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Article

Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review

by
Phepy G. A. Dawod
1,2,
Jasna Jancic
1,3,
Ana Marjanovic
1,
Marija Brankovic
1,
Milena Jankovic
4,
Janko Samardzic
5,
Ayman Gamil Anwar Dawod
6,
Ivana Novakovic
1,
Fayda I. Abdel Motaleb
2,
Vladimir Radlovic
1,7,
Vladimir S. Kostic
1,4 and
Dejan Nikolic
1,8,*
1
Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia
2
Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Ain Shams University, Cairo 11591, Egypt
3
Clinic of Neurology and Psychiatry of Children and Youth, 11000 Belgrade, Serbia
4
Neurology Clinic, Clinical Center of Serbia, 11000 Belgrade, Serbia
5
Institute of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia
6
Internal Medicine, Hepatogastroenterology and Endoscopy Department, Faculty of Medicine, Ain Shams University, Cairo 11591, Egypt
7
Pediatric Surgery Department, University Children’s Hospital, 11000 Belgrade, Serbia
8
Physical Medicine and Rehabilitation Department, University Children’s Hospital, Tirsova 10, 11000 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Diagnostics 2021, 11(11), 1969; https://doi.org/10.3390/diagnostics11111969
Submission received: 5 September 2021 / Revised: 19 October 2021 / Accepted: 21 October 2021 / Published: 23 October 2021
(This article belongs to the Special Issue Cyto-Histopathological Correlations in Pathology Diagnostics)
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Abstract

:
Mitochondrial encephalomyopathies (MEMP) are heterogeneous multisystem disorders frequently associated with mitochondrial DNA (mtDNA) mutations. Clinical presentation varies considerably in age of onset, course, and severity up to death in early childhood. In this study, we performed molecular genetic analysis for mtDNA pathogenic mutation detection in Serbian children, preliminary diagnosed clinically, biochemically and by brain imaging for mitochondrial encephalomyopathies disorders. Sanger sequencing analysis in three Serbian probands revealed two known pathogenic mutations. Two probands had a heteroplasmic point mutation m.3243A>G in the MT-TL1 gene, which confirmed mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome (MELAS), while a single case clinically manifested for Leigh syndrome had an almost homoplasmic (close to 100%) m.8993T>G mutation in the MT-ATP6 gene. After full mtDNA MITOMASTER analysis and PhyloTree build 17, we report MELAS’ association with haplogroups U and H (U2e and H15 subclades); likewise, the mtDNA-associated Leigh syndrome proband shows a preference for haplogroup H (H34 subclade). Based on clinical–genetic correlation, we suggest that haplogroup H may contribute to the mitochondrial encephalomyopathies’ phenotypic variability of the patients in our study. We conclude that genetic studies for the distinctive mitochondrial encephalomyopathies should be well-considered for realizing clinical severity and possible outcomes.

