| BackgroundMitochondria are organelles with bilayer membranes in eukaryote cells.The basic role of mitochondria is to produce adenosine triphosphate(ATP)through oxidative phosphorylation(OXPHOS).Mitochondria also play important regulatory roles in cell signaling,calcium homeostasis,apoptosis and autophagy.Mitochondria contain more than 1,500 proteins,and some of them are expressed in a tissue-specific manner.Human mitochondrial DNA(mtDNA)consists of 16569 base pairs and encodes the 13 important peptides in OXPHOS,the 12S and 16S rRNAs and 22 tRNAs.Most proteins required for mitochondrial function are encoded by nuclear DNA(nDNA),which are introduced and classified into each mitochondrial chamber to form respiratory chain complex or assembly factors,mitochondrial membrane proteins,transporters,network proteins,etc.No matter mtDNA or nDNA defects could cause mitochondrial disease.Mitochondrial disease is an inherited metabolic disease characterized by energy production disorders with clinical and genetic heterogeneity.Multi-organs are usually involved in mitochondrial disease varying in severity,especially those with high energy requirements,such as muscles and brain;while there are mitochondrial diseases affecting a single organ.The clinical manifestations of some mitochondrial diseases can be highly suggestive of a specific phenotype,such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes(MELAS).However,many patients present a set of clinical symptoms that suggest mitochondrial disease but weren't fully conformed to any particular disease phenotype.To date,more than 300 disease-causing genes have been identified in mitochondrial diseases.The mutant and wild mtDNA can coexist in the same cell,known as heterogeneity.The proportion of mutant mtDNA must exceed the tissue-specific threshold level to cause biochemical defects in cells and develop disease phenotypes.The heterogeneity level is one of the main reasons for the clinical variability in different patients,or different individuals within the same family.In addition,some neutral mtDNA mutations,even without pathogenicity,can modulate the effects of disease-causing mtDNA mutations.About 70%-85%mitochondrial disease cases were due to nDNA mutations,which usually led to mitochondrial dysfunction by disrupting the integrity of the mtDNA structure and function.However,more than 50%of adult patients with mitochondrial disease,even a larger proportion of children,have not yet been made a molecular genetic diagnosis.The study of mitochondrial diseases is therefore complicated because the same genetic and biochemical defect could cause various phenotypes,and vice versa.Leigh syndrome(LS)is the most frequent mitochondrial encephalopathy in infancy and childhood.The incidence was estimated at 1/40,000.It has phenotypic and genotypic heterogeneity.The common clinical features include development retardation or regression,feeding difficulties,hypotonia,ophthalmoplegia,ataxia,seizures,etc.The prognosis is poor with a high mortality.Patients could die within 2 years since the onset of symptoms.However,there were also late onset patients or cases with a relatively slow progression.To date,the pathogenic mutations have been identified in more than 75 genes,consistent with maternal,autosomal or X-linked inheritance.The development of molecular genetics has led to the discovery of more and more disease-causing genes.The clinicians may misdiagnose LS as something else,or misdiagnose other similar conditions as LS due to the lack of understanding of the correlation between genotypes and phenotypes.Currently,there is no definite cure for LS,and clinical management was mainly directed to relieving the symptoms.Children with LS impose considerable psychological and financial burdens on their parents.In addition,we found a special case carrying mtDNA G14459A mutation.The G14459A mutation have been reported in phenotypes of Leber hereditary optic neuropathy(LHON),dystonia,LHON plus dystonia(LDYT),LS or asymptomatic,But,our proband had specific clinical manifestations,neuroimaging and molecular genetic characteristics.We made the study on these two aspects.Part ? Clinical,neuroimaging,pathological and genetic analyses of Leigh syndrome patientsObjectiveWe aimed to analyze the clinical symptoms,neuroimaging,biochemical examinations,muscular histopathology,and genotypes of LS patients to provide clinical experience for the early diagnosis of LS.Subjects and MethodsWe retrospectively analyzed the patients diagnosed with LS between January 2000 and December 2018 in Qilu Hospital of Shandong University.The subjects met the diagnostic criteria proposed by Baertling et al:(1)progressive neurological symptoms of LS,(2)mitochondrial dysfunction resulted from a genetic defect,and(3)bilateral central nervous system lesions that can be associated with further abnormalities in diagnostic imaging(symmetrical lesions located in the basal ganglia and/or brain stem).Participants were excluded