Keywords mitochondrial DNA mutation - mitochondrial diseases - dystonia - epilepsy
Introduction
Dystonia is a group of movement disorders characterized by distorted, repetitive,
abnormal movements, and postures caused by involuntary, sustained muscle contractions.[1 ] Depending on the etiology, dystonia can be classified as acquired, genetic, or idiopathic.[2 ] Previous studies have reported several genes associated with primary dystonia, but
a proportion of dystonia is secondary to trauma, infection, and inherited neurodegenerative
diseases such as Wilson's disease[3 ] or Huntington's disease.[4 ]
Mitochondria are key organelles for the generation of cellular energy. Mitochondrial
diseases are a large group of clinically and genetically heterogeneous disorders caused
by mitochondrial dysfunction.[5 ]
[6 ] Mitochondrial dysfunction affects organs with high energy demands, especially muscles,
as well as the brain. Primary mitochondrial diseases are defined as pathogenic mutations
in mitochondrial DNA or nuclear DNA (mtDNA or nDNA, respectively), usually affecting
proteins involved in oxidative phosphorylation (OXPHOS), and include Friedreich's
ataxia, Leber's hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic
acidosis, and stroke-like episodes (MELAS) syndrome, and myoclonic epilepsy with ragged
red fibers.[7 ] Pathogenic mutations can induce different types of deleterious phenotypes manifested
as neurodevelopmental delay, seizures, blindness, hearing loss, stroke, and premature
death.[8 ]
In this study, we report a case of an infant with dystonia who died with the 5816
A > G mutation in the mitochondrial transfer RNA (mt-tRNA) gene carried by both her
mother and maternal grandmother. This case is the first child in China to carry the
5816 A > G mutation in the mt-tRNA gene. We also found that the mitochondrial OXPHOS
function was significantly decreased in the primary cells of the previous patients
carrying this mutation.
Methods
Ethics Compliance
The study was approved by the Ethics Committee of Wuhan Children's Hospital (ethics
number: 2022R093-F02). Informed written consent was obtained from all participants
and the parents of the proband.
Whole Exome Sequencing
Genomic DNA samples were extracted from peripheral blood using QIAamp Blood Mini Kit
(Qiagen, Hilden, Germany). Quality of genomic DNA was evaluated by agarose gel analysis
and quantity was measured by NanoDrop2000 and Qubit3.0. DNA is sheared with M220 Focused-ultrasonicator
(Covaris, Woburn, Massachusetts, United States). DNA target regions were captured
by hybridizing the genomic DNA sample library with the xGen Exome Research Panel v1.0
(IDT, United States). The captured and amplified DNA samples were sequenced using
Illumina NovaSeq6000 (Illumina, San Diego, California, United States) with 150 base-paired
end reads.
Mitochondrial Genome Sequencing and Analysis
Genome was extracted from urine and blood samples using QIAamp DNA Micro Kit and TLANamp
Blood DNA Kit, respectively. TaKaRa LA Taq Hot Start Version was used to amplify the
full-length mitochondrial genome using the following amplification procedure: 95°C
for 2 minutes, 95°C for 20 seconds, and 68°C for 18 minutes (30). After fragmentation
of the mitochondrial genome by Covaris M220, the library was constructed using NanoPrepDNA
kit (for Illumina). The size of nucleic acid fragments in the library was checked
by Agilent 2100 to ensure that the fragment size was around 260 to 400 bp.
Next, bioinformatics analysis was performed by public software and packages. First,
preprocessing of reads was carried out using fastp to remove low-quality reads.[9 ] Second, the Burrows–Wheeler Aligner tool[10 ] and default parameters were compared with the revised Cambridge Reference Sequence,[11 ] the generated BAM file, sorted by SAMtools[10 ]; finally, VarDict[12 ] detected single nucleotide variant (SNV) and indels (<50 bp), and ANNOVAR[13 ] and MSeqDR[14 ] annotate the database for SNV.
Primary Skin Fibroblast Cell Culture
The patient's skin on the medial side of the large arm is first disinfected and locally
anesthetized. The loop drill is placed perpendicular to the skin and then pressed
firmly downward while rotating. The core area was fully elevated with forceps or the
tip of a needle, and a small amount of tissue mass was excised at the bottom of the
core area with small scissors and immediately placed in phosphate buffer. The tissue
mass was cut into 1 mm three pieces and added to DMEM complete medium containing 15%
fetal bovine serum, 1% penicillin and streptomycin, and incubated at 37°C and 5% CO2 . When skin fibroblasts grew around the tissue block (usually about 1 week), 0.25%
trypsin was added to digest the cells, and then 1 mL of culture medium was added and
cultured at 37°C and 5% CO2 .
