Expanding the phenotypic and genetic spectrum of GTPBP3  deficiency: findings from nine Chinese pedigrees

Expanding the phenotypic and genetic spectrum of GTPBP3 deficiency: findings from nine Chinese pedigrees | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expanding the phenotypic and genetic spectrum of GTPBP3 deficiency: findings from nine Chinese pedigrees Yaojun Xie, Keyi Li, Yang Li, Xiaofei Zeng, Zhehui Chen, Xue Ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4634652/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Dec, 2024 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted 5 You are reading this latest preprint version Abstract Background GTPBP3 catalyzes τm 5 (s 2 ) U biosynthesis at the 34th wobble position of mitochondrial tRNAs, the hypomodification of τm 5 U leads to mitochondrial disease. While twenty-three variants of GTPBP3 have been reported worldwide, the genetic landscape in China remains uncertain. Methods By using whole-exome sequencing, the candidate individuals carrying GTPBP3 variants were screened and identified. Pathogenicity analysis of variants was biochemically verified by patients-derived immortalized lymphocytes and cell models. Results Through whole-exome sequencing, thirteen variants associated with GTPBP3 were identified in nine Chinese pedigrees, with eight of these variants being newly reported. Affected individuals displayed classic neurologic phenotypes and heart complications including developmental delay, seizures, hypotonia, exercise intolerance, and hypertrophic cardiomyopathy. Additionally, they displayed new symptoms such as eye problems like strabismus and heart issues related to valve function. Studies conducted on patient-derived cells provided evidence of reduced levels of GTPBP3 and impairment in mitochondrial energetic biogenesis. Re-expressing GTPBP3 variants in knockout cell lines further defined the pathogenicity of the novel variants. Analysis of the genetic spectrum in the Chinese population highlighted a concentration in exons 4 and 6, with c.689A > C being the prominent hotspot. Conclusion Our findings emphasize the extensive clinical and genetic implications of GTPBP3 -related mitochondrial disorders, particularly within the Chinese population, but further investigations are needed to explore the phenotype-genotype correlation. Mitochondrial diseases oxidative phosphorylation GTPBP3 genetic hotspot τm5(s2)U modification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mitochondria contain over 1,500 proteins, with the majority being encoded by the nuclear genome. The mitochondrial genome encodes 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system and is further transcribed and translated into proteins by the mitochondria’s internal system 1,2 . Initially, mitochondrial DNA (mtDNA) transcriptions produce mitochondrial ribosomal RNAs (mt-rRNAs) and mitochondrial transfer RNAs (mt-tRNAs) required for translation. According to the mt-mRNA template, mature mt-tRNAs are enzymatically catalyzed by aminoacyl tRNA synthase to transport amino acid to the mitochondrial ribosome, facilitating the synthesis of new polypeptide chains 3 . The maturation of mt-tRNA involves endonuclease cleavage, 3 'end addition of CCA, and nucleobase modification. Post-transcriptional nucleobase modification plays a crucial role in preserving the stability and spatial conformation of mt-tRNA molecules, ensuring efficient and accurate decoding 4–6 . Presently, 18 types of nucleobase modifications have been identified on 22 different mt-tRNAs. Distinct modifications at various locations may correspond to diverse physiological outcomes 3 . GTPBP3 is a highly conserved mt-tRNA modifying enzyme that plays a crucial role in the biosynthesis of τm 5 (s 2 ) U, which modifies the 34th nucleobase of mt-tRNALeu (UUR) , mt-tRNA Trp , mt-tRNA Glu , mt-tRNA Gln and mt-tRNA Lys7–9 . This modified nucleobase, also referred to as "wobble base", is essential for limiting the range of wobble base pairs, which helps to maintain an efficient decoding rate 10 . Defects in GTPBP3 often result in combined oxidative phosphorylation deficiency 23 (COXPD23), with patients clinically manifesting a series of symptoms, such as hypotonia, seizures, dyspnea, feeding difficulties, developmental retardation, fatigue, and limited vision. The examination results often suggest myocardial hypertrophy, lactic acidosis, and T2 hypersignal in the bilateral thalamus, basal ganglia, and brain stem 11–13 . Currently, only 21 cases of GTPBP3 deficiency have been reported 11–17 . Despite the collection of over 300 variants of ClinVar ( www.ncbi.nlm.nih.gov/clinvar ), the pathogenicity of the majority of these remains unknown, and there is a lack of clinical information to facilitate accurate clinical diagnosis. Consequently, there is a need to enhance our understanding of the clinical spectrum and genetic spectrum associated with GTPBP3 deficiency. This study involved the recruitment of nine Chinese patients with GTPBP3 defects. We conducted in-depth analyses of clinical information and carried out cytological function experiments to confirm the pathogenicity of the novel variants and broaden the genetic spectrum of GTPBP3 . These findings will contribute to a deeper understanding of the intricate structure and function of the GTPBP3 protein, while also offering valuable insights for clinical diagnosis. Results Clinical presentations Patient 1(P1, F-1 in Fig. 1 A) is a female who was hospitalized at the age of 1-year-9-month-old due to experiencing fever and seizures three times in a single day. Upon physical examination, she exhibited developmental delay and left ankle clonus. Laboratory tests revealed elevated levels of lactate in the blood (16mM). Brain MRI indicated abnormalities in the bilateral dorsal thalamus, cerebellar dentate nucleus, and superior cerebellar peduncle. Echocardiography (ECG) revealed left ventricle enlargement and left ventricular wall thickening, which indicates hypertrophic cardiomyopathy. Genetic analysis revealed a GTPBP3 compound heterozygous mutation: c.689A > C inherited from the father and c.424G > A inherited from the mother. Patient 2 (P2, F-2 in Fig. 1 A) is a female born through full-term natural delivery. She exhibited delayed motor milestones and could not stand on her own at 9 months of age. At 1-year-1-month old, she was hospitalized due to vomiting for 4 consecutive days. Physical examination revealed hypotonia and hyperreflexia in the knee tendons. Blood lactate levels were elevated to 8.58mM. Brain MRI showed abnormal signals in the bilateral thalamus and peduncle. Electroencephalogram (EEG) indicated diffuse delta waves as the predominant slow wave pattern, and echocardiography revealed decreased left ventricular function (ejection fraction (EF) = 54%) and mild regurgitation of the second, tricuspid, and pulmonary valves. Genetic testing confirmed compound heterozygous variants in GTPBP3 : c.934_957del inherited from her father and c.689A > C inherited from her mother. Patient 3 (P3, F-3 in Fig. 1 A) experienced a seizure with coma lasting 2 hours at the age of 3 years and 8 months. Her condition rapidly deteriorated, presenting with unconsciousness, abnormal breathing patterns, decreased oxygen saturation, and impaired liver function accompanied by uncorrectable metabolic acidosis. Laboratory examination revealed a blood glucose level of 27 mM, blood lactate level of 16mM, blood ammonia level of 94µM, alanine aminotransferase of 283.3U/L, aspartate aminotransferase of 716.6 U/L. Brain MRI revealed abnormal signals in the bilateral thalamus, peduncle, and bilateral temporoparietal cortex. EEG showed a slowing of basic waves, and ECG revealed that the left atrium and left ventricle were slightly enlarged, and the left ventricular systolic function was normal (EF = 55%, FS = 28%). Genetic examination revealed compound heterozygous variants in GTPBP3 : c.689A > C (paternally inherited) and c.127C > T (maternally inherited). Patient 4 (P4, F-4 in Fig. 1 A) is a young boy who was admitted to the hospital at the age of 4 years and 8 months due to weakness that had persisted for over 2 years. He experienced fatigue easily, had poor endurance. Upon physical examination, it was noted that his growth and development were borderline normal, and he had astigmatism. Laboratory tests revealed a high-sensitive troponin-I level of 0.026 ng/mL, NT-proBNP of 894pg/ml, CK-MB of 21 U/L, and blood lactate level of 9.18mM. Ultrasonography showed left ventricular enlargement, ventricular wall hypertrophy, and an EF of 38%, consistent with a diagnosis of heart failure with grade III cardiac function. Genetic testing revealed a homozygous variant in GTPBP3 (c.473T > G). Patient 5 (P5, F-5 in Fig. 1 A) is a male infant who presented with rapid, shallow breathing and intermittent moaning starting at 20 hours after birth. His blood lactate level was significantly elevated at 26mM, indicating metabolic acidosis along with respiratory acidosis. Additionally, neonatal disease screening using LC/MS revealed a marked increase in alanine. Genetic examination revealed GTPBP3 compound heterozygous mutation: c.413C > T inherited from her father and c.509_510del inherited from her mother. Patient 6 (P6, F-6 in Fig. 1 A) is a female child born to healthy, unrelated parents. Her sibling passed away at 7 months due to a “brain disease”. Shortly after birth, developmental delays were noted in the child, presenting with symptoms of unsteady running, speech disorder, and limited comprehension. By the age of 2, she experienced sporadic seizures associated with colds and fever once or twice a year. Physical examination revealed increased muscle tone in the limbs, left eye esotropia, and restricted external rotation. Elevated blood lactic acid levels at 6.94 mmol/L were observed, along with abnormal signals in the bilateral thalamus and left cortex on brain MRI. Additionally, the ECG displayed two generalized spikes and slow spikes during sleep. Cardiac ultrasound indicated mild regurgitation in the tricuspid valve, main arteries, and pulmonary arteries. Genetic testing identified a compound heterozygous mutation in GTPBP3 : c.187C > T (paternally inherited) and c.776A > G (maternally inherited). Patient 7 (P7, F-7 in Fig. 1 A) is a 3-year-old girl who was hospitalized due to multiple seizures. Genetic examination revealed GTPBP3 compound heterozygous mutation c.848C > A (paternally inherited) and c.680_691dup (maternally inherited). Patient 8 (P8, F-8 in Fig. 1 A), a male, was hospitalized at the age of 2 due to fever and seizures. He exhibited delayed motor development, weak muscle tone, hypotonia, and fatigue. Elevated blood lactate levels were recorded at 8.3 mM. Brain MRI displayed abnormal hyperintensity signals in the basal ganglia. Genetic examination revealed GTPBP3 compound heterozygous mutation c.689A > C (paternally inherited) and c.774_775insC (maternally inherited). Patient 9 (P9, F-9 in Fig. 1 A) is the third child born to unrelated parents. His sister is normal, and his brother passed away at the age of 1 year and two months of unknown etiology. He presented with language development delay and hypotonia of both lower limbs. Brain MRI revealed suspicious white matter abnormality. Genetic examination revealed GTPBP3 compound heterozygous mutation: c.689A > C (paternally inherited) and c.1092_1103del (maternally inherited). Detailed clinical presentations and other examination results are summarized in Table 1 . Table 1 Clinical features of 9 patients with GTPBP3 deficiency. *LS, Leigh syndrome; MD, mitochondrial disease; NA, not available. Patient P1 P2 P3 P4 P5 P6 P7 P8 P9 Gender female female female male male female female male male Age of onset 1.8 years 1.1 years 3.7 years 4.7 years At bitrh (20h) 7 months 3 years 2 years 1 years Variant c.689A > C; c.424G > A c.689A > C; c.934_957del c.689A > C; c.127C > T c.473T > G, hom c.413C > T; c.509_510del c.187C > T; c.776A > G c.680_691dup; c.848C > A c.774_775insC; c.689A > C c.689A > C; c.1092_1103del Clinical diagnosis LS LS LS MD MD LS LS MD MD Clinical features developmental delay; seizure; fatigue developmental delay; hypotonia; knee tendon hyperreflexes Seizure; fatigue developmental critical state; fatigue; exercise intolerance rapid shallow breathing followed by intermittent moaning developmental delay; epilepsy; hypertonia; knee