1. Introduction

Mitochondrial encephalomyopathies (MEMP) are clinically and genetically heterogeneous group of neurometabolic disorders resulting from abnormal mitochondrial function [1]. They are represented with various clinical syndromes sharing the oxidative phosphorylation deficiency due to alteration in the enzymes essential to the production of ATP in mitochondria. In general, MEMPs are caused by mutations in genes that control mitochondrial function (mitochondrial or nuclear genome), and can be expressed in many tissues [2]. The unifying feature of MEMP is progressive neurodegeneration of the central nervous system, causing encephalopathy that affects cognition, movement, strength, coordination, sensation, vision, or autonomic control. Non-nervous system tissues such as muscle tissue may also be prominently affected [3,4,5]. These disorders include mitochondrial encephalopathy, lactic acidosis, and stroke like-episodes (MELAS) syndrome, myoclonic epilepsy with ragged red fibers (MERRF) syndrome, Kearns–Sayre syndrome (KSS), maternally inherited Leigh syndrome (MILS), and neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome. The mtDNA mutations that cause these disorders are generally heteroplasmic, and the age of disease onset is relatively early [6,7,8,9]. More than 200 mtDNA mutations were discovered associated with MEMP and novel mutations are still being reported [10]. Pathogenic mtDNA changes could be maternally inherited or arise de novo [11,12,13].
Leigh syndrome (also called Leigh disease and sub-acute necrotizing encephalomyelopathy, OMIM 25600, LS) is considered the most common progressive and severe neurodegenerative disorder in children with onset within the first months or years of life. LS is characterized by psychomotor regression with progressive loss of mental and movement abilities and may result in fatal encephalopathy [14,15]. The disorder could be associated with mutations in more than 75 genes that have been identified in both the nuclear and mitochondrial genome; about 20% of LS cases are caused by mtDNA mutations [16,17,18]. Point mutations at m.8993T>G or the less severe m.8993T>C in MT-ATP6 gene in the complex V are the most frequent LS-associated mtDNA changes. LS occurs when mutation load is greater than 90%, with subsequent substitution of the highly evolutionary conserved leucine at position 156 to either an arginine or a proline [19,20,21], affecting the protein component of the F1F0-ATPase that directly blocks ATP generation [22]. Furthermore, both mutations are associated with NARP syndrome when mutation load is around 50–60% [23,24].
MELAS (OMIM# 540000) is an example of MEMPs related to mitochondrial tRNA gene changes. In this syndrome, the predominant mtDNA mutations are in mitochondrial tRNALeu, invariably as heteroplasmics [25,26]. More than 80% of all cases of MELAS are caused by a substitution of m.3243A>G in the MT-TL1 gene. This nucleotide replacement disrupts the correct 3D folding structure of tRNALeu by affecting the anticodon wobble base pair of mt-tRNA molecules by reducing the capacity for amino-acylation and methylation. Besides that, this mutation interferes with the 16S RNA molecule transcription and termination site with subsequent accumulation of unprocessed RNA [27,28,29]. Percentages of mutant mtDNA cause different degrees of the energetic defects which are presented clinically by variable phenotypes of MELAS, with central nervous system involvement when the mutant is present at higher percentages including stroke-like episodes, seizures, cortical blindness, and dementia. MELAS is also accompanied by features of myopathy, recurrent headaches, short stature, and episodic vomiting resulting from lactic acidosis [30,31,32].
Several specific mtDNA haplogroups have been associated with different neurodegenerative mitochondriopathies, among them LHON, which is caused by mtDNA mutations [33,34], as well as Parkinson’s disease [35], and Alzheimer’s disease [36], which are associated with mitochondrial dysfunction. Furthermore, haplogroup studies have shown significant roles of both diagnostic features [37] and therapeutic susceptibility [38,39] in various conditions, encouraging targeted mtDNA mutations testing jointly with mtDNA haplogroup determination. However, previous studies did not confirmed the association with MELAS and Leigh syndromes [40].
Here, we present results of the molecular-genetic study for pathogenic mtDNA primary mutations in Serbian probands preliminary diagnosed as MEMP and correlations of genetic data with clinical phenotypes of the probands. Additionally, we analysed mtDNA haplotypes of detected mutations and constructed the phylogenic tree according to previously described concepts [41,42].

2. Patients and Methods

2.1. Patients

Three Serbian unrelated children included in this study showed features for MEMP syndromes revealed by their clinical evaluation; two of them were suspected for MELAS (proband 1 and proband 2) and one for LS (proband 3). In all cases family history was negative. Probands were recruited from child neurology units at the Clinic for Neurology and Psychiatry for Children and Youth, and Institute for Health Protection of Mother and Child of Serbia, Belgrade, Serbia. Detailed neurological assessments including standardized testing procedures and biochemical and neuroimaging investigations were conducted. All molecular genetic analyses were performed in the laboratory for genetic and molecular diagnostics of neurological disorders, Neurology Clinic, Clinical Centre of Serbia, Belgrade, Serbia.

2.2. Ethical Considerations

Conveniently informed consent was obtained from probands’ parents which was then reviewed by the ethical committee (Number: 2650/VI-1, approved on 26 June 2018) of the Faculty of Medicine, University of Belgrade, who provided ethical approval for this study.