Cellular Oxygen Consumption Rate Assay
Primary skin fibroblasts were inoculated into XF24 cell culture microtiter plates
(5 × 104 cells/well). Prior to testing, the medium was changed to specific XF assay
medium (pH 7.4) and the cells were allowed to stand for 1 hour at 37°C in a CO2 -free incubator. According to the instructions, oligomycin (2 μM), trifluoromethoxy
carbonylcyanide phenylhydrazone (2 μM), rotenone (1 μM), and antimycin (1 μM) were
prepared and added to the wells of the assay plate. The test plates were placed on
the pallets of the Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies
Ltd., Santa Clara, California, United States) and the oxygen consumption rate (OCR)
was measured three times (3 minutes/time).
Results
Clinical Phenotype of the Proband
In April 2021, the presentee (2 years and 10 months old) had episodes of vomiting
during sleep at night without apparent cause, and the vomit consisted of gastric contents
of medium volume; 1 hour after vomiting, she had decreased consciousness response,
did not respond to calls, eyes were closed, mouth twitched, and she had been given
bath, and her limbs continued to shake for about 1 hour, without fever or diarrhea;
her parents urgently took her to a hospital near her place of residence (the specific
treatment strategy was not clear), and her symptoms subsided. In August 2021, while
sleeping at night, the subject again vomited several times, followed by salivation,
closed eyes, and reluctance to speak, and her symptoms subsided after 1 hour. After
the symptoms subsided, the patient was very tired and was again taken to a hospital
near her home for treatment (the exact treatment strategy was not known). After being
discharged from the hospital, the patient gradually developed abnormal behavior: reluctance
to play with other children, reluctance to speak, slurred speech, choking on water,
poor sleep, noisy at night, unstable walking, easy to fall, and gradually increasing
symptoms.
The preexisting patient was admitted to Wuhan Children's Hospital on September 21,
2021, for further definitive diagnosis and treatment. The mother reported that she
had one pregnancy and one delivery, and the perinatal period was safe and uneventful
l without hypoxic asphyxia. The mother had symptoms of double eyelid blinking and
was diagnosed with possible epilepsy by the hospital near her home and was treated
with oral oxcarbazepine. The grandmother had hand tremors.
Physical examination of the patient revealed a delirious state of confusion and poor
mental responsiveness. She had limited abduction of the left eye, along with salivation,
irritability and agitation, unsteady gait, and persistent hand tremors. The proband
had unrestricted limb movement, normal muscle tone, normal bilateral knee tendon reflexes,
and both Babinski, cervical resistance, and Kerning signs were negative. Electrocardiogram,
five-category blood count, parathyroid hormone, blood ammonia, liver function, renal
function, cardiac enzyme profile, electrolytes, lipids, blood glucose, immune panel,
25-hydroxyvitamin D, erythrocyte sedimentation rate, calcitoninogen, and high-sensitivity
C-reactive protein were all normal. Cerebrospinal fluid was negative for autoimmune-related
antibodies.
The electroencephalogram (EEG) results were abnormal in the children with a large
number of multifocal spikes, multispikes, spikes and slow waves, slow waves and low
amplitude fast waves, more pronounced in the occipital region bilaterally, and occurring
continuously during sleep. The cranial magnetic resonance imaging (MRI) scan showed
no abnormal signal in the brain parenchyma, and the arterial spin labeling (ASL) suggested
a symmetrical increase in perfusion in the bilateral parieto-occipital lobes; the
cranial MRI + diffusion-weighted imaging scan showed no obvious abnormal signs ([Fig. 1 ]).
Fig. 1 The magnetic resonance imaging of the proband. (A ) ASL, (B ) MRS, (C ) T1, (D ) T2, (E ) T2 fluid-attenuated inversion recovery, and (F ) diffusion-weighted imaging.