tendon hyperreflexes seizure developmental delay; seizure; fatigue developmental delay; hypotonia Echocardiography (EF ≧ 50%;25%≦FS ≦ 45%) cardiac hypertrophy (EF = 72%; FS = 40%) low left ventricular function (EF = 54%; FS = 27%) slightly larger atria and ventricles, low left ventricular function (EF = 55%; FS = 28%) ventricular hypertrophy, slightly reduced left ventricular systolic function, mitral regurgitation (EF = 38–60%; FS = 28.5–45%) NA mild regurgitation of tricuspid valve and main and pulmonary valve NA NA NA Brain MRI Bilateral thalamus, cerebellar dentate nucleus and local subcortical brain abnormal signals Abnormal signals in the bilateral thalamus and peduncle Bilateral high signal in thalamus, midbrain and peduncle No obvious change NA Multiple asymmetric signal foci in the bilateral thalamus, brainstem and medulla oblongata NA Basal ganglia lesion Suspected white matter abnormality Plasma lactate level (mM) 16 8.58 16 9.18 NA 5.92 NA 8.3 NA Others NA Abnormal electroencephalogram Abnormal electroencephalogram NA NA Abnormal electroencephalogram NA NA NA Pathogenicity prediction of variants Cross-species amino acid conservative analysis (Fig. 1 B) was carried out among different variants. Except for c.689A > C (p.Q230P), other residues are highly conserved during evolution. The summary of pathogenicity analysis for genetic variants is presented in Table 2 . gnomAD ( http://gnomad.broadinstitute.org ) was an allele frequency annotations database 18 . The variants were either absent or the frequency was extremely low in the population. SIFT ( http://sift-dna.org ) and PolyPhen-2 ( http://genetics.bwh.harvard.eduy/pph2 ) were the typical pathogenicity prediction tools for non-synonymous single nucleotide substitution 19,20 . MutationTaster ( https://www.mutationtaster.org ) works on the DNA level, also suitable for indels. The score of c.413C > T, c.424G > A, c.473T > G, c.776A > G, and c.848C > A in SIFT all greater than 0.05. And the score of c.413C > T, c.424G > A, c.473T > G, c.776A > G and c.848C > A in PolyPhen-2 all greater than 0.909. Almost variants are predicted to be disease-causing in MutationTaster. Moreover, MUpro ( http://mupro.proteomics.ics.uci.edu/ ) was used to predict the change in protein stability 21 . It seems that c.413C > T, c.424G > A, c.473T > G, c.689A > C, c.776A > G, and c.848C > A were more likely to lead to decreased protein stability. Table 2 The annotations of variants. Variant (NM_032620.4) Exon Amino acid change gnomAD SIFT Polyphen-2 MutationTaster MUpro c.127C > T 2 p.Q43* NA NA NA D NA c.187C > T 2 p.R63* NA NA NA D NA c.413C > T 4 p.A138V A 4 p.E142K NA 0.00 1.000 D decreased c.473T > G 4 p.V158G NA 0.05 0.999 D decreased c.509_510del 4 p.E170Gfs*42 C 6 p.Q230P < 1‰ 0.25 0.091 D decreased c.774_775insC 6 p.N259Qfs*28 G 6 p.N259S A 7 p.T283N NA 0.00 0.999 D decreased c.934_957del 7 p.G312_V319del < 1‰ NA NA D NA c.1092_1103del 8 p.D364_R368del NA NA NA D NA * SIFT score: 0.0-0.05 means deleterious and 0.05-1.0 means tolerated; PolyPhen-2 score: 0.0-0.446 is Benign, 0.447–0.908 is possibly damaging and 0.909-1.0 for probably damaging; D-prediction disease causing, P-prediction polymorphism; NA: not found. In summary, a total of thirteen variant sites were involved in nine pedigrees, with five have been reported and eight novel variants. While predictive analyses indicate potential defects in all variants, confirmation through additional biological functional verification is required. Decreased GTPBP protein levels and impaired mitochondrial function were observed in patient-derived immortalized lymphocytes Four patients (P1-P4) and three age-matched healthy children as controls were included in the immortalized lymphocyte experiments. Initial validation of the immortalized lymphocytes was conducted through Sanger sequencing, confirming consistency with the previous genetic examination ( Supplementary Fig. 1A ). Analysis comparing the steady-state GTPBP3 protein levels in patients P1-P4 with the normal control group revealed significant decreases of 82.8% (p < 0.001), 79.1% (p < 0.001), 74.4% (p < 0.001), and 25.5% (p = 0.036) respectively (Fig. 2 A-B). These findings highlight the substantial reduction in GTPBP3 protein levels in patient-derived lymphocytes. As previously mentioned, GTPBP3 is a highly conserved mt-tRNA modifying enzyme essential for the mitochondrial protein translation process 7 . To further elucidate its impact on mitochondrial function, BN-PAGE is a common technique optimized for the analysis of the five complexes (CI-CV) of OXPHOS (Fig. 2 C-F) 22,23 . Compared with the control, the content of CI, CIII, CIV, and CV of P1 was decreased in P1 and P2. Additionally, in P4, the content of complex CIII was decreased, while no significant differences were observed in the abundance of mitochondrial complexes in P3. To further detect the mitochondrial respiratory capacity, the oxygen consumption levels in lymphocytes were measured 24,25 . The basal respiration rate was measured under normal conditions. Oligomycin was used to inhibit ATP synthase, allowing for the calculation of the corresponding OXPHOS-related oxygen consumption rate (OCR). FCCP was used to disrupt the proton gradient and mitochondrial membrane potential, stimulating cells to reach their maximum respiration potential 26 . As a result, basal OCR of P1was decreased by 23.7% (p < 0.001), P2 decreased by 30.2% (p < 0.001), P3 decreased by 20.4% (p < 0.001) and P4 decreased by 14.4% (p = 0.0012). The OCR of oxidative phosphorylation decreased by 57.8% (p < 0.001) in P1, 54.0% (p < 0.001) in P2, 47.4% (p < 0.001) in P3, and 57.4% (p < 0.001) in P4. The maximum respiration potential of P1 was decreased by 31.3% (p < 0.001), P2 was decreased by 29.9% (p < 0.001), P3 was decreased by 20.1% (p = 0.0018) and P4 was decreased by 19.4% (p = 0.0024) (Fig. 2 E). In summary, the OXPHOS function of P1-P4 was impaired to varying degrees. Re-expression of wild-type vectors rescues the deficit in GTPBP3 expression level and OXPHOS complexes To further investigate the impact of GTPBP3 on mitochondrial functions, we utilized CRISPR-Cas9 technology to generate a HEK-293T GTPBP3 knockout (KO) cell model, which was then rescued by re-expressing wild-type GTPBP3. Western blot analysis confirmed the reduced level of GTPBP3 protein in the KO cell model (Fig. 3 A). Additionally, blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed significant decreases in Complexes I, III, IV, and V (Fig. 3 B), consistent with findings in patient-derived lymphocyte models. Subsequent analysis of the re-expressed cells through WB and BN-PAGE demonstrated partial recovery of the observed defects (Fig. 3 C-D). Protein abundance decreased in GTPBP3 site-directed mutagenesis cell model According to instructions of the Standard and guidelines for the interpretation of sequence (2015) published by the American Society for Medical Genetics and Genomics (ACMG) 27 , nonsense mutations, frameshifts, ± 1 or 2 canonical splice sites, initiation codons, and large deletions, all have pathogenic very strong evidence, combined with extremely low population frequency, they can be distributed to Likely pathogenic (LP) at least. Cytological function experiments can provide strong evidence for pathogenicity analysis, which was a milestone significance for VUS variants 28,29 . Re-expressing GTPBP3 carrying mutant vectors of c.127C > T, c.187C > T, c.473T > G, c.776A > G, c.848C > A and mutation hot spot (c.689A > C) vectors in GTPBP3 KO cells, mutations were identified by Sanger sequencing ( Supplementary Fig. 1B ). To eliminate the interference of wild-type GTPBP3 protein, the GTPBP3 vectors carrying different mutations were transfected into KO cell lines. As shown in Fig. 4 A, when compared with KO + GTPBP3 cell line, KO + c.127C > T decreased by 97.8% (p T decreased by 99.6% (p A decreased by 94.8% (p G decreased by 32.9% (p = 0.0064) and decreased by 45.7% (p C. No significant decrease was found in KO + c.776A > G and KO + c.848C > A. It needs to be considered that there are differences in vector copy number among cell lines 30,31 . We designed primers targeting to the 3' end of the CDS and PGK promoter which was a conserved region on vectors to assess the relative level of vectors. As shown in Fig. 4 B. The vector levels of each cell lines were compared with KO + GTPBP3 cell line, KO + c.127C > T was 3.2 times (p T was 2.4 times (p A was 1.8 times (p = 0.0118), KO + c.473T > G was 3.7 times (p C was 2.8 times (p G was 2.5 times (p A was 3.2 times (p G was 4.8 times (p G and KO + c.848C > A can be uncovered (Fig. 4 C). Analysis of genetic variants spectrum and phenotype-genotype correlation of GTPBP3 The initial report by Robert Kopajtich et al in 2014 documented 11 cases of GTPBP3 mutations 11 . To date, in total of 21 cases with 23 distinct variants of GTPBP3 have been reported. By incorporating these reported variants with the 8 novel variants identified in our study, the genetic spectrum of the GTPBP3 gene was expanded (Fig. 5 A). Notably, variants identified in the Chinese population are marked in yellow (Fig. 5 A), we noted that c.689A > G is most common in the Chinese population. Moreover, our analysis revealed that the variant sites are predominantly concentrated in exon 4 and exon 6, with c.689A > C showing high frequencies of 8/51, indicating it as a hot spot mutation site within this population. Based on the protein’s functional domains, the mutations can be roughly categorized into four regions, which are mitochondrial targeting sequence (MTS, M), GTP-box (G), C-terminal (C), and other undetermined (U) regions 11 . Combined with previous cases, the characteristics were summarized as follows (Fig. 5 B-D). Firstly, the onset age for all cases was under 10 years old, with a trend to earlier onset in the M/U region (Fig. 5 B). Secondly, the clinical outcomes of patients in the M/U region more servere, with a higher proportion mortality ratio (Fig. 5 C). In terms of muscle involvement, individuals in the M/G/U region were usually affected by dual involvement. Those in the C region tended to exhibit muscle involvement, along with energy deficiency along with energy deficiency symptoms like fatigue and mild myocardial hypertrophy (limited by the small number of case samples) (Fig. 5 D). Overall, it appears that symptoms in the C region were relatively mild, may due to the smaller sample size. Lastly, all patients exhibited hyperlactatemia, and the majority experienced developmental delay along with muscle and/or nerve involvement (such as cardiac hypertrophy, seizures, hypermyotonia, or hypotonia) and abnormal brain MRI. They may also present with dyspnea, feeding difficulties, short stature, and occasionally visual impairment, as well as cardiac abnormalities. These cardiac abnormalities can include conduction and heart valve issues. Notably, valvular insufficiency was initially linked to GTPBP3 deficiency in our study. Discussion GTPBP3 is a catalytic enzyme involved in the synthesis of τm 5 (s 2 ) U in mitochondria and has been associated with mitochondrial diseases. Since Robert Kopajtich first described the phenotypes associated with 11 cases of GTPBP3 deficiency 11 . At present, there are only several sporadic cases. However, restricted by the small number of cases, the disease-related phenotypes are incomplete, and the mutation spectrum still needs to improve. The correlation between genotype and phenotype remains to be further studied. According to previous studies, changes in oxidative phosphorylation complex enzyme activity of skeletal muscle and fibroblasts, several mitochondrial subunits protein levels as well as mitochondrial oxygen consumption rate under different culture conditions in patient-derived fibroblasts have been presented 11 . However, there are still few studies on patient-derived cell models, and the studies are only for individuals, and the subjects are inconsistent. We constructed patient-derived immortalized lymphocyte cell lines and performed BN-PAGE to preserve their native structure of OXPHOS complexes and subsequently oxygen consumption rates detection. Our results agree with the conclusion that GTPBP3 deficiency led to mitochondrial dysfunction. However, the new phenotypes including heart valve involvement and strabismus were first proposed in this study. The level of residual steady-state GTPBP3 protein