for the trans-membrane regions that are shown as cylinders. The phosphoethanolamine phospholipid is shown in gray. Inset shows a zoomed view of Tim29 Trp172. Figure made with the PyMol program (https://pymol.org)

Remarkably, the knockdown of CG14270, the Drosophila TIMM29 orthologue, by RNA interference (RNAi) led to the recapitulation of the clinical, biochemical and histopathological phenotype of the human S.S-TIMM29 participants. The knock-down flies exhibited the major constituents of the mitochondrial cytopathy exhibited in the S.S-TIMM29 participants. This resulted in them having short lifespan, lethargy and severe loss of locomotion activity and climbing abilities that likely reflect severe muscle hypotonia. Moreover, reduced activity of several mitochondrial respiratory chain complexes was demonstrated in dissected muscle preparations (Fig. 7, Xls4- Human Genomics), and ultrastructural analysis of flight muscles revealed abnormal mitochondrial structure mimicking the mitochondrial phenotype seen in S.S- TIMM29 patients (Fig. 6). These findings demonstrate that the Drosophila model recapitulates the human S.S-TIMM29 participants’ phenotype. Interestingly, in a a systematic RNAi-based screen for genes involved in muscle morphogenesis and function in Drosophila, the fly AGK ortholog Mulk was identified as one of 78 genes whose knockdown in muscles led to abnormal locomotor behavior [35]. To the best of our knowledge, no biochemical or ultrastructural phenotypic data related to Mulk is available.

Delving into the clinical and biochemical features of both S.S-AGK and S.S-TIMM29 highlights additional clinical findings which allow us to expand the phenotype of S.S.

Cardiac arrythmia: One S.S-TIMM29 participant (VII-11) died at home suddenly. We assume that cardiac arrythmia was the cause of his "sudden death" although he did not display cardiomyopathy. Arrythmia was suggested to be the underlying cause of "sudden death" in one S.S-AGK patient who did not feature previous cardiac decompensation at the age of 2 years [11]. Interestingly, the ANT1 homozygous mutant mice model, also featured cardiac arrhythmia [36].

In the S.S-AGK participants, motor developmental delay, hypotonia, hyporeflexia, and impaired physical mobility were commonly attributed to muscular weakness due to the mitochondrial myopathy. However, these symptoms could express also neurological abnormalities secondary to central-nervous-system (CNS) involvement. Long-term follow-up of the S.S-AGK participants revealed several developmental challenges, such as intellectual disability, psychomotor delay and language acquisition delay or absence [5, 11]. In three S.S-TIMM29 participants (VI-20, VII-4 & VII-5), we suspected developmental delay due to brain involvement. These three S.S-TIMM29 participants did not achieve any developmental milestone, nor social eye contact. Also, two of them had elevated level of lactate in their CSF, suggesting mitochondrial encephalopathy. Brain imaging in participant VII-5 revealed periventricular leukomalacia, partial agenesis of corpus callosum and vermis hypoplasia depicted by brain ultrasound and CT scan, respectively. Neuro-radiological examinations were performed in only 30% of the reported S.S-AGK cases. This is probably because most of them died within the first or second year of their life and they were assumed to have developmental delay, due to the mitochondrial myopathy [4, 7, 43]. The most common neuroradiological manifestations in the S.S-AGK participants include hypoplasia of the brainstem and inferior cerebellar vermis, impaired myelination of the cerebral hemispheres, and brainstem, and cortical infarctions. However, these infarctions follow a vascular pattern different from typical metabolic strokes, which do not adhere to any specific arterial distributions [10]. The developmental delay, neuroradiological findings and elevated CSF lactate in the S.S participants suggest that both S.S types can be described as mitochondrial Encephalo-Cardio-Myopathies. In order to substantiate this possibility, brain MRI and MRS should be added routinely to the study of S.S patients.

The most striking biochemical difference between S.S-TIMM29 and S.S-AGK patients was the constant finding of elevated levels of serum creatine phosphokinase (CK) (HyperCPkemia) in S.S-TIMM29, which could progress to rhabdomyolysis (Table 1) [44, 45]. HyperCPKemia has not been reported in S.S-AGK patients, except for one recently reported S.S-AGK patient [12]. CK, both the mitochondrial CK and the cytosolic CK isoenzymes, play a critical role in the buffering of energy to maintain cellular energy homeostasis [46, 47]. Thus, the hyperCPKemia found in S.S-TIMM29 patients could denote a central role of Tim29 in the CK metabolism. Many mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) variants cause mitochondrial cytopathies, but only sporadic cases have been reported to be associated with rhabdomyolysis [45]. Thus, the role of Tim29 deficiency in the causation of rhabdomyolysis remains to be elucidated.