with any of the followings:(1)incomplete clinical history or physical examination data,(2)definite diagnosis with other diseases other than LS,(3)refusing informed consent or pedigree verification.The total DNA was derived from specimens of patients' peripheral venous blood or biopsied skeletal muscles.The"3243","8344","8993" and "10191" mutations of mtDNA were targeted for Sanger sequencing.The whole mtDNA or exon related to mitochondrial diseases sequencing were carried out for some patients directly or patients with negative Sanger sequencing.Genotype analysis was performed.The clinical manifestations,brain magnetic resonance imaging(MRI)and biochemical examinations were analyzed.The levels of urinary organic acids,plasma amino acids,and acylcarnitines were examined with gas chromatography-mass spectrometry and tandem mass spectrometry.The histopathological characteristics of biopsied muscles were observed under microscope.ResultsA total of 16 patients(10 males;6 females)were involved.The onset ages ranged from 3 months to 10 years,with 6 patients younger than 2 years(1 male:5 females)and 10 late-onset patients older than 2 years(9 males:1 female).There was statistical difference about sex between the early-onset and late-onset patients.The 2 patients with nDNA mutations had onset ages less than 1 year.There were 10 late-onset and 4 early-onset patients with mtDNA mutations,including 1 conformed to LS/MELAS overlap syndrome.The initial symptoms were developmental delay,strabismus,tremor,walking unstable,ptosis,feeding difficulties,exercise intolerance and abdominal pain.The clinical manifestations included weakness(10/16,62.5%),abnormal ophthalmology(10/16,62.5%),hypotonia or hypertonia(9/16,56.3%),abnormal tendon reflex(7/16,43.8%),ataxia(6/16,37.5%),tremor(6/16,37.5%),mental retardation(5/16,31.3%),short stature(4/16,25%),respiratory distress(4/1 6,25%),seizures(1/16,6.5%),etc.There were 8 cases with strabismus,4 with ptosis,3 with nystagmus and 1 with optic nerve atrophy among the 10 patients with abnormal ophthalmology;and the early-onset and late-onset patients were 2 and 8 respectively.Among the 4 patients with respiratory disorder,3 had early-onset ages less than 1 year.However,there was no significant difference about these clinical manifestations between the early-onset and late-onset patients except that all 6 patients with ataxia were late-onset.Renal tubular acidosis,diabetes,left ventricular noncompaction,preexcitation syndrome,atrioventricular block were also found in these subjects.All the participants received cranial MRI scan with lesions in brainstem(14/16,87.5%),especially midbrain(14/16,87.5%),followed by basal ganglia(8/16,50%),thalamus(8/16,50%),cerebellum(5/16,31.3%),supratentorial white matter(2/16,12.5%)and cortex(2/16,2.5%).Four patients had lesions located in thalamus without basal ganglia involvement.There was no significant difference in the distribution of lesion sites between the early-onset and late-onset patientsThe levels of blood lactate were increased in 8 among the 16 patients(8/16,50%).Cerebrospinal fluid(CSF)lactic acid levels were measured in 8 patients,and elevated in 7(7/8,87.5%).Urine organic acid examination in 5 patients showed that 1 patient had increased levels of 3-hydroxypropionic acid and 3-methyIpentenedioic acid(1/5).The results were normal in all the 4 patients examined for plasma anino acids and acylcarnitinesA total of 9 patients accepted muscular biopsy.The pathological findings showed 2 patients with ragged red fibers(RRF)and 1 patient with subsarcolemmal mitochondrial proliferation,and they were all older than 9 years at biopsy.Cytochrome c oxidase(COX)deficiency was found in 2 patients.Lipid accumulation was common.Gene sequencing demonstrated 3 patients with T10191C mutation in MT-ND3 gene,1 with C11777A mutation in MT-ND4 gene,1 with G11778A mutation in MT-ND4 gene,3 with G13513A mutation in MT-ND5 gene,and 1 with G14459A mutation in MT-ND6 gene.Four patients had mutations in MT-ATP6 gene,including3 with T8993C mutation and 1 with T9185C mutation.A3243G mutation in MT-TL1 gene was found in 1 patient.Two patients had nDNA mutations in NDUFAF5 gene(c.145C>T and c.238G>C)and SDHA gene(c.163 DC and c.934C>T)respectively,and all the 4 nucleotide variations were previously unreported.There were 10 patients with follow-up data.Four patients died 1 month,3 months,6 months and 6 years,respectively after the diagnosis;in addition,the former 3 were all early-onset patients with respiratory distress at their first visit.The condition was stable or aggravated in the rest 6 patients with follow-up period from 3 months to 4 years.Conclusions1.In our study,patients with mtDNA mutations were mainly late onset.Females predominated in the early-onset LS patients,while males predominated in the late onset.2.Weakness and ophthalmological abnormality were the most common clinical manifestations,and strabismus was the most frequent in the latter.Seizure wasn't common.Ataxia