seems to be correlated with the degree of mitochondrial impairment, except for P3, which may be due to the following hypotheses: ( 1 ) The produced mitochondrial complex protein is useless in this patient, so the protein level does not change significantly, but the activity of complex enzyme is severely impaired 25 ; ( 2 ) The disadvantage of immortalized lymphocytes is that the phenotype is not obvious 32,33 ; ( 3 ) Cells were cultured in a high-nutrient environment, and excessive reliance on glycolysis can compensate for the deficiency. In other words, stress culture can make the difference obviously 33,34 . In the results of GTPBP3 protein level detection on site-directed mutagenesis cell models, c.776A > G and c.848C > A were inconsistent with the predicted results. The possible reasons were as follows: ( 1 ) The mutations did not affect the protein level but affected the enzyme catalytic activity 25 ; ( 2 ) Different transfection and replication efficiency of plasmids would affect the protein expression level 35,36 . The results showed that the plasmids expression level in all site-mutant cell lines was higher than control. As a result, the pathogenicity of c.776A > G and c.848C > A cannot be ruled out. The reduction of protein level may be due to the decrease in protein production and/or the acceleration of protein degradation 37–39 . Previous studies have shown that the GTPBP3 protein is extensively modified by ubiquitination and degraded through the proteasome pathway 7 . To avoid rapid protein degradation, we first treated the cells with MG-132 for 6h, then inhibited cell protein synthesis by CHX treatment, the degree of degradation of target protein can be observed within 24h 38,40–42 ( Supplementary Fig. 2A ). It was found that compared with the control group, c.127C > T and c.187C > T significantly decreased after 6h of treatment, suggesting that c.127C > T and c.187C > T accelerated the degradation of GTPBP3 protein and reduced the stability of the protein ( Supplementary Fig. 2B ). In summary, we enrolled 9 individuals with GTPBP3 deficiency. We identified 8 novel variants, which were c.127C > T, c.187C > T, c.473T > G, c.680_691dup, c.774_775insC, c.776A > G, c.848C > A and c.1092_1103del, respectively. 7 mutations were screened to construct site-directed mutagenesis cell models and cytological function experiments were performed. It was confirmed that c.127C > T, c.187C > T, c.424G > A, c.473T > G, and c.689A > C were pathogenic mutations. GTPBP3 mutation spectrum was expanded, and it was found that mutations in the Chinese population were mostly concentrated in exon 4 and exon 6, and c.689A > C and c.424G > A were hot spots in the population. This study highlights the important role of mt-tRNA modification defects in mitochondrial diseases and provides a reference for the diagnosis of diseases related to GTPBP3 deficiency, as well as subsequent prenatal diagnosis and genetic counseling. Methods Study participant Patients were born from 9 non-consanguineous families. Patients 5 and 6 underwent evaluation at Xiangya Hospital, Central South University, while other patients were recruited and assessed at Peking University First Hospital. Approval for this study was obtained from the Ethics Committees of both Peking University First Hospital (2017 − 217) and Xiangya Hospital (No. 201605585, 2016/03/20). Moreover, informed consent was obtained from all participants or guardians. Genetic analysis DNA was extracted from peripheral blood samples of probands and their parents. Whole exome sequencing (WES) and mitochondrial genome sequencing were performed using the HiSeq 2000 sequencer (Illumina, USA). Sanger sequencing was then carried out as a follow-up to validate the identified mutations 43,44 . The specific primers used are detailed in Supplementary Table 1 . Immortalized lymphocytes construction As previously outlined 45,46 , mononuclear cells were isolated from the peripheral blood using the lymphocyte separation medium (Solarbio, China). These isolated cells were continuously stimulated by Epstein-Barr virus (EBV). Furthermore, 0.5 mg/mL phytohemagglutinin (Sigma-Aldrich, USA) and 1 mg/mL cyclosporin A (Sigma-Aldrich) were also added to the culture medium. Plasmids construction and transfection As previously described 46,47 , Knockout (KO) plasmids were constructed using the CRISPR/Cas9 technology, and gRNAs were annealed to duplexes and inserted into pX330 vector. As for overexpression (OE) plasmid, GTPBP3 was synthesized by Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) and cloned into lentiviral pLVX vector by ClonExpression® II One Step Cloning Kit (Vazyme), and site-specific mutant vectors were obtained from Tsingke (Tsingke Biotechnology, China). All constructions were confirmed by Sanger sequencing. Transfection was performed with Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. KO cell lines were selected by limiting dilution, and OE cell lines and site-mutant cell lines were generated by infection of the cells with lentiviral particles and puromycin (Sangon Biotech, China) selection. Cell culture Immortalized lymphocytes from patients were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (GIBCO, USA) and 1% penicillin/streptomycin, as well as 50 mg/mL uridine (Sigma-Aldrich). HEK-293T was a gift from Dr. Haihua Gu (Wenzhou Medical University). HEK-293T and other cell models generated from HEK-293T were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, USA) containing 12% calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin. 2 µg/mL puromycin (Sangon, China) was extremely added to cell models generated from HEK-293T. All cells were cultured with 5% CO2 at 37°C in an incubator. Quantitative real-time PCR (qPCR) detection RNA was extracted according to the TRIzol Reagent protocol (Thermo Fisher Scientific) 48 . Complementary DNA (cDNA) was synthesized by PrimeScript RT reagent Kit (Takara Biotechnology, Japan). For quantification transcripts expression level, qPCR was performed with 2xChamQ SYBR qPCR Master Mix (Vazyme, China). Primers are provided in Supplementary Table 1 . Immunoblotting For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a total protein isolated from whole cell using RIPA lysis buffer (Cell Signaling Technology, USA) with 1 mM phenylmethylsulfonyl fluoride (PMSF, Sangon Biotech, China). For Blue native polyacrylamide gel electrophoresis (BN-PAGE), as previously described 22 , mitochondrial membrane protein was extracted from a whole cell using 2% Triton-X100 (Sigma-Aldrich) and subsequently separated by a 3.5%-16% gradient gel. Proteins were electroblotted onto 0.22 um PVDF membranes (Bio-rad, USA) and blocked with 5% milk powder solution, incubated with primary and secondary horseradish peroxidase-conjugated antibodies ( Supplementary Table 2 ). Signals detected with clarity ECL western blotting (WB) substrate (Bio-Rad). Mitochondrial respiration measurement The detection of oxygen consumption rate (OCR) was conducted as described before 44,49 . Concisely, about 5 × 10 6 immortalized lymphocytes were harvested and added to the chamber of Oxygraph-2k (Oroboros, Austria). The respiration was recorded under normal conditions and with subsequent injection with inhibitors, including oligomycin (0.1mM, Sigma-Aldrich) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.1mM, Sigma-Aldrich). Cycloheximide (CHX) Chase Assay Cells were seeded on 24-well cell culture plates (10000 cells per well). Once the confluence reached 90%, cells were pretreated with a complete medium containing 10uM MG132 for 6 h. After washing cells twice with PBS, the culture medium was converted to a complete medium containing 20uM cycloheximide (CHX). Samples were harvested at 0, 3, 6, 12, and 24 h after CHX treatment for SDS-PAGE detection. Statistical analysis All quantitative data were performed for three or more times, independently. Statistical analysis and graphs were plotted using Prism 8.4.0. The results were shown with mean ± SD. When the data conforms to a normal distribution, independent double-tailed student t was used. If not, the Mann-Whitney U test is used. Three or more groups of data were compared using one-way analysis of variance (ANOVA), p < 0.05 indicates statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001. Declarations Contributors Statement: Xiaoting Lou, Yanling Yang, and Liqin Jin conceptualized the study, developed the experimental designs, interpreted the data, and revised the manuscript. Yaojun Xie designed and conducted the experiments, analyzed the data, and drafted the manuscript. Keyi Li, Li Yang, Luyi Zhang, Xiaofei Zeng, and Yuwei Zhou conducted the experiments. Zhehui Chen and Xue Ma coordinated the clinical assessment of the patients and their families. All authors provided final approval and consented to take responsibility for all aspects of the research. Ethical Approval This ethical approval was obtained from the Ethics Committee of Peking University First Hospital (Ethics approval number: 2017 − 217), the Ethics Committee of Xiangya Hospital of Central South University, China (Ethics approval number: 201605585) Informed consent Consent was obtained from the participants’ guardians to publish this document. Conflict of interest The authors have declared that no conflict of interest exists. Funding This work was supported by grants from the National Natural Science Foundation of China (82302618, X.L. 82072366, L.J.), the National Key Research and Development Program of China (2021YFC2700900, 2017YFC1001700, Y.Y.), China Postdoctoral Science Foundation (2023M743124, X.L.), the Key Discipline of Zhejiang Province in Medical Technology (First Class, Category A), Wenzhou Medical University, and the Key Discipline of Zhejiang Province in Public Health and Preventive Medicine (First Class, Category A), Hangzhou Medical College. Acknowledgments We express our gratitude to the patients and their families for their participation in this study. Additionally, we extend our thanks for the generous gift of HEK293T cells from Dr. Haihua Gu at Wenzhou Medical University. Data availability The datasets generated and/or analyzed during the current study are not publicly accessible due to the confidentiality and ethical considerations associated with patient data. 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A membrane arm of mitochondrial complex I sufficient to promote respirasome formation. Cell Rep 35 , 108963 (2021). https://doi.org:10.1016/j.celrep.2021.108963 Supplementary Files Fig.2aGTPBP3.tiff Fig.2aTOM70.tiff Fig.2a.tiff Fig.2cCICII.tiff Fig.2cCIIICIVCV.tiff Fig.2cTOM70.tiff Fig.2dCI.tiff Fig.2dCIICIIICIVCV.tiff Fig.2dTOM70.jpeg Fig.2eCI.tiff Fig.2eCIICIIICVTOM70.jpeg Fig.2eCIV.tiff Fig.3aGTPBP3.tiff Fig.3aTOM70.tiff Fig.3bCICIICIIICIVCV.tiff Fig.3bTOM70.tiff Fig.3cGTPBP3.tiff Fig.3cTOM70WB.tiff Fig.3dCICIII.tiff Fig.3dCIVCIICV.tiff Fig.3dTOM70BNG.tiff Fig.4aGTPBP3TOM70.tiff Fig3bsupple1.tiff Fig3bsupple2.tiff SFig.2asupple1.tiff SFig.2asupple2.tiff Supplementaryfigures.pdf Cite Share Download PDF Status: Published Journal Publication published 24 Dec, 2024 Read the published version in Orphanet Journal of Rare Diseases → Version 1 posted Editorial decision: Accept 19 Nov, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers invited by journal 06 Aug, 2024 Editor assigned by journal 30 Jun, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4634652","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336843877,"identity":"1614c225-c67b-4b1a-abf6-d8fa9a80e679","order_by":0,"name":"Yaojun Xie","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yaojun","middleName":"","lastName":"Xie","suffix":""},{"id":336843878,"identity":"d51abf6a-731d-4993-b068-4bcb8ddd8d86","order_by":1,"name":"Keyi Li","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keyi","middleName":"","lastName":"Li","suffix":""},{"id":336843879,"identity":"a63a5260-b3f1-4e22-996e-a82ac4275655","order_by":2,"name":"Yang Li","email":"","orcid":"","institution":"Xiangya Hospital Central South University Department of Pediatrics","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Li","suffix":""},{"id":336843880,"identity":"139538a3-a42a-46b3-83a6-80e4cdafc3a8","order_by":3,"name":"Xiaofei Zeng","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofei","middleName":"","lastName":"Zeng","suffix":""},{"id":336843881,"identity":"18b835c5-bae9-4093-9899-0f361ed12ea3","order_by":4,"name":"Zhehui