The phenotypic resemblance of the S.S-TIMM29 studied cohort to the adenine nucleotide translocator 1 (ANT1) deficient mice model was astonishing two decades ago. Both shared the hypertrophic cardiomyopathy, elevated serum lactate and strikingly abnormal mitochondrial structure in skeletal and cardiac muscle (Fig. 2) [10, 23, 36]. ANT1 deficiency was demonstrated also in skeletal and cardiac muscles tissues derived from S.S-AGK [13, 23] and in a patient with compound heterozygous variants in the TIMM22 gene [24].

For many years, ANT1, encoded by the SLC25A4 gene, was known for its canonical function of forming a channel which moves ADP into ATP out of the mitochondrion to be used as energy source of the cell. However, over the years, ANT1 has been shown to be a moonlighting protein, a biological phenomenon in which a protein with a canonical function can acquire additional roles during evolution [48]. In addition to its canonical function, ANT1 was shown to drive mitophagy, independent of the nucleotide exchange [36]. Thus, the profound abnormal mitochondrial changes shown in the ANT1 mice model [35], in previously reported S.S-AGK tissues [10, 23], and in skeletal and cardiac muscle tissues derived from the S.S-TIMM29 cohort (Fig. 2a,b c and c), could be attributed to the blunted mitophagy associated with ANT1 deficiency [49]. ANT1 was also shown to be involved in the assembly of the respiratory chain complexes (RCC), a function which could be involved in the multiple mitochondrial respiratory chain complexes (MMRCC) deficiency depicted in the S.S patients (see below) [50, 51]. Recently, variants in the ANT1 were shown to be associated with clogging of cytosolic synthesized proteins targeted to the inner-mitochondrial membrane at the translocase of the outer membrane (TOM) complex [52]. Tim29 was shown to connect between the inner mitochondrial membrane and TOM complex [19, 22]. Combining these facts thus lead us to hypothesize that Tim29 deficiency may also affect indirectly protein transport from the cytosol into the mitochondria. This hypothesis needs to be addressed in future studies.

Multiple Mitochondrial Respiratory Chain Complexes (MMRCC) deficiency has been reported in ~ 49% of patients with mitochondrial disorders [53]. Combined defects of complexes I, III, IV and V in various combinations could be due to variants in genes that encode enzymes of mtDNA replication machinery, or maintain a balanced mitochondrial nucleotide pool or genes encoding proteins involved in mitochondrial fusion [54]. While most of the studied S.S-TIMM29 participants showed MMRCC deficiency in various combinations (Table 2), it was not constantly reported in the S.S-AGK participants [4,5,6, 12].This difference should be further explored.

The pyruvate dehydrogenase complex (PDHc) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDHc deficiency demonstrated in most of the S.S-TIMM29 studied participants (Table 2) was previously reported in only two S.S-AGK cases [23]. The resulting phenotype of PDHc deficiency mainly affects the central nervous system [55]. This raises the hypothesis that PDHc deficiency could contribute to the neurological symptoms and brain malformation in S.S individuals.

Recently, mitochondrial pyruvate carriers MPC1 and MPC2 were shown to constitute novel substrates of TIM22 in yeasts and humans [56]. The MPCs reside at a central metabolic point by transporting cytosolic pyruvate across the inner mitochondrial membrane (IMM), thereby linking glycolysis with oxidative phosphorylation [57, 58]. Thus, MPCs deficiencies could be involved in causing PDHc deficiency in S.S. However, it cannot explain the difference in the frequency of PDHc deficiency in S.S-TIMM29 compared to S.S-AGK individuals. Interestingly, individuals with MPC1 and MPC2 deficiency feature mainly neurological abnormalities [57, 58]. Thus, MPCs deficiency can be added to the list of factors which could contribute to the abnormal neurological phenotype depicted in some of the S.S participants. We still do not know if the difference in the frequency of PDHc deficiency in the two S.S types uncovers a specific, not yet defined function, whereby Tim29 differs from AGK. It also remains unclear the extent to which the biochemical differences between S.S-TIMM29 and S.S-AGK individuals uncover novel specific functions of Tim29.

Conclusion

In summary, we report a novel form of S.S caused by a biallelic variant in TIMM29. Its phenotype resembles to large extent the severe infantile fatal form of S.S caused by biallelic variants in the AGK (S.S-AGK). Nevertheless, the present study uncovers several biochemical differences between the two S.S types, including the hyperCPKemia being almost unique for S.S-TIMM29 cohort, the different frequency of MMRCC and PDHc deficiencies among the two S.S types. We propose to designate the S.S associated with TIMM29 homozygous variant as S.S-TIMM29. The precise molecular pathomechanism underlying these differences is yet to be elucidated.