2.3. Molecular Genetic Methods

DNA for genetic analyses was extracted from 5 mL peripheral blood samples following the manufacturer’s protocol (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA. Targeted Sanger sequencing to check for m.3243A>G and m.8993T>G pathogenic variants as major causes of MELAS and Leigh syndrome, respectively, was initially performed. PCR was used to amplify specific mitochondrial DNA fragments by using appropriated primers according to Taylor et al., 2001 [43]. MELAS point mutation m.3243A>G is within the mtDNA fragment encompassed with forward primer 5′-GGATCAGGACATCCCGATG-3′ (MT 5F), and a reverse primer 5′-CACCTCTAGCCTAGCCGTT-3′ (MT 5R). Leigh syndrome point mutation m.8993T>G is located inside the mtDNA fragment surrounded with forward primer 5′- ACAATCCTAGGCCTACCCG-3′ (MT 14F), and reverse primer 5′-CCACCAATCACATGCCTATC-3′ (MT 14R). PCR was performed by adding 1 µL of patient DNA sample to a total volume of 12.5 µL solution containing 10× DreamTaq Buffer, 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.5 mM of appropriate both forward and reverse primers, 0.5 U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and 15 µg of Bovine Serum Albumin (BSA). The purified fragments were cleaned up by ExoSAP-enzymatic reaction. Fluorescence-based cycle sequencing was performed by applying a BigDye Terminator v3.1 Cycle Sequencing Kit according to the standard protocols, and more purification was done by alcohol-based nucleic acid ethanol precipitation. Capillary electrophoresis has been used for automated DNA sequencing on ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The more detailed description of procedures is previously provided in Dawod et al., 2020 [44].

2.4. Haplogroups Analysis and Phylogenetic Tree Reconstruction

Our probands with detected mtDNA primary mutations m.3243A>G and m.8993T>G pathognomonic for LS and MELAS, respectively, underwent entire mtDNA sequencing by using the appreciated primers mentioned in previous studies [44]. We determined the predicted haplogroups for all included probands by MITOMASTER analysis (https://www.mitomap.org/foswiki/bin/view/MITOMASTER/WebHome, accessed on 15 August 2021) [45]. All defined haplotypes were assigned regarding the PhyloTree build 17 for Phylogenetic tree reconstruction (https://www.phylotree.org/ accessed on 25 August 2021) [46].

2.5. Bioinformatics Analysis

Alignment and comparison of mtDNA variants were adapted with rCRS “the Revised Cambridge Reference Sequence” (accession NC_012920) by Sequencer DNA Sequence Analysis Software [47]. The MITOMAP database system for the human mitochondrial genome (http://www.mitomap.org/MITOMAP accessed on 15 August 2021) and GenBank for Human Mitochondrial Genome Database (http://www.ncbi.nlm.nih.gov/Genbank/index.html, accessed 15 August 2021) were used for analysis of the detected variants [48,49].
The pathogenicity of nonsynonymous mtDNA sequence changes in mtDNA coding regions have been determined by protein-based metrics using in silico predictive software. We used the Polymorphism PolyPhen-2 database (Polymorphism Phenotyping v2, http://genetics.bwh.harvard.edu/pph2/, accessed on 19 August 2021) and PROVEAN (Protein Variation Effect Analyzer, http://provean.jcvi.org accessed on 19 August 2021) for predicting effects of substitution of amino acids on protein function [50,51]. Meanwhile, PANTHER (Protein ANalysis THrough Evolutionary Relationships, (http://pantherdb.org/panther/summaryStats.jsp accessed on 19 August 2021) was tested as a source for evolutionary history classification of protein sequences [52]. The pathogenic characteristics of mutations in tRNA of mtDNA were evaluated by the MitoTIP scoring system (https://www.mitomap.org/MITOMAP/MitoTipInfo accessed on 19 August 2021) [53]. Furthermore, the Mamit-tRNA database that contains mammalian mitochondrial tRNAs was tested as it provides extensive documentation of polymorphisms and mutations in mitochondrial tRNA genes related to human mitochondrial disorders and deciphering the 2D cloverleaf secondary structures of mitochondrial tRNA (http://mamit-tRNA.u-strasbg.fr accessed on 19 August 2021) [54].