Chen","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhehui","middleName":"","lastName":"Chen","suffix":""},{"id":336843882,"identity":"d492628e-1c17-429c-bb54-2b8804ba26de","order_by":5,"name":"Xue Ma","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Ma","suffix":""},{"id":336843883,"identity":"218981c7-5406-44bc-b2e9-4be846406eb9","order_by":6,"name":"Luyi Zhang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Luyi","middleName":"","lastName":"Zhang","suffix":""},{"id":336843884,"identity":"2d3b1e06-a121-4185-9b8b-afccc4b786f1","order_by":7,"name":"Yuwei Zhou","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Zhou","suffix":""},{"id":336843885,"identity":"28408ad6-dbfc-4f6c-8be1-45189c21e4b5","order_by":8,"name":"Liqin Jin","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liqin","middleName":"","lastName":"Jin","suffix":""},{"id":336843886,"identity":"80095ba1-026a-4e79-a941-e1d3c15be5e1","order_by":9,"name":"Yanlin Yang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanlin","middleName":"","lastName":"Yang","suffix":""},{"id":336843887,"identity":"70d949d4-d5cb-489b-809e-067793337769","order_by":10,"name":"Xiaoting Lou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie2PPQrCQBBGJwzsNsG0ipcYEIJCSK6iLGxlIdhYBtJ6AI+hJ3B1URvB1sJCEaxTiYWFG3+wS7YU3AczTPE9ZgbA4fhFsGhkipvKKYrtFEWvwZsMpLDbpN4u+vnSS6vStMHTOR8c4hRx3YxIIXC9mpYpjYy1SNFFpMhks0+HGvhS7suUACGsK9LCXBUa5YJQ98NShSG/fpU2aS+tUgKTLJT4qYCN0sj8IW1JdwGZ6IxJClb1C+02s+PorhPg2WJ/u0dxwPW6VPnQm6vPdzbxgsQ26HA4HH/IAx2DQNOzoMlAAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiaoting","middleName":"","lastName":"Lou","suffix":""}],"badges":[],"createdAt":"2024-06-25 08:09:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4634652/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4634652/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13023-024-03469-3","type":"published","date":"2024-12-24T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64308502,"identity":"0687798d-5580-411b-897e-e44b1bb8a46b","added_by":"auto","created_at":"2024-09-11 13:06:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":309720,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePedigree diagram of patients and amino acid conservative analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Pedigree map of family 1-9. The black arrow indicates the proband, the box indicates the male, the circle indicates the female, the black solid shape indicates the patient, the black slash indicates the death of unknown cause, and the red font indicates the newly discovered variants of \u003cem\u003eGTPBP3. \u003c/em\u003e(B)\u003cem\u003e \u003c/em\u003eThe amino acid conservation analysis of variants across species.\u003c/p\u003e","description":"","filename":"Figures1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/6f82554d4ed06834b8a6822f.png"},{"id":64309417,"identity":"1021fa11-f6d8-466f-a756-512af62f1b76","added_by":"auto","created_at":"2024-09-11 13:14:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115593,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of GTPBP3 amount in patient-derived immortalized lymphocytes. Analysis of mitochondrial complex content and OCR in patient-derived immortalized lymphocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) WB analysis of GTPBP3 in immortalized lymphocytes of normal controls and patients. (B) Quantitative analysis of GTPBP3 abundance in immortalized lymphocytes of normal controls and patients (Fig.2A). TOM70 was used as an internal control (n=3). The asterisk indicates the target strip. Data are presented as the means ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003cstrong\u003e \u003c/strong\u003e(C-F) Abundance of OXPHOS complexes in immortalized lymphocytes of normal controls and patients. TOM70 was used as a loading control. (G) Oxygen consumption rate analysis in immortalized lymphocytes of normal controls and patients. Basal indicates basal respiration, Basal-Oligo indicates ATP-link OCR, FCCP indicates maximal respiration. The absolute OCR was normalized against the cell number (n=4). Data are presented as the means ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures2.png","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/d56a375749cfe887bf04cd16.png"},{"id":64309419,"identity":"dfadc4fa-e779-4f86-83b7-90100e38bb82","added_by":"auto","created_at":"2024-09-11 13:14:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of HEK-293T \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGTPBP3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e KO and re-expression cell model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The abundance of GTPBP3 in HEK-293T of KO and control (Ctrl) cells. TOM70 was used as a loading control. (B) The BN-PAGE analysis of GTPBP3 in HEK-293T of KO and Ctrl. TOM70 was used as a loading control.\u003cstrong\u003e \u003c/strong\u003e(C) The abundance of GTPBP3 in HEK-293T of KO, KO transfected with \u003cem\u003eGTPBP3\u003c/em\u003e and Ctrl. TOM70 was used as a loading control. (D) The BN-PAGE analysis of GTPBP3 in HEK-293T of KO, transfected with \u003cem\u003eGTPBP3\u003c/em\u003eand Ctrl. TOM70 was used as a loading control.\u003c/p\u003e","description":"","filename":"Figures3.png","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/cf8f67a8a4735fa4b4132aa3.png"},{"id":64308506,"identity":"2d49ac62-54f8-4b94-8873-827b8c784022","added_by":"auto","created_at":"2024-09-11 13:06:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGTPBP3 protein expression level analysis of HEK-293T cell lines carrying different vatiants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The abundance of GTPBP3 in HEK-293T with KO, KO transfected with \u003cem\u003eGTPBP3\u003c/em\u003e, KO transfected with \u003cem\u003eGTPBP3\u003c/em\u003e carrying different variants (sites can be seen in the panel). TOM70 was used as a loading control. (B) Relative plasmids copy number levels in HEK-293T with KO transfected with \u003cem\u003eGTPBP3 \u003c/em\u003eand KO transfected with \u003cem\u003eGTPBP3\u003c/em\u003e carrying different variants. β-Actin was used as an internal control (n = 3). (C) Quantitative results of relative abundance were corrected by relative plasmid copy number levels. Data are presented as the means ± SEM (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figures4.png","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/f8ee250fffa153c879659080.png"},{"id":64310745,"identity":"413bae21-168b-42ce-8670-e1b23cb4419f","added_by":"auto","created_at":"2024-09-11 13:30:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMutations spectrum of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGTPBP3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand phenotype-genotype correlation analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Updated mutation map of \u003cem\u003eGTPBP3. \u003c/em\u003eThe orange font represents the mutations found in Chinese population, and the gray font represents the mutations reported in other populations; The number in the circle indicates the frequency of occurrence, and the unmarked frequency is 1; The asterisk indicates the hot spots of the population; The blue box indicates the exon, and the number in the blue box indicates the number of exon; Purple, yellow, and green boxes represent different functional domains of the corresponding proteins, respectively. (B) The age of onset distribution of patients deficit in \u003cem\u003eGTPBP3\u003c/em\u003e. (C) The percentage of death outcomes of patients deficit in \u003cem\u003eGTPBP3\u003c/em\u003e. (D) The distribution of patients presented with nerve and/or muscle systems.\u003c/p\u003e","description":"","filename":"Figures5.png","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/f5ce67f6b8ea29939a2b44d9.png"},{"id":72641789,"identity":"0f28a647-9ff3-4fab-824f-e5545441e67d","added_by":"auto","created_at":"2024-12-30 16:17:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1975456,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/f5005a69-bb0a-4d8e-b576-74cc4fd366e7.pdf"},{"id":64309420,"identity":"3ee1efc5-140c-47e9-9d11-7a71db4de821","added_by":"auto","created_at":"2024-09-11 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13:06:57","extension":"tiff","order_by":31,"title":"","display":"","copyAsset":false,"role":"supplement","size":372966,"visible":true,"origin":"","legend":"","description":"","filename":"SFig.2asupple1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/7f0a232831a767f0c29e0fd6.tiff"},{"id":64308525,"identity":"07ed52f6-594f-44bb-9cbe-c754d657052b","added_by":"auto","created_at":"2024-09-11 13:06:56","extension":"tiff","order_by":32,"title":"","display":"","copyAsset":false,"role":"supplement","size":254798,"visible":true,"origin":"","legend":"","description":"","filename":"SFig.2asupple2.tiff","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/62975c50bed145c1dd35056c.tiff"},{"id":64308535,"identity":"99906a9b-d2b6-43b8-9e7c-b66847af7880","added_by":"auto","created_at":"2024-09-11 13:06:58","extension":"pdf","order_by":33,"title":"","display":"","copyAsset":false,"role":"supplement","size":494736,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634652/v1/e3b9e8e6faca499d27c3c64d.pdf"}],"financialInterests":"","formattedTitle":"Expanding the phenotypic and genetic spectrum of GTPBP3 deficiency: findings from nine Chinese pedigrees","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMitochondria contain over 1,500 proteins, with the majority being encoded by the nuclear genome. The mitochondrial genome encodes 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system and is further transcribed and translated into proteins by the mitochondria\u0026rsquo;s internal system\u003csup\u003e1,2\u003c/sup\u003e. Initially, mitochondrial DNA (mtDNA) transcriptions produce mitochondrial ribosomal RNAs (mt-rRNAs) and mitochondrial transfer RNAs (mt-tRNAs) required for translation. According to the mt-mRNA template, mature mt-tRNAs are enzymatically catalyzed by aminoacyl tRNA synthase to transport amino acid to the mitochondrial ribosome, facilitating the synthesis of new polypeptide chains\u003csup\u003e3\u003c/sup\u003e. The maturation of mt-tRNA involves endonuclease cleavage, 3 'end addition of CCA, and nucleobase modification. Post-transcriptional nucleobase modification plays a crucial role in preserving the stability and spatial conformation of mt-tRNA molecules, ensuring efficient and accurate decoding\u003csup\u003e4\u0026ndash;6\u003c/sup\u003e. Presently, 18 types of nucleobase modifications have been identified on 22 different mt-tRNAs. Distinct modifications at various locations may correspond to diverse physiological outcomes\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGTPBP3 is a highly conserved mt-tRNA modifying enzyme that plays a crucial role in the biosynthesis of τm\u003csup\u003e5\u003c/sup\u003e (s\u003csup\u003e2\u003c/sup\u003e) U, which modifies the 34th nucleobase of mt-tRNALeu\u003csup\u003e(UUR)\u003c/sup\u003e, mt-tRNA\u003csup\u003eTrp\u003c/sup\u003e, mt-tRNA\u003csup\u003eGlu\u003c/sup\u003e, mt-tRNA\u003csup\u003eGln\u003c/sup\u003e and mt-tRNA\u003csup\u003eLys7\u0026ndash;9\u003c/sup\u003e. This modified nucleobase, also referred to as \"wobble base\", is essential for limiting the range of wobble base pairs, which helps to maintain an efficient decoding rate\u003csup\u003e10\u003c/sup\u003e. Defects in \u003cem\u003eGTPBP3\u003c/em\u003e often result in combined oxidative phosphorylation deficiency 23 (COXPD23), with patients clinically manifesting a series of symptoms, such as hypotonia, seizures, dyspnea, feeding difficulties, developmental retardation, fatigue, and limited vision. The examination results often suggest myocardial hypertrophy, lactic acidosis, and T2 hypersignal in the bilateral thalamus, basal ganglia, and brain stem\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e. Currently, only 21 cases of \u003cem\u003eGTPBP3\u003c/em\u003e deficiency have been reported\u003csup\u003e11\u0026ndash;17\u003c/sup\u003e. Despite the collection of over 300 variants of ClinVar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.ncbi.nlm.nih.gov/clinvar\" target=\"_blank\"\u003ewww.ncbi.nlm.nih.gov/clinvar\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/clinvar\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the pathogenicity of the majority of these remains unknown, and there is a lack of clinical information to facilitate accurate clinical diagnosis. Consequently, there is a need to enhance our understanding of the clinical spectrum and genetic spectrum associated with \u003cem\u003eGTPBP3\u003c/em\u003e deficiency.