The proband was admitted to our hospital for treatment several times between September
2021 and May 2022. During this period, the proband's multiple EEG findings suggested
persistent bilateral occipital discharges. We adjusted the treatment for dystonia,
including benzhexol, levodopa, and clonazepam, but the results were poor. Throughout
the course of the disease, the patient's lactate level was higher than normal, and
the results are shown in [Table 1 ]. At the end of May 2022, during the final course of the disease, the previous patient
developed a convulsive state with convulsions lasting 1 to 2 hours, manifested by
double-eye gaze, cyanosis of the lips, rhythmic shaking of the extremities, and profuse
salivation, and then died.
Table 1
The lactate level of proband
Time
September 27, 2021
February 7, 2021
December 12, 2021
January 11, 2022
February 3, 2022
March 22, 2022
June 1, 2022
Reference (mmol/L)
Lactate
4.04
2.68
2.54
2.10
2.32
1.77
2.63
0.5–2.22
Note: bold signifies reference values higher than (2.22).
Sequencing
In October 2021, we first performed whole-exome and copy number variant sequencing
of the blood of the proband; however, no pathogenic or potentially pathogenic variants
highly correlated with the clinical presentation of the patient were identified. Considering
that both the mother and grandmother of the proband had symptoms of epilepsy, we obtained
blood and urine samples from the proband for mitochondrial genome sequencing. The
results are shown in [Fig. 2 ], where the proband carried a novel mutation m.5816A > G. In addition, we also sequenced
the mitochondrial genomes of the blood and urine samples from the mother and grandmother
of the proband. The results showed that both the mother and grandmother carried m.5816A > G.
The family tree of the proband is shown in [Fig. 3 ].
Fig. 2 Sequence chromatogram confirming the m.5816A > G mutation in urine and blood from
proband. (A ) rCRS of blood, (B ) rCRS of urine, and (C ) sequence of m.5816A > G. mtDNA, mitochondrial DNA; rCRS, revised Cambridge Reference
Sequence.
Fig. 3 The family tree of proband showing all affected family members known to harbor the
m.5816A > G mutation (closed symbols).
OCR to Detect Changes in Mitochondrial Function
Next, we obtained primary cells from the proband, her mother, and grandmother, and
performed OCR experiments to observe the changes in mitochondrial function in the
cells after the m.5816A > G mutation. [Fig. 4A ] shows that the function of the mitochondrial respiratory chain was more vigorous
in the primary cells of children compared with adults. [Fig. 4B ] shows that there was no significant change in mitochondrial respiratory chain function
in progenitor cells from the proband's mothers and grandmothers compared with adults
who did not carry the m.5816A > G mutation; however, the progenitor cells from the
proband had only 28.6% basal OCR at rest compared with healthy children of the same
age who did not carry the m.5816A > G mutation ([Fig. 4C ]). We also found that adenosine triphosphate (ATP) production, proton leak, maximal
respiration, and reserve capacity OCR were reduced to 28.6, 20.1, 24.1, and 11.1%,
respectively, in the progenitor cells.
Fig. 4 The oxygen consumption rate (OCR) experiments of (A ) healthy child, proband, (B ) her mother, and (C ) her grandmother.
Discussion
This case is the first Chinese family with a mutation in the mitochondrial m.5816A > G
locus. In this case study, the proband was diagnosed with dystonia. The EEG findings
showed a large number of multifocal spikes, multispike, spikes and slow waves, and
slow waves and low amplitude fast waves bilaterally in the posterior head. The proband's
mother and grandmother also had partial epilepsy symptoms. Mitochondrial genomic analysis
revealed the presence of the m.5816A > G mutation in the mitochondrial tRNA (MTT)
gene, and both her mother and grandmother carried this mutation. We also analyzed
the mitochondrial OXPHOS function in the primary cells of the proband, her mother,
and her grandmother. Interestingly, the mitochondrial OXPHOS function was significantly
decreased in the primary cells of the proband, whereas no significant changes were
observed in her mother and grandmother.