was more common in the late-onset patients.Early-onset LS patients with respiratory disorder had a more serious condition and poorer prognosis.3.Brainstem,especially midbrain,was the most frequent lesion site,followed by basal ganglia and thalamus.Thalamic lesions may not be accompanied with lesions in basal ganglia.The raised CSF lactate levels might help to make a suspect of mitochondrial disease,although not a mandatory requirement for LS diagnosis.4.The typical RRF was rare in muscular pathology.For the suspected diagnosis of LS,whole mitochondrial and/or high-throughput exon sequencing was a recommendation.MT-ATP6,MT-ND3 and MT-ND5 genes were the most frequent disease-causing genes in mtDNA for LS;in addition,T8993C,T10191C and G13513A mutations were mutant hotspots,especially for the late-onset patients.The early molecular diagnosis and timely treatment of LS will hopefully extend the life span of patients and improve their quality of life.Part ? Mechanism of the special phenotype caused by mtDNA G14459A mutationObjectiveIt was designed to report the special phenotype of a patient wit'h mtDNA G14459A and A6064T mutations and to explore the pathogenicity of G14459A mutation.Materials and MethodsWe described the clinical features of the young female proband.She accepted muscle biopsy.Whole-length mtDNA sequencing from blood sample was performed.The lymphoblastic cell lines derived from the proband's blood were immortalized by transformation with the Epstein-Barr virus.The cybrids were constructed by fusing mitochondria from the probanda platelets to mtDNA-deficient(pO)cells.Cybrids harboring homogeneous G14459A mutation were classified as the mutant type cybrids,while those without G14459A mutation were classified as the wild type cybrids.The activities of mitochondrial respiratory chain complex in immortalized lymphoblasts were tested by spectrophotometer.The oxygen consumption rates(OCR)of the cybrids were measured with a Seahorse Bioscience XF-24 extracellular flux analyzer.The mitochondrial membrane potential levels and the production of ROS in cybrids were also detected.ResultsThe 22-year-old female proband was born to an unrelated couple.She was in good health until the occurrence of painless vision decline in both eyes at age 15.She developed tonic-clonic seizures,dystonia in right upper limb and left lower limb,unsteady gait since 18 years old.Neurological examinations showed basically normal intelligence with mini-mental state examination(MMSE)scores of 28,dysarthria,dystonia in the right upper extremity and left lower extremity and ataxia.Ophthalmic examination showed bilateral visual acuity at 0.1,pale temporal quadrant of optic discs and thinning retinal nerve fiber layer(RNFL)in both eyes.Laboratory examination revealed an elevated level of lactate(2.7 mmol/L,normal range 1.2-2.1 mmol/L)in CSF.The brain MRI showed abnormal signals in bilateral precentral gyri without basal ganglia involvement at age 18.The lesions located in bilateral putamen on MRI were found at age 21.Histopathology of biopsied muscle showed no mitochondrial myopathy change.Whole-length mtDNA sequencing identified heterogeneous G14459A mutation in MT?ND3 gene,and previously unreported and nearly homogenous A6064T mutation in MT-COI gene.One brother of the proband died with "fever,weakness and dyspnea" at age 4.The 7-year-old little brother was asymptomatic.The fundus examination of her mother revealed optic atrophy.Both the mother and little brother had G14459A and A6064T mutations.The activity of respiratory chain complex ? in immortalized lymphoblasts of the proband was decreased by 50%relative to that of the normal control.There was no difference about the activities of complex ?,? and IV between the proband and normal control.The basal OCR,maximal OCR,and the ATP-linked OCR of the mutant type cybrid cells were reduced by 55.8%,56.3%and 60.3%respectively compared with those of the wild type cybrid cells.The mutant type cybrids had a 64%reduction in the levels of mitochondrial membrane potential compared with the wild type cybrids.The production of ROS in mutant type cybrids was a 104%increase relative to the wild type cybrids.Conclusions1.We reported the special phenotype with mtDNA G14459A mutation characterized by vision decline,non-childhood onset dystonia and seizures showing bilateral lesions in the frontal cortex preceding those in the basal ganglia.Our study further expanded the spectrum of clinical phenotype associated with G14459A mutation.2.The decreased activity of mitochondrial respiratory chain complex I,declined mitochondrial membrane potential level,increased production of ROS,and defects in mitochondrial respiration were the mechanism of mtDNA G14459A mutation causing the particular clinical phenotype.Additionally,the previously unreported mtDNA A6064T mutation might affect the clinical phenotype with G14459A mutation,which requires further study. |
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