3. Results

3.1. Mutational Genetic Analysis

Heteroplasmic mtDNA mutation m.3243A>G in the MT-TL1 gene, which encodes mitochondrial tRNALeu, has been detected in two probands which were clinically corresponding to MELAS diagnosis (proband 1 and proband 2). Mutation m.8993T>G in the MT-ATP6 gene (F-ATPase protein 6) specific for Leigh disease was detected in proband 3; this mutation was almost homoplasmic (close to 100%) (Figure 1).
In silico predictive software was used for determination of the pathogenic characteristics of mtDNA mutations, nonsynonymous change m.8993T>G in MT-ATP6 gene causing substitution of a hydrophobic leucine residue into a charged arginine residue (L156R) in a highly conserved part of the ATP6 subunit that it has probably damaging effect on the protein function with a score of 0.998 on HumVar Polymorphism PolyPhen-2 database, as well on evolutionary history classification of protein sequences by PANTHER software. Concurrently, PROVEAN showed this amino acid substitution is deleterious on protein function. On another side, different specified informatics predictors were used for assessment of mitochondrial tRNA variant m.3243A>G in the MT-TL1 gene such as MitoTIP is accessed within MITOMAP, besides, Mamit-tRNA databases, both of them have proven m.3243A>G is a pathogenic mutation in the D-loop of the mt-tRNALeu with a probably damaging impact on its structure (Table 1).

3.2. MITOMASTER Analysis

In our study, entire mtDNA sequencing for all included Serbian probands was carried out for haplogroup analysis, and fasta-formatted files were submitted to MITOMASTER. The results showed that the most frequent MELAS m.3243A>G mutation was associated with both haplogroup H and U, while a single case of mtDNA-associated Leigh syndrome showed predilection for haplogroup H. Furthermore, our analysis reported forty-four polymorphic variants that scattered all over mtDNA fragments at m.55T>C, m.56insC, m.73A>G, m.143G>A, m.152T>C, m.263A>G, m.315insC, m.508A>G, m.739C>T, m.750A>G, m.1438A>G, m.1811A>G, m.2706A>G, m.3116C>T, m.3720A>G, m.3847T>C, m.4769A>G, m.5390A>G, m.5426T>C, m.6045C>T, m.6152T>C, m.6253T>C, m.7028C>T, m.8860A>G, m.10876A>G, m.11197C>T, m.11467A>G, m.11719G>A, m.12308A>G, m.12372G>A, m.13020T>C, m.13359G>A, m.14766C>T, m.15326A>G, m.15519T>C, m.15907A>G, m.15948A>G, m.16051A>G, m.16093T>C, m.16129G>C, m.16183A>C, m.16189T>C, m.16291C>T and m.16519T>C; most of them presented in different frequencies in different haplogroups (Table 2).

3.3. Phylogenetic Tree Construction

All detected variants are represented for more explication and construction of the phylogenetic tree compared with the rCRS haplogroup (H2a2a) for establishment of the haplogroup affiliation and motifs, following the nomenclature of mtDNA tree Build 17 (Figure 2).

3.4. Genotype-Phenotype Relationship

Clinical evaluation, laboratory data and brain imaging of positive m.3243A>G and m.8993T>G probands revealed phenotype features of mitochondrial encephalomyopathies (Table 3). Neurological impairment was the most common feature indicating that two probands (P1 and P2) meet the clinical diagnostic criteria for MELAS with onset during the second decade of life and one single case for Leigh syndrome (P3) with age of onset being within a few months of birth. MELAS probands experienced epileptic seizures, psychosis, muscle weakness, hemiparesis, altered conscious, dementia and associated headache and vomiting, while the Leigh proband presented with symptoms of psychomotor retardation preceded by respiratory viral infection, severe early onset of series epileptic attacks with breaks of 10 min between the attacks up to 30 epileptic attacks per day associated with twitches of the left-sided extremities, sometimes followed by twitches of the left half of the face with deviation of the eyes and the head to the right side, other symptoms of speech delay and muscle weakness.
Neurological assessments were done to estimate frequencies of epileptic attacks, sluggishness of the motor system, muscle weakness, generalized dystonia, dementia, and marked irritability in behaviour. Milestone developmental progression for early childhood assessment was also carried out. Furthermore, laboratory assessment of lactate in blood and cerebrospinal fluid (CSF) was estimated, which showed marked elevation. Brain magnetic resonance imaging (MRI) revealed an expended right ventricle of the brain with an extensive zone of the oedematous cortex and areas of abnormal high signal on Fluid Attenuated Inversion Recovery (FLAIR) images, with changes of T2-weight (T2W) which corresponded to MELAS diagnosis, whereas the specific imaging finding for LS is reported as subcortical necrotizing encephalopathy with symmetrical lesions of basal ganglia, and the brain stem as mesencephalon, tectum, substantia nigra and hypoplasia pons with atrophy of the vermis and cerebellar hemisphere. Necrosis was accompanied by mild lateral ventricular dilatation; moreover, cortex hyperintensity on T2- weighted MR imaging was recorded.
Leigh patients had a severe early onset of epileptic attacks with breaks of 10 min between attacks. Up to 30 epileptic attacks per day had been recorded, which were associated with twitches of the left-sided extremities and sometimes followed by twitches of the left half of the face with deviation of the eyes and the head to the right side. Meanwhile, the MELAS probands experienced psychiatric symptoms, epileptic attacks, muscle weakness and hemiparesis. All probands showed significant laboratory and brain image changes.