Mitochondria are important organelles in cells and their main function is to produce
ATP to supply energy to the cell. The distribution of mitochondria in cells from different
tissues depends on energy requirements. Mitochondrial dysfunction can affect several
tissues, particularly those with high energy demands, especially the brain. Compared
with other tissues, the brain consumes almost 10 times more oxygen and glucose. Given
the high demand and consumption of ATP in the brain, most mitochondrial mutations
affect brain function and lead to neurological pathologies.[15 ]
Mitochondrial diseases are clinically heterogeneous disorders characterized by mitochondrial
dysfunction,[16 ]
[17 ] mainly caused by Mendelian inherited mutations in nDNA and/or maternally inherited
mtDNA.[18 ]
[19 ] Mutations in mtDNA are found in most mitochondrial diseases, and most of these mutations
are located in the mt-tRNA gene (MTT gene).[20 ] MTT gene is essential for protein synthesis, is a hotspot for mtDNA mutations, and
leads to involvement of tissues and organs with greater aerobic metabolism.[21 ] Today, more than 250 MTT mutations causing disease have been reported.[22 ] The same tRNA mutation may lead to different mutational loads and various types
of clinical manifestations, ranging from asymptomatic to severe phenotypes.[23 ] For example, the m.1A > G mutation in MTT gene 3243 causes MELAS syndrome, chronic
progressive external oculomotor paralysis, maternally inherited diabetes mellitus
and deafness syndrome, and two or more of these phenotypes may occur together in the
same family.[24 ] In addition, Scuderi et al reported another adjacent mutation (m.5814T > C) that
disrupts Watson–Crick base pairing in the D-stem and is associated with mitochondrial
encephalopathy.[25 ]
Fewer studies have been conducted on mutations at the m.5816A > G locus, which shows
a high degree of evolutionary conservation ([Fig. 5 ]). We used the mtDNA polymorphic variant database MITOMAP (https://www.mitomap.org/cgi-bin/mitotip?pos=5816&alt=G&quart=2 ) to predict the pathogenicity of the m.5816A > G locus mutation, and the results
showed that the m.5816A > G locus mutation was pathogenic with a probability of 59.9%
([Fig. 6 ]).[26 ] The possible reasons for this are, first, that the structural integrity of mt-tRNACys
is essential for maintaining the helical conformation as well as the interactions
between the translation components.[27 ] Therefore, the m.5816A > G mutation may disrupt the mt-tRNACys structure and affect
Watson–Crick base pairing in the dihydrouridine arm of mt-tRNACys.[28 ] Second, the abnormal structure may lead to a significant decrease in the steady-state
level of mt-tRNACys and consequently to a mitochondrial translation defect and subsequent
OXPHOS dysfunction.[23 ] Both of these points support the deleterious nature of the m.5816A > G locus mutation.
Fig. 5 Evolutionary conservation of the T-A base pairing in the dihydrouridine (DHU) stem
of mt-tRNACys.
Fig. 6 The prediction of pathologic finding for m.5816A > G.
Data from the OCR assay showed a significant decrease in ATP production and maximal
respiration in the cells of the proband compared with healthy children of the same
age who did not carry the m.5816A > G mutation, accompanied by an increase in serum
lactate, suggesting that OXPHOS in the mitochondria may be severely impaired. However,
it is interesting to note that ATP production and maximal respiration were not significantly
reduced in the cells of the proband's mother and grandmother. More surprisingly, mitochondrial
ATP production, proton leak, maximal respiration, and OCR reserve capacity were much
higher in healthy children than in normal adults ([Fig. 4A ]). We hypothesized that the mitochondrial OXPHOS function is more vigorous in children
compared with adults due to their growth and developmental stage. In this case, the
proband had an onset at 3 years and 3 months of age and died less than a year later,
while her mother and grandmother had no life-threatening symptoms and only partial
epilepsy. McFarland et al reported a family in which progressive dystonia in family
members was also caused by the m.5816A > G mutation. The patients in this case were
all adults and adolescents with good survival. Therefore, we speculate that the m.5816A > G
mutation is likely to cause severe mitochondrial dysfunction, which is more harmful
in children.
There are several limitations to this report. First, we only obtained blood and urine
from the subject, mother, and grandmother for mitochondrial genome sequencing, and
further serial muscle sections should be performed to clarify the pathological features
of mitochondrial disease; second, multiple perspectives are needed to evaluate the
mitochondrial dysfunction caused by the m.5816A > G mutation, especially tRNA-Cys
steady-state level, mitochondrial protein expression, mitochondrial ROS level, etc.
This study will encourage us to combine mitochondrial genome sequencing approaches
with clinical symptoms to facilitate accurate genetic diagnosis and correct pathogenicity
attribution of childhood dystonia, and to provide appropriate genetic counseling and
transmission prevention techniques.