\u003c/p\u003e \u003cp\u003eThis study involved the recruitment of nine Chinese patients with \u003cem\u003eGTPBP3\u003c/em\u003e defects. We conducted in-depth analyses of clinical information and carried out cytological function experiments to confirm the pathogenicity of the novel variants and broaden the genetic spectrum of \u003cem\u003eGTPBP3\u003c/em\u003e. These findings will contribute to a deeper understanding of the intricate structure and function of the GTPBP3 protein, while also offering valuable insights for clinical diagnosis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical presentations\u003c/h2\u003e \u003cp\u003ePatient 1(P1, F-1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a female who was hospitalized at the age of 1-year-9-month-old due to experiencing fever and seizures three times in a single day. Upon physical examination, she exhibited developmental delay and left ankle clonus. Laboratory tests revealed elevated levels of lactate in the blood (16mM). Brain MRI indicated abnormalities in the bilateral dorsal thalamus, cerebellar dentate nucleus, and superior cerebellar peduncle. Echocardiography (ECG) revealed left ventricle enlargement and left ventricular wall thickening, which indicates hypertrophic cardiomyopathy. Genetic analysis revealed a \u003cem\u003eGTPBP3\u003c/em\u003e compound heterozygous mutation: c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C inherited from the father and c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A inherited from the mother.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePatient 2 (P2, F-2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a female born through full-term natural delivery. She exhibited delayed motor milestones and could not stand on her own at 9 months of age. At 1-year-1-month old, she was hospitalized due to vomiting for 4 consecutive days. Physical examination revealed hypotonia and hyperreflexia in the knee tendons. Blood lactate levels were elevated to 8.58mM. Brain MRI showed abnormal signals in the bilateral thalamus and peduncle. Electroencephalogram (EEG) indicated diffuse delta waves as the predominant slow wave pattern, and echocardiography revealed decreased left ventricular function (ejection fraction (EF)\u0026thinsp;=\u0026thinsp;54%) and mild regurgitation of the second, tricuspid, and pulmonary valves. Genetic testing confirmed compound heterozygous variants in \u003cem\u003eGTPBP3\u003c/em\u003e: c.934_957del inherited from her father and c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C inherited from her mother.\u003c/p\u003e \u003cp\u003ePatient 3 (P3, F-3 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) experienced a seizure with coma lasting 2 hours at the age of 3 years and 8 months. Her condition rapidly deteriorated, presenting with unconsciousness, abnormal breathing patterns, decreased oxygen saturation, and impaired liver function accompanied by uncorrectable metabolic acidosis. Laboratory examination revealed a blood glucose level of 27 mM, blood lactate level of 16mM, blood ammonia level of 94\u0026micro;M, alanine aminotransferase of 283.3U/L, aspartate aminotransferase of 716.6 U/L. Brain MRI revealed abnormal signals in the bilateral thalamus, peduncle, and bilateral temporoparietal cortex. EEG showed a slowing of basic waves, and ECG revealed that the left atrium and left ventricle were slightly enlarged, and the left ventricular systolic function was normal (EF\u0026thinsp;=\u0026thinsp;55%, FS\u0026thinsp;=\u0026thinsp;28%). Genetic examination revealed compound heterozygous variants in \u003cem\u003eGTPBP3\u003c/em\u003e: c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C (paternally inherited) and c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T (maternally inherited).\u003c/p\u003e \u003cp\u003ePatient 4 (P4, F-4 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a young boy who was admitted to the hospital at the age of 4 years and 8 months due to weakness that had persisted for over 2 years. He experienced fatigue easily, had poor endurance. Upon physical examination, it was noted that his growth and development were borderline normal, and he had astigmatism. Laboratory tests revealed a high-sensitive troponin-I level of 0.026 ng/mL, NT-proBNP of 894pg/ml, CK-MB of 21 U/L, and blood lactate level of 9.18mM. Ultrasonography showed left ventricular enlargement, ventricular wall hypertrophy, and an EF of 38%, consistent with a diagnosis of heart failure with grade III cardiac function. Genetic testing revealed a homozygous variant in \u003cem\u003eGTPBP3\u003c/em\u003e (c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G).\u003c/p\u003e \u003cp\u003ePatient 5 (P5, F-5 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a male infant who presented with rapid, shallow breathing and intermittent moaning starting at 20 hours after birth. His blood lactate level was significantly elevated at 26mM, indicating metabolic acidosis along with respiratory acidosis. Additionally, neonatal disease screening using LC/MS revealed a marked increase in alanine. Genetic examination revealed \u003cem\u003eGTPBP3\u003c/em\u003e compound heterozygous mutation: c.413C\u0026thinsp;\u0026gt;\u0026thinsp;T inherited from her father and c.509_510del inherited from her mother.\u003c/p\u003e \u003cp\u003ePatient 6 (P6, F-6 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a female child born to healthy, unrelated parents. Her sibling passed away at 7 months due to a \u0026ldquo;brain disease\u0026rdquo;. Shortly after birth, developmental delays were noted in the child, presenting with symptoms of unsteady running, speech disorder, and limited comprehension. By the age of 2, she experienced sporadic seizures associated with colds and fever once or twice a year. Physical examination revealed increased muscle tone in the limbs, left eye esotropia, and restricted external rotation. Elevated blood lactic acid levels at 6.94 mmol/L were observed, along with abnormal signals in the bilateral thalamus and left cortex on brain MRI. Additionally, the ECG displayed two generalized spikes and slow spikes during sleep. Cardiac ultrasound indicated mild regurgitation in the tricuspid valve, main arteries, and pulmonary arteries. Genetic testing identified a compound heterozygous mutation in \u003cem\u003eGTPBP3\u003c/em\u003e: c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T (paternally inherited) and c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G (maternally inherited).\u003c/p\u003e \u003cp\u003ePatient 7 (P7, F-7 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is a 3-year-old girl who was hospitalized due to multiple seizures. Genetic examination revealed \u003cem\u003eGTPBP3\u003c/em\u003e compound heterozygous mutation c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A (paternally inherited) and c.680_691dup (maternally inherited).\u003c/p\u003e \u003cp\u003ePatient 8 (P8, F-8 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), a male, was hospitalized at the age of 2 due to fever and seizures. He exhibited delayed motor development, weak muscle tone, hypotonia, and fatigue. Elevated blood lactate levels were recorded at 8.3 mM. Brain MRI displayed abnormal hyperintensity signals in the basal ganglia. Genetic examination revealed \u003cem\u003eGTPBP3\u003c/em\u003e compound heterozygous mutation c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C (paternally inherited) and c.774_775insC (maternally inherited).\u003c/p\u003e \u003cp\u003ePatient 9 (P9, F-9 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is the third child born to unrelated parents. His sister is normal, and his brother passed away at the age of 1 year and two months of unknown etiology. He presented with language development delay and hypotonia of both lower limbs. Brain MRI revealed suspicious white matter abnormality. Genetic examination revealed \u003cem\u003eGTPBP3\u003c/em\u003e compound heterozygous mutation: c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C (paternally inherited) and c.1092_1103del (maternally inherited).\u003c/p\u003e \u003cp\u003eDetailed clinical presentations and other examination results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eClinical features of 9 patients with\u003c/b\u003e \u003cb\u003eGTPBP3\u003c/b\u003e \u003cb\u003edeficiency.\u003c/b\u003e *LS, Leigh syndrome; MD, mitochondrial disease; NA, not available.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP9\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge of onset\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.7 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAt bitrh (20h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1 years\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVariant\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec.689A\u0026thinsp;\u0026gt;\u0026thinsp;C; c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ec.689A\u0026thinsp;\u0026gt;\u0026thinsp;C; c.934_957del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ec.689A\u0026thinsp;\u0026gt;\u0026thinsp;C; c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ec.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, hom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ec.413C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.509_510del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ec.187C\u0026thinsp;\u0026gt;\u0026thinsp;T; c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ec.680_691dup; c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ec.774_775insC; c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003ec.689A\u0026thinsp;\u0026gt;\u0026thinsp;C; c.1092_1103del\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical diagnosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eLS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eMD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eClinical features\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edevelopmental delay;\u003c/p\u003e \u003cp\u003eseizure;\u003c/p\u003e \u003cp\u003efatigue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edevelopmental delay;\u003c/p\u003e \u003cp\u003ehypotonia;\u003c/p\u003e \u003cp\u003eknee tendon hyperreflexes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSeizure;\u003c/p\u003e \u003cp\u003efatigue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003edevelopmental critical state; fatigue; exercise intolerance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003erapid shallow breathing followed by intermittent moaning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edevelopmental delay;\u003c/p\u003e \u003cp\u003eepilepsy;\u003c/p\u003e \u003cp\u003ehypertonia; knee tendon hyperreflexes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eseizure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003edevelopmental delay;\u003c/p\u003e \u003cp\u003eseizure; fatigue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003edevelopmental delay;\u003c/p\u003e \u003cp\u003ehypotonia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEchocardiography (EF\u0026thinsp;≧\u0026thinsp;50%;25%≦FS\u0026thinsp;≦\u0026thinsp;45%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecardiac hypertrophy (EF\u0026thinsp;=\u0026thinsp;72%; FS\u0026thinsp;=\u0026thinsp;40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elow left ventricular function (EF\u0026thinsp;=\u0026thinsp;54%; FS\u0026thinsp;=\u0026thinsp;27%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eslightly larger atria and ventricles, low left ventricular function (EF\u0026thinsp;=\u0026thinsp;55%; FS\u0026thinsp;=\u0026thinsp;28%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eventricular hypertrophy, slightly reduced left ventricular systolic function, mitral regurgitation (EF\u0026thinsp;=\u0026thinsp;38\u0026ndash;60%;\u003c/p\u003e \u003cp\u003eFS\u0026thinsp;=\u0026thinsp;28.5\u0026ndash;45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emild regurgitation of tricuspid valve and main and pulmonary valve\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBrain MRI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBilateral thalamus, cerebellar dentate nucleus and local subcortical brain abnormal signals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbnormal signals in the bilateral thalamus and peduncle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBilateral high signal in thalamus, midbrain and peduncle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo obvious change\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMultiple asymmetric signal foci in the bilateral thalamus, brainstem and medulla oblongata\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eBasal ganglia lesion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSuspected white matter abnormality\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePlasma lactate level (mM)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOthers\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbnormal electroencephalogram\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbnormal electroencephalogram\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAbnormal electroencephalogram\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity prediction of variants\u003c/h2\u003e \u003cp\u003eCross-species amino acid conservative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) was carried out among different variants. Except for c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Q230P), other residues are highly conserved during evolution. The summary of pathogenicity analysis for genetic variants is presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. gnomAD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gnomad.broadinstitute.org\u003c/span\u003e\u003cspan address=\"http://gnomad.broadinstitute.