4. Discussion

Disorders of mitochondrial encephalomyopathies are the most frequent group of inherited neurogenetic disorders, caused by point mutations in mtDNA that disrupt the formation of mitochondrially encoded respiratory chain subunits and therefore cause respiratory chain dysfunction. MEMP mainly presented clinically by different phenotypes [55,56,57]. By direct Sanger sequencing of mtDNA in this study, our genetic analysis reported the first sporadic case of mtDNA-associated Leigh syndrome disclosed in a four year old Serbian girl (P3) who was diagnosed according to the criteria declared by Rahman et al. for a neurodegenerative disease with psychomotor developmental retardation, sluggishness of the motor system, muscle weakness, sever epileptic attacks, nystagmus, dystonia and regression in infancy as a results of basal ganglia and/or brainstem damage accompanied by the characteristic features of hyperintense lesions on T2-weighted on MRI and biochemical lactosidosis [58,59]. By our molecular genetic sequencing, we precisely diagnosed mtDNA-associated Leigh syndrome by our finding of a nearly homoplasmic m.8993T>G mutation that correlates the severity of the disease; m.8993T>G was detected in protein-coding gene MT-ATP6 causing replacement of the strongly conserved leucine to an arginine at position 156 in complex V with subsequent blocking of the terminal step in oxidative phosphorylation. The pathogenicity of this amino acid substitution in MT-ATP6 (UniProt ID P00846) was checked according to in silico software predictors; PolyPhen-2 and PANTHER considered m.8993T>G probably damaging, along with PROVEAN L156R prediction, which was deleterious (−5.18). Herein, our case showed irrelevant family history and we could not proceed with complete mtDNA sequence analysis for her family. As it is known, heteroplasmic mutation can be transmitted with different mutation loads between generations, exhibiting inter-individual variation of symptoms in the same family [13,60]. Moreover, phenotypic heterogeneity has been recorded in m.8993T>G carriers [61,62]. Our study supports the literature which displays the occurrence of m.8993T>G mutation in sporadic cases with rapid segregation toward homoplasmy [63,64,65]; it is noticeable in a single generation and reported in about 1/5 LS cases [66]. Other differential diagnoses for LS were excluded [67,68,69].
Furthermore, our results revealed a pathogenic heteroplasmic m.3243A>G mutation in the MT-TL1 gene with a defect in the protein synthesis of mitochondrial tRNALeu in two teenage sporadic Serbian children (P1 and P2) who experienced MELAS symptoms. Upon our diagnosis for MELAS cases, it fully fit the Japanese criteria for phenotypic and laboratory findings required for definitive MELAS diagnosis through our reporting of more than two clinical findings of stroke-like episodes, including headache with vomiting, seizures and hemiplegia, which appeared in childhood following a period of normal development with undistinguished family history, plus two evidences of mitochondrial dysfunction detected by high lactate levels in plasma and the decisive molecular finding of m.3243A>G mutation [70]. Otherwise, our MELAS diagnosis did not followed Hirano’s diagnostic criteria by lacking clinical–brain imaging correlation for definite diagnosis of stroke-like episodes [71], which can sometimes be missed within variable ages as mentioned in previous literatures [72,73]; the other systemic symptoms and signs of mitochondrial disorder, such as short stature, diabetes mellitus, deafness, ophthalmoplegia or heart failure were not observed. On the other hand, “silent” m.3243A>G mutation carriers were reported in previous studies; they commonly present with autonomic dysfunction [74] and less neuropsychiatric symptoms [75,76], without an overt full MELAS clinical picture. They have a lower mortality than MELAS probands [77]; for that, strict follow up of their metabolite biomarkers is recommended for predicting their potentiality for MELAS [78] and encouraging a stress-free life and ketogenic diet for keeping healthy mitochondria [79]. In our study, relatives of the probands were unavailable for analysis. The pathogenicity of m.3243A>G in MT-TL1 was confirmed by software specific for mitochondrial tRNA mutations, MitoTIP recorded m.3243A>G as possibly pathogenic (54.30%), and Mamit-tRNA databases for more detailed tRNA 2-D structures also substantiated m.3243A>G pathogenicity.
