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.