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was an allele frequency annotations database\u003csup\u003e18\u003c/sup\u003e. The variants were either absent or the frequency was extremely low in the population. SIFT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sift-dna.org\u003c/span\u003e\u003cspan address=\"http://sift-dna.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and PolyPhen-2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genetics.bwh.harvard.eduy/pph2\u003c/span\u003e\u003cspan address=\"http://genetics.bwh.harvard.eduy/pph2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were the typical pathogenicity prediction tools for non-synonymous single nucleotide substitution\u003csup\u003e19,20\u003c/sup\u003e. MutationTaster (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mutationtaster.org\u003c/span\u003e\u003cspan address=\"https://www.mutationtaster.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) works on the DNA level, also suitable for indels. The score of c.413C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G, and c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A in SIFT all greater than 0.05. And the score of c.413C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G and c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A in PolyPhen-2 all greater than 0.909. Almost variants are predicted to be disease-causing in MutationTaster. Moreover, MUpro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mupro.proteomics.ics.uci.edu/\u003c/span\u003e\u003cspan address=\"http://mupro.proteomics.ics.uci.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the change in protein stability\u003csup\u003e21\u003c/sup\u003e. It seems that c.413C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G, and c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A were more likely to lead to decreased protein stability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe annotations of variants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariant\u003c/p\u003e \u003cp\u003e(NM_032620.4)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmino acid change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003egnomAD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSIFT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePolyphen-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMutationTaster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMUpro\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.127C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.Q43*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.187C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.R63*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.413C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.A138V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.424G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.E142K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.473T\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.V158G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.509_510del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.E170Gfs*42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.680_691dup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.Q230_V231insGALQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.689A\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.Q230P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.774_775insC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.N259Qfs*28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.776A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.N259S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.848C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.T283N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edecreased\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.934_957del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.G312_V319del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u0026permil;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ec.1092_1103del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep.D364_R368del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003cb\u003e*\u003c/b\u003eSIFT score: 0.0-0.05 means deleterious and 0.05-1.0 means tolerated; PolyPhen-2 score: 0.0-0.446 is Benign, 0.447\u0026ndash;0.908 is possibly damaging and 0.909-1.0 for probably damaging; D-prediction disease causing, P-prediction polymorphism; NA: not found.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn summary, a total of thirteen variant sites were involved in nine pedigrees, with five have been reported and eight novel variants. While predictive analyses indicate potential defects in all variants, confirmation through additional biological functional verification is required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDecreased GTPBP protein levels and impaired mitochondrial function were observed in patient-derived immortalized lymphocytes\u003c/h2\u003e \u003cp\u003eFour patients (P1-P4) and three age-matched healthy children as controls were included in the immortalized lymphocyte experiments. Initial validation of the immortalized lymphocytes was conducted through Sanger sequencing, confirming consistency with the previous genetic examination (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). Analysis comparing the steady-state GTPBP3 protein levels in patients P1-P4 with the normal control group revealed significant decreases of 82.8% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 79.1% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 74.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 25.5% (p\u0026thinsp;=\u0026thinsp;0.036) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). These findings highlight the substantial reduction in GTPBP3 protein levels in patient-derived lymphocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs previously mentioned, GTPBP3 is a highly conserved mt-tRNA modifying enzyme essential for the mitochondrial protein translation process\u003csup\u003e7\u003c/sup\u003e. To further elucidate its impact on mitochondrial function, BN-PAGE is a common technique optimized for the analysis of the five complexes (CI-CV) of OXPHOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-F)\u003csup\u003e22,23\u003c/sup\u003e. Compared with the control, the content of CI, CIII, CIV, and CV of P1 was decreased in P1 and P2. Additionally, in P4, the content of complex CIII was decreased, while no significant differences were observed in the abundance of mitochondrial complexes in P3. To further detect the mitochondrial respiratory capacity, the oxygen consumption levels in lymphocytes were measured\u003csup\u003e24,25\u003c/sup\u003e. The basal respiration rate was measured under normal conditions. Oligomycin was used to inhibit ATP synthase, allowing for the calculation of the corresponding OXPHOS-related oxygen consumption rate (OCR). FCCP was used to disrupt the proton gradient and mitochondrial membrane potential, stimulating cells to reach their maximum respiration potential\u003csup\u003e26\u003c/sup\u003e. As a result, basal OCR of P1was decreased by 23.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), P2 decreased by 30.2% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), P3 decreased by 20.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and P4 decreased by 14.4% (p\u0026thinsp;=\u0026thinsp;0.0012). The OCR of oxidative phosphorylation decreased by 57.8% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in P1, 54.0% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in P2, 47.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in P3, and 57.4% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in P4. The maximum respiration potential of P1 was decreased by 31.3% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), P2 was decreased by 29.9% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), P3 was decreased by 20.1% (p\u0026thinsp;=\u0026thinsp;0.0018) and P4 was decreased by 19.4% (p\u0026thinsp;=\u0026thinsp;0.0024) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In summary, the OXPHOS function of P1-P4 was impaired to varying degrees.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRe-expression of wild-type vectors rescues the deficit in GTPBP3 expression level and OXPHOS complexes\u003c/h2\u003e \u003cp\u003eTo further investigate the impact of GTPBP3 on mitochondrial functions, we utilized CRISPR-Cas9 technology to generate a HEK-293T GTPBP3 knockout (KO) cell model, which was then rescued by re-expressing wild-type GTPBP3. Western blot analysis confirmed the reduced level of GTPBP3 protein in the KO cell model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed significant decreases in Complexes I, III, IV, and V (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), consistent with findings in patient-derived lymphocyte models. Subsequent analysis of the re-expressed cells through WB and BN-PAGE demonstrated partial recovery of the observed defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein abundance decreased in\u003c/b\u003e \u003cb\u003eGTPBP3\u003c/b\u003e \u003cb\u003esite-directed mutagenesis cell model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to instructions of the Standard and guidelines for the interpretation of sequence (2015) published by the American Society for Medical Genetics and Genomics (ACMG)\u003csup\u003e27\u003c/sup\u003e, nonsense mutations, frameshifts, \u0026plusmn;\u0026thinsp;1 or 2 canonical splice sites, initiation codons, and large deletions, all have pathogenic very strong evidence, combined with extremely low population frequency, they can be distributed to Likely pathogenic (LP) at least. Cytological function experiments can provide strong evidence for pathogenicity analysis, which was a milestone significance for VUS variants\u003csup\u003e28,29\u003c/sup\u003e. Re-expressing \u003cem\u003eGTPBP3\u003c/em\u003e carrying mutant vectors of c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G, c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A and mutation hot spot (c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C) vectors in \u003cem\u003eGTPBP3\u003c/em\u003e KO cells, mutations were identified by Sanger sequencing (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo eliminate the interference of wild-type GTPBP3 protein, the \u003cem\u003eGTPBP3\u003c/em\u003e vectors carrying different mutations were transfected into KO cell lines. