Discussion
In this study, we described two novel heterozygous mutations ofTMEM126B(c.82-2A>G and c.290dupT) from a Chinese patient manifested with LS.In silico predictions, minigene splicing assays and patients’ RNA analyses were combined to determine that the c.82-2A>G mutation resulted in exon 2 completely skipped, and that the c.290dupT mutation caused an increase of partial and complete exon 3 deletion transcripts, which would lead to frameshift and premature termination. Patient-derived immortalized lymphocytes carrying biallelic mutations exhibited complex I content and assembly defect and mitochondrial dysfunction. To the best of our knowledge, this is the first report thatTMEM126B mutations cause LS.
TMEM126B was identified as the part of MCIA complex to co-migrate with other MCIA complex components (NDUFAF1, ECSIT, and ACAD9) by complexome profiling (Heide et al., 2012). The defect of each components can cause heterogeneous clinical phenotypes. For example, mutations inNDUFAF1 mostly associated with cardiological symptoms (Dunning et al., 2007; Elisa Fassone et al., 2011), including Wolff-Parkinson-White syndrome and hypertrophic cardiomyopathy. Genetically deficit inACAD9 commonly linked to cardiac symptoms, neurological symptoms, and severe lactic acidosis (E. Fassone et al., 2011; Schiff et al., 2015). Mendelian mutations in ECSIT gene has not yet reported. Individuals carrying TMEM126B mutations mainly presented with exercise intorlance, muscle weakness, hyperlactic acidemia, pure myopathy, chronic renal failure and cardiomyopathy (Alston et al., 2016; Sánchez-Caballero et al., 2016; Theunissen et al., 2017). Notably, the clinical phenotypes of our patient are consistent with those of patients carrying TMEM126Bmutations previously reported, except for chronic renal failure and cardiomyopathy (Table 1). Chronic renal failure is not a necessary symptom to diagnose mitochondrial diseases. Significantly, our patient show a more severe neurological symptoms with clinical presentation consistent with LS. Overall, we believe that patients withTMEM126B mutations may exhibit high clinical heterogenicity. The combination of clinical and molecular diagnosis are required for the diagnosis of TMEM126B mutation‐related mitochondrial diseases.
In silico analysis indicateed that the c.82-2A>G mutation located 2bp before exon 2 could lead to the loss of the original 3’splice acceptor site, while the c.290dupT mutation located exon 3 might create a new constitutive splice acceptor site. In vitroexperiments verified that c.82-2A>G mutation led to complete exon 2 skipping and c.290dupT caused an increase of transcripts with partial and complete deletion of TMEM126B exon 3. Mutations that located in exonic splicing enhancer (ESE) region are thought to prevent serine and arginine-rich (SR) proteins from binding to ESE sequence motifs, which induced exon skipping. Results of ESE finder software suggested that c.290dupT mutation may disrupt a putative SRp55 binding site, thus increasing the proportion of abnormal splicing transcripts. Further functional study indicated that patient-derived immortalized lymphocytes exhibited a global mitochondrial dysfunction with decreased mitochondrial respiratory capacity, reduced ATP content and increased mitochondrial ROS levels due to the complex I content and assembly defect. Unlike previous reports, biallelic mutations in our patient caused the complete deletion of exon 2 and the partial truncation of exon 3 of TMEM126B , which resulted in a more severeTMEM126B defect, leading to a more severe complex I deficiency and brain phenotypes.
In summary, we identify TMEM126B as a novel disease-causing gene resulting in LS with obvious neurological symptoms, and report two novelTMEM126B mutations (c.82-2A>G and c.290dupT) that cause splicing defects and lead to mitochondrial dysfunction due to the severe complex I deficiency. Our study expands the genetic mutation spectrum of LS and the clinical spectrum caused by TMEM126Bmutations.