Clinical manifestation and genetics analysis
The individual (II-1) was born into nonconsanguineous healthy Chinese family with a normal gestational and delivery record. She fell the development milestone, with instability in walking at the age of 12 months, and received recovery training for two months, however, no improvement in her symptoms. She developed uninterrupted strabismus in her left eye at the age of 2 years. Imaging tests showed negative brain magnetic resonance imaging (MRI) and electroencephalogram (EEG). She was consulted in the Department of Genetic Metabolism at Peking University First Hospital when she was three years and two months old. The candidate walked unsteadily, with weak muscular strength, reduced muscle tension, malnutrition but well developed reflexes and mental response. The face of patient was yellow, with yellow palms and feet, and the back of the neck was ciliated. Metabolic investigation showed increased blood lactate (3.14, normal range 0.50-2.20 mM) and β- hydroxybutyric acid (1.18, normal range 0.02-0.27 mM). Amino acid and acylcarnitine profile analysis showed there were no significant abnormal findings. Other tests showed increased aspartate amino acid transferase (42.2, normal range 0.0-35.0 U/L), lactate dehydrogenase (247.3, normal range 109.0-245 U/L) and α-hydrobutyrate dehydrogenase (260.8, normal range 90.0-250.0 U/L), indicating an abnormal liver function. MRI demonstrated symmetrical abnormal signals in bilateral cerebral peduncles, and abnormal signals in the cerebral bridge with adenoid hypertrophy (Figure 1A). Electromyogram/evoked potentials test (EMG/EP) revealed that left gastrocnemius nerve had sensory conduction abnormalities. Echocardiogram was normal. She was established the diagnosis of LS according to the standard criteria (Rahman et al., 1996). Next-generation sequencing was performed to detect the disease-causing gene of the probing, after the filtering with established criteria (Wei et al., 2020), a novel splice site variant (c.82-2A>G, intron 1) and a novel insertion variant (c.290dupT, exon 3) in TMEM126B (NM_018480.7) were identified and no clinically significant mitochondrial genomic-related variants were detected. Segregation analysis confirmed that c.82-2A>G was maternal inherited while c.290dupT was paternal (Figure 1B and 1C).
A comprehensivein silico analysis ofc.82-2A>G and c.290dupT
To investigate the potential pathogenicity of the above two variants (c.82-2A>G and c.290dupT), we performed a series of bioinformatics analyses. As shown in Figure 2A, both exon 2 and exon 3 of TMEM126B are conserved across the species and mutations in exon 3 is prone to intolerance by using the MetaDome web tool (Wiel et al., 2019), and the p.98K seems intolerance and highly conservative. The prediction for the pathogenicity in several databases showed c.82-2A>G was like pathogenic and c.290dupT was pathogenic, the allele frequency of c.82-2A>G in gnomAD was extremely low (0.0006), and neither of them had record in several population variation frequency and pathogenicity prediction databases (Figure 2B). ASSP (Alternative Splice Site Predictor) (Wang & Marín, 2006) indicates that the c.82-2A>G mutation may destroy original splice constitutive acceptor, while the frameshift mutation c.290dupT may create a new splice constitutive acceptor (Figure 2C). ESE finder (exonic splicing enhancers) predicts the c.82-2A>G variant resulted in a decrease in the SRp40 binding sequence score, and the c.290dupT variant leads to increased SRp40 binding sequence score and loss of SRp55 binding sequence (Figure 2D). Taken together, these results indicate that both of these mutations (c.82-2A>G and c.290dupT) are disease-causing and affect pre-mRNA splicing.
Identification of variants affecting TMEM126B splicing by using a minigene splicing assay
To explore whether the c.82-2A>G and c.290dupT variants influence mRNA splicing, we conducted an exon trapping assay based on pSPL3 plasmids (Figure 3A). RT-PCR and Sanger sequencing showed that both the empty pSPL3 control and c.82-2G mutant constructs gave rise to a 263-bp PCR fragment missing exon 2 ofTMEM126B gene, whereas the wild-type c.82-2A yielded a RT-PCR product of 385-bp containing exon 2 (Figure 3B and 3C), which indicated that c.82-2A>G mutation destroyed the original splice acceptor site and resulted in full exon 2 skipping. The plasmid constructs of both wild-type c.290T and mutant c.290dupT expressed three transcripts, including a transcript without exon 3, a transcript with 103-bp deletion of exon 3 and a transcript containing exon 3 but with one base (T) duplication (Figure 3D and 3E). Quantitative analysis showed that natural partial and complete exon 3 skipping were weak in wild-type c.290T construct, but significantly increased in mutant c.290dupT (Figure 3F). Altogether, our data suggested that c.82-2A>G mutation caused complete exon 2 skipping and c.290dupT induced partial and complete exon 3 skipping.
Confirmation of variant-induced spliceogenicity inpatient-derivedlymphocytes
To assess the physiologic relevance of the splicing defects revealed by the minigene assay, we analyzed the splicing pattern of TMEM126B in patient-derived lymphocytes. RT-PCR using primers specific to exons 1 and 3 spanning the variant c.82-2A>G generated a PCR product with complete exon 2 skipping in patient-derived sample (Figure 4A and 4B), resulting in a 40 amino-acid deletion with a subsequent frame-shift from codon 28 and premature termination at position 58 in exon 3 (Figure 4C), thus leading to nonsense-mediated mRNA decay. Agarose gel analysis and Sanger sequencing of the RT-PCR products generated from patient-derived mRNA using primers specific to exons 2 and 4 detected a full-length transcript carrying the c.290dupT in exon 3 and a shorter mRNA with the c.290dupT and 103-bp deletion of exon 3 (Figure 4D and 4E). Both of the above transcripts were predicted to cause frameshift and premature termination (Figure 4F), which would likely lead to transcript elimination via the nonsense-mediated decay pathway. Whereas very little levels ofTMEM126B cDNA lacking 103-bp from exon 3 could be detected in control lymphocytes, further suggesting c.290dupT induced splicing defects. These results obtained from patient-derived RNA samples were in agreement with the minigene data.
Mitochondrial complex I content and assembly defect and mitochondrial dysfunction in patient- derived lymphocytes
To validate the pathogenic role of c.82-2A>G and c.290dupT variants in TMEM126B , OXPHOS supercomplexes and complexes were tested in patient-derived lymphocytes. The results indicated that the content of complex I was markedly decreased in patient-derived immortalized lymphocytes compared with normal controls. Moreover, supercomplex CI/III2/IV assembly was blocked, while lower assembly intermediate appeared to accumulate notably (Figure 5A and 5B). Mitochondrial respiratory chain complexes are involved in maintaining proper mitochondrial function, and we then investigated mitochondrial functions in patient-derived immortalized lymphocytes. As shown in Figure 5C, patient-derived lymphocytes showed a general decrease in cellular respiratory capacity, including basal, ATP-linked respiration, maximal respiration, and spare respiration capacity compared to controls. Cellular ATP content of patient-derived lymphocytes was significantly decreased, whereas the mitochondrial ROS level was increased (Figure 5D and 5E). Together, these results demonstrated that mitochondrial OXPHOS function was severely impaired in patient‐derived lymphocytes carrying mutations of c.82-2A>G and c.290dupT.