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, when compared with KO\u0026thinsp;+\u0026thinsp;\u003cem\u003eGTPBP3\u003c/em\u003e cell line, KO\u0026thinsp;+\u0026thinsp;c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T decreased by 97.8% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T decreased by 99.6% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A decreased by 94.8% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G decreased by 32.9% (p\u0026thinsp;=\u0026thinsp;0.0064) and decreased by 45.7% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in KO\u0026thinsp;+\u0026thinsp;c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C. No significant decrease was found in KO\u0026thinsp;+\u0026thinsp;c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G and KO\u0026thinsp;+\u0026thinsp;c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A. It needs to be considered that there are differences in vector copy number among cell lines\u003csup\u003e30,31\u003c/sup\u003e. We designed primers targeting to the 3' end of the CDS and PGK promoter which was a conserved region on vectors to assess the relative level of vectors. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. The vector levels of each cell lines were compared with KO\u0026thinsp;+\u0026thinsp;\u003cem\u003eGTPBP3\u003c/em\u003e cell line, KO\u0026thinsp;+\u0026thinsp;c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T was 3.2 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T was 2.4 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A was 1.8 times (p\u0026thinsp;=\u0026thinsp;0.0118), KO\u0026thinsp;+\u0026thinsp;c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G was 3.7 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and KO\u0026thinsp;+\u0026thinsp;c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C was 2.8 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G was 2.5 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A was 3.2 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), KO\u0026thinsp;+\u0026thinsp;c.1384C\u0026thinsp;\u0026gt;\u0026thinsp;G was 4.8 times (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Once the relative efficiency level further corrected the protein content, the masked differences of KO\u0026thinsp;+\u0026thinsp;c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G and KO\u0026thinsp;+\u0026thinsp;c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A can be uncovered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of genetic variants spectrum and phenotype-genotype correlation of\u003c/b\u003e \u003cb\u003eGTPBP3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe initial report by Robert Kopajtich et al in 2014 documented 11 cases of GTPBP3 mutations\u003csup\u003e11\u003c/sup\u003e. To date, in total of 21 cases with 23 distinct variants of \u003cem\u003eGTPBP3\u003c/em\u003e have been reported. By incorporating these reported variants with the 8 novel variants identified in our study, the genetic spectrum of the \u003cem\u003eGTPBP3\u003c/em\u003e gene was expanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Notably, variants identified in the Chinese population are marked in yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), we noted that c.689A\u0026thinsp;\u0026gt;\u0026thinsp;G is most common in the Chinese population. Moreover, our analysis revealed that the variant sites are predominantly concentrated in exon 4 and exon 6, with c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C showing high frequencies of 8/51, indicating it as a hot spot mutation site within this population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the protein\u0026rsquo;s functional domains, the mutations can be roughly categorized into four regions, which are mitochondrial targeting sequence (MTS, M), GTP-box (G), C-terminal (C), and other undetermined (U) regions\u003csup\u003e11\u003c/sup\u003e. Combined with previous cases, the characteristics were summarized as follows (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). Firstly, the onset age for all cases was under 10 years old, with a trend to earlier onset in the M/U region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Secondly, the clinical outcomes of patients in the M/U region more servere, with a higher proportion mortality ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In terms of muscle involvement, individuals in the M/G/U region were usually affected by dual involvement. Those in the C region tended to exhibit muscle involvement, along with energy deficiency along with energy deficiency symptoms like fatigue and mild myocardial hypertrophy (limited by the small number of case samples) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Overall, it appears that symptoms in the C region were relatively mild, may due to the smaller sample size. Lastly, all patients exhibited hyperlactatemia, and the majority experienced developmental delay along with muscle and/or nerve involvement (such as cardiac hypertrophy, seizures, hypermyotonia, or hypotonia) and abnormal brain MRI. They may also present with dyspnea, feeding difficulties, short stature, and occasionally visual impairment, as well as cardiac abnormalities. These cardiac abnormalities can include conduction and heart valve issues. Notably, valvular insufficiency was initially linked to \u003cem\u003eGTPBP3\u003c/em\u003e deficiency in our study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGTPBP3 is a catalytic enzyme involved in the synthesis of τm\u003csup\u003e5\u003c/sup\u003e(s\u003csup\u003e2\u003c/sup\u003e) U in mitochondria and has been associated with mitochondrial diseases. Since Robert Kopajtich first described the phenotypes associated with 11 cases of \u003cem\u003eGTPBP3\u003c/em\u003e deficiency\u003csup\u003e11\u003c/sup\u003e. At present, there are only several sporadic cases. However, restricted by the small number of cases, the disease-related phenotypes are incomplete, and the mutation spectrum still needs to improve. The correlation between genotype and phenotype remains to be further studied. According to previous studies, changes in oxidative phosphorylation complex enzyme activity of skeletal muscle and fibroblasts, several mitochondrial subunits protein levels as well as mitochondrial oxygen consumption rate under different culture conditions in patient-derived fibroblasts have been presented\u003csup\u003e11\u003c/sup\u003e. However, there are still few studies on patient-derived cell models, and the studies are only for individuals, and the subjects are inconsistent. We constructed patient-derived immortalized lymphocyte cell lines and performed BN-PAGE to preserve their native structure of OXPHOS complexes and subsequently oxygen consumption rates detection. Our results agree with the conclusion that \u003cem\u003eGTPBP3\u003c/em\u003e deficiency led to mitochondrial dysfunction. However, the new phenotypes including heart valve involvement and strabismus were first proposed in this study.\u003c/p\u003e \u003cp\u003eThe level of residual steady-state GTPBP3 protein seems to be correlated with the degree of mitochondrial impairment, except for P3, which may be due to the following hypotheses: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) The produced mitochondrial complex protein is useless in this patient, so the protein level does not change significantly, but the activity of complex enzyme is severely impaired\u003csup\u003e25\u003c/sup\u003e; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) The disadvantage of immortalized lymphocytes is that the phenotype is not obvious\u003csup\u003e32,33\u003c/sup\u003e; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Cells were cultured in a high-nutrient environment, and excessive reliance on glycolysis can compensate for the deficiency. In other words, stress culture can make the difference obviously\u003csup\u003e33,34\u003c/sup\u003e. In the results of GTPBP3 protein level detection on site-directed mutagenesis cell models, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G and c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A were inconsistent with the predicted results. The possible reasons were as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) The mutations did not affect the protein level but affected the enzyme catalytic activity \u003csup\u003e25\u003c/sup\u003e; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Different transfection and replication efficiency of plasmids would affect the protein expression level\u003csup\u003e35,36\u003c/sup\u003e. The results showed that the plasmids expression level in all site-mutant cell lines was higher than control. As a result, the pathogenicity of c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G and c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A cannot be ruled out.\u003c/p\u003e \u003cp\u003eThe reduction of protein level may be due to the decrease in protein production and/or the acceleration of protein degradation\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. Previous studies have shown that the GTPBP3 protein is extensively modified by ubiquitination and degraded through the proteasome pathway\u003csup\u003e7\u003c/sup\u003e. To avoid rapid protein degradation, we first treated the cells with MG-132 for 6h, then inhibited cell protein synthesis by CHX treatment, the degree of degradation of target protein can be observed within 24h\u003csup\u003e38,40\u0026ndash;42\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). It was found that compared with the control group, c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T and c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T significantly decreased after 6h of treatment, suggesting that c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T and c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T accelerated the degradation of GTPBP3 protein and reduced the stability of the protein (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn summary, we enrolled 9 individuals with \u003cem\u003eGTPBP3\u003c/em\u003e deficiency. We identified 8 novel variants, which were c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, c.680_691dup, c.774_775insC, c.776A\u0026thinsp;\u0026gt;\u0026thinsp;G, c.848C\u0026thinsp;\u0026gt;\u0026thinsp;A and c.1092_1103del, respectively. 7 mutations were screened to construct site-directed mutagenesis cell models and cytological function experiments were performed. It was confirmed that c.127C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.187C\u0026thinsp;\u0026gt;\u0026thinsp;T, c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.473T\u0026thinsp;\u0026gt;\u0026thinsp;G, and c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C were pathogenic mutations. \u003cem\u003eGTPBP3\u003c/em\u003e mutation spectrum was expanded, and it was found that mutations in the Chinese population were mostly concentrated in exon 4 and exon 6, and c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C and c.424G\u0026thinsp;\u0026gt;\u0026thinsp;A were hot spots in the population. This study highlights the important role of mt-tRNA modification defects in mitochondrial diseases and provides a reference for the diagnosis of diseases related to \u003cem\u003eGTPBP3\u003c/em\u003e deficiency, as well as subsequent prenatal diagnosis and genetic counseling.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStudy participant\u003c/h2\u003e \u003cp\u003ePatients were born from 9 non-consanguineous families. Patients 5 and 6 underwent evaluation at Xiangya Hospital, Central South University, while other patients were recruited and assessed at Peking University First Hospital. Approval for this study was obtained from the Ethics Committees of both Peking University First Hospital (2017 − 217) and Xiangya Hospital (No. 201605585, 2016/03/20). Moreover, informed consent was obtained from all participants or guardians.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis\u003c/h2\u003e \u003cp\u003eDNA was extracted from peripheral blood samples of probands and their parents. Whole exome sequencing (WES) and mitochondrial genome sequencing were performed using the HiSeq 2000 sequencer (Illumina, USA). Sanger sequencing was then carried out as a follow-up to validate the identified mutations\u003csup\u003e43,44\u003c/sup\u003e. The specific primers used are detailed in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmortalized lymphocytes construction\u003c/h2\u003e \u003cp\u003eAs previously outlined\u003csup\u003e45,46\u003c/sup\u003e, mononuclear cells were isolated from the peripheral blood using the lymphocyte separation medium (Solarbio, China). These isolated cells were continuously stimulated by Epstein-Barr virus (EBV). Furthermore, 0.5 mg/mL phytohemagglutinin (Sigma-Aldrich, USA) and 1 mg/mL cyclosporin A (Sigma-Aldrich) were also added to the culture medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids construction and transfection\u003c/h2\u003e \u003cp\u003eAs previously described\u003csup\u003e46,47\u003c/sup\u003e, Knockout (KO) plasmids were constructed using the CRISPR/Cas9 technology, and gRNAs were annealed to duplexes and inserted into pX330 vector. As for overexpression (OE) plasmid, \u003cem\u003eGTPBP3\u003c/em\u003e was synthesized by Phanta Max Super-Fidelity DNA Polymerase (Vazyme, China) and cloned into lentiviral pLVX vector by ClonExpression® II One Step Cloning Kit (Vazyme), and site-specific mutant vectors were obtained from Tsingke (Tsingke Biotechnology, China). All constructions were confirmed by Sanger sequencing. Transfection was performed with Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. KO cell lines were selected by limiting dilution, and OE cell lines and site-mutant cell lines were generated by infection of the cells with lentiviral particles and puromycin (Sangon Biotech, China) selection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eImmortalized lymphocytes from patients were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (GIBCO, USA) and 1% penicillin/streptomycin, as well as 50 mg/mL uridine (Sigma-Aldrich).\u003c/p\u003e \u003cp\u003eHEK-293T was a gift from Dr. Haihua Gu (Wenzhou Medical University). HEK-293T and other cell models generated from HEK-293T were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, USA) containing 12% calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin. 2 µg/mL puromycin (Sangon, China) was extremely added to cell models generated from HEK-293T. All cells were cultured with 5% CO2 at 37°C in an incubator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qPCR) detection\u003c/h2\u003e \u003cp\u003eRNA was extracted according to the TRIzol Reagent protocol (Thermo Fisher Scientific)\u003csup\u003e48\u003c/sup\u003e. Complementary DNA (cDNA) was synthesized by PrimeScript RT reagent Kit (Takara Biotechnology, Japan). For quantification transcripts expression level, qPCR was performed with 2xChamQ SYBR qPCR Master Mix (Vazyme, China). Primers are provided in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eFor sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a total protein isolated from whole cell using RIPA lysis buffer (Cell Signaling Technology, USA) with 1 mM phenylmethylsulfonyl fluoride (PMSF, Sangon Biotech, China).\u003c/p\u003e \u003cp\u003eFor Blue native polyacrylamide gel electrophoresis (BN-PAGE), as previously described\u003csup\u003e22\u003c/sup\u003e, mitochondrial membrane protein was extracted from a whole cell using 2% Triton-X100 (Sigma-Aldrich) and subsequently separated by a 3.5%-16% gradient gel. Proteins were electroblotted onto 0.22 um PVDF membranes (Bio-rad, USA) and blocked with 5% milk powder solution, incubated with primary and secondary horseradish peroxidase-conjugated antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e). Signals detected with clarity ECL western blotting (WB) substrate (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial respiration measurement\u003c/h2\u003e \u003cp\u003eThe detection of oxygen consumption rate (OCR) was conducted as described before\u003csup\u003e44,49\u003c/sup\u003e. Concisely, about 5 × 10\u003csup\u003e6\u003c/sup\u003e immortalized lymphocytes were harvested and added to the chamber of Oxygraph-2k (Oroboros, Austria). The respiration was recorded under normal conditions and with subsequent injection with inhibitors, including oligomycin (0.1mM, Sigma-Aldrich) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.1mM, Sigma-Aldrich).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCycloheximide (CHX) Chase Assay\u003c/h2\u003e \u003cp\u003eCells were seeded on 24-well cell culture plates (10000 cells per well). Once the confluence reached 90%, cells were pretreated with a complete medium containing 10uM MG132 for 6 h. After washing cells twice with PBS, the culture medium was converted to a complete medium containing 20uM cycloheximide (CHX). Samples were harvested at 0, 3, 6, 12, and 24 h after CHX treatment for SDS-PAGE detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll quantitative data were performed for three or more times, independently. Statistical analysis and graphs were plotted using Prism 8.4.0. The results were shown with mean ± SD. When the data conforms to a normal distribution, independent double-tailed student t was used. If not, the Mann-Whitney U test is used. Three or more groups of data were compared using one-way analysis of variance (ANOVA), \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 indicates statistical significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eContributors Statement:\u003c/h2\u003e\n\u003cp\u003eXiaoting Lou, Yanling Yang, and Liqin Jin conceptualized the study, developed the experimental designs, interpreted the data, and revised the manuscript. Yaojun Xie designed and conducted the experiments, analyzed the data, and drafted the manuscript. Keyi Li, Li Yang, Luyi Zhang, Xiaofei Zeng, and Yuwei Zhou conducted the experiments. Zhehui Chen and Xue Ma coordinated the clinical assessment of the patients and their families. All authors provided final approval and consented to take responsibility for all aspects of the research.\u003c/p\u003e\n\u003ch2\u003eEthical Approval\u003c/h2\u003e\n\u003cp\u003eThis ethical approval was obtained from the Ethics Committee of Peking University First Hospital (Ethics approval number: 2017\u0026thinsp;\u0026minus;\u0026thinsp;217), the Ethics Committee of Xiangya Hospital of Central South University, China (Ethics approval number: 201605585)\u003c/p\u003e\n\u003ch2\u003eInformed consent\u003c/h2\u003e\n\u003cp\u003eConsent was obtained from the participants\u0026rsquo; guardians to publish this document.\u003c/p\u003e\n\u003ch2\u003eConflict of interest\u003c/h2\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82302618, X.L. 82072366, L.J.), the National Key Research and Development Program of China (2021YFC2700900, 2017YFC1001700, Y.Y.), China Postdoctoral Science Foundation (2023M743124, X.L.), the Key Discipline of Zhejiang Province in Medical Technology (First Class, Category A), Wenzhou Medical University, and the Key Discipline of Zhejiang Province in Public Health and Preventive Medicine (First Class, Category A), Hangzhou Medical College.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe express our gratitude to the patients and their families for their participation in this study. Additionally, we extend our thanks for the generous gift of HEK293T cells from Dr. Haihua Gu at Wenzhou Medical University.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are not publicly accessible due to the confidentiality and ethical considerations associated with patient data. However, these datasets can be obtained from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSayyed, U. M. H. \u0026amp; Mahalakshmi, R. Mitochondrial protein translocation machinery: From TOM structural biogenesis to functional regulation. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e298\u003c/strong\u003e, 101870 (2022). https://doi.org:10.1016/j.jbc.2022.101870\u003c/li\u003e\n\u003cli\u003eFilograna, R., Mennuni, M., Alsina, D. \u0026amp; Larsson, N. G. 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S. 2\u0026apos;-5\u0026apos; Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e277\u003c/strong\u003e, 25601-25608 (2002). https://doi.org:10.1074/jbc.M200211200\u003c/li\u003e\n\u003cli\u003eFang, H.\u003cem\u003e et al.\u003c/em\u003e A membrane arm of mitochondrial complex I sufficient to promote respirasome formation. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 108963 (2021). https://doi.org:10.1016/j.celrep.2021.108963\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mitochondrial diseases, oxidative phosphorylation, GTPBP3, genetic hotspot, τm5(s2)U modification","lastPublishedDoi":"10.21203/rs.3.rs-4634652/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4634652/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGTPBP3 catalyzes τm\u003csup\u003e5\u003c/sup\u003e(s\u003csup\u003e2\u003c/sup\u003e) U biosynthesis at the 34th wobble position of mitochondrial tRNAs, the hypomodification of τm\u003csup\u003e5\u003c/sup\u003eU leads to mitochondrial disease. While twenty-three variants of \u003cem\u003eGTPBP3\u003c/em\u003e have been reported worldwide, the genetic landscape in China remains uncertain.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eBy using whole-exome sequencing, the candidate individuals carrying \u003cem\u003eGTPBP3\u003c/em\u003e variants were screened and identified. Pathogenicity analysis of variants was biochemically verified by patients-derived immortalized lymphocytes and cell models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThrough whole-exome sequencing, thirteen variants associated with \u003cem\u003eGTPBP3\u003c/em\u003e were identified in nine Chinese pedigrees, with eight of these variants being newly reported. Affected individuals displayed classic neurologic phenotypes and heart complications including developmental delay, seizures, hypotonia, exercise intolerance, and hypertrophic cardiomyopathy. Additionally, they displayed new symptoms such as eye problems like strabismus and heart issues related to valve function. Studies conducted on patient-derived cells provided evidence of reduced levels of GTPBP3 and impairment in mitochondrial energetic biogenesis. Re-expressing \u003cem\u003eGTPBP3\u003c/em\u003e variants in knockout cell lines further defined the pathogenicity of the novel variants. Analysis of the genetic spectrum in the Chinese population highlighted a concentration in exons 4 and 6, with c.689A\u0026thinsp;\u0026gt;\u0026thinsp;C being the prominent hotspot.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings emphasize the extensive clinical and genetic implications of \u003cem\u003eGTPBP3\u003c/em\u003e-related mitochondrial disorders, particularly within the Chinese population, but further investigations are needed to explore the phenotype-genotype correlation.\u003c/p\u003e","manuscriptTitle":"Expanding the phenotypic and genetic spectrum of GTPBP3 deficiency: findings from nine Chinese pedigrees","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-11 13:06:50","doi":"10.21203/rs.3.rs-4634652/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2024-11-19T11:54:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-08-06T21:20:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-06T18:14:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-30T19:52:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Orphanet Journal of Rare Diseases","date":"2024-06-27T02:06:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"orphanet-journal-of-rare-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ojrd","sideBox":"Learn more about [Orphanet Journal of Rare Diseases](http://ojrd.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ojrd/default.aspx","title":"Orphanet Journal of Rare Diseases","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"55ba964e-929e-43de-bd51-0a3125ed0b17","owner":[],"postedDate":"September 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-30T16:04:41+00:00","versionOfRecord":{"articleIdentity":"rs-4634652","link":"https://doi.org/10.1186/s13023-024-03469-3","journal":{"identity":"orphanet-journal-of-rare-diseases","isVorOnly":false,"title":"Orphanet Journal of Rare Diseases"},"publishedOn":"2024-12-24 15:57:28","publishedOnDateReadable":"December 24th, 2024"},"versionCreatedAt":"2024-09-11 13:06:50","video":"","vorDoi":"10.1186/s13023-024-03469-3","vorDoiUrl":"https://doi.org/10.1186/s13023-024-03469-3","workflowStages":[]},"version":"v1","identity":"rs-4634652","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4634652","identity":"rs-4634652","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}