The strong genotype–phenotype correlations in MELAS and Leigh disease have been discussed in the literature. Our LS proband’s phenotype is consistent with Sofou et al.’s 2018 study regarding the onset of disease and severity of m.8993T>G mutation in MT-ATP6 which is preferentially presented with repeated epileptic attacks [80]. Phenotype diversity of Leigh disease has been reported for m.8993T>C mutation in the same MT-ATP6 gene [81]; noticeably, other unusual presentations rather than neurological ones were found with other mitochondrial and nuclear-encoded genes, causing Leigh disease to be ocular and gastrointestinal with MT-ND mutations [82,83], cardiac with NDUF [84,85] and renal with SURF1 and ACAD9 gene defects [86,87]. Previous studies have reported many mitochondrial mutations causing classic MELAS without any detectable phenotypic specificity [88,89]; exceptional renal diseases were detected in association with m.3243 and m.13513G>A as a first manifestation of MELAS [90,91]. Furthermore, MELAS m.3243 was associated with uncommon presentations such as cardiac and ketoacidosis [92,93].
Evolutionary European mtDNA haplogroups were detected by MITOMASTER analysis in our patients, in which MELAS showed preference for U and H haplogroups with collection of their associated haplotypes. In Proband one, thirty three different homoplasmic sequence variants were identified; twenty eight of them are haplotypes for haplogroup U2e regarding PhyloTree build 17, included defining mutational haplogroup U markers at m.11467A>G, m.12308A>G and m.12372G>A, and ancestral markers motifs at m.73A>G, m.263A>G, m.750A>G, m.1438A>G, m.2706A>G, m.4769A>G, m.7028C>T, m.8860A>G, m.11719G>A, m.14766C>T and m.15326A>G. All were found to be widely distributed across our sample. Furthermore, the characteristic mutation m.1811A>G for U’2’3’4’7’8’9 the common ancestor of haplogroup U was detected, with subdivision to European U2e on the basis of our finding of characteristic non-coding variants at m.152T>C, m.508A>G, m.15907A>G, m.16051A>G, m.16129G>C and m.16189T>C and the synonymous SNPs at m.3720A>G, m.5390A>G, m.5426T>C, m.6045C>T, m.6152T>C, m.10876A>G and m.13020T>C. The other five variants were detected in our analysis at m.739C>T, m.3116C>T, m.11197C>T, m.13359G>A and m.16183A>C, which are considered nonspecific to haplogroup U2e regarding PhyloTree build 17, although all of them with the exception of m.13359G>A have lower frequencies in haplogroup U2e by Mitomaster analysis (0.29%, 22.71%, 22.42% and 75.52%, respectively) (GenBank ID KY930472.1 and AY339545.1); interestingly, three of them, m.739C>T, m.3116C>T and m.13359G>A, were detected as polymorphisms and pathogenic mutations in different other diseases associated with aminoglycoside-induced hearing loss [94,95,96,97], whereas haplotypes for haplogroup H, a subclass of haplogroup HV, were shown to be associated with the second MELAS proband. Ancestral marker motifs were detected at m.263A>G, m.750A>G, m.1438A>G, m.4769A>G, m.8860A>G, m.15326A>G, and m.16519C>T; haplogroup H selected markers were detected at m.2706G>A and m.7028C>T. This MELAS proband was characterized as the H15 subclass, and we detected variants for haplogroup H15 at m.55T>C and m.6253T>C which further subdivided to H15b in the presence of m.3847T>C. In addition we detected two other non-coding variants: insertion C at m.56, which has very low frequency in association to haplogroup H15b on MITOMASTER (GenBank ID KF162889.1), and m.143G>A that did not reported previously in association to that haplogroup. Our results are consistent with European haplogroups [98], and also in agreement with Caucasian and Spanish population studies for MELAS m.3243A>G mutation which reported its association with both the most represented haplogroup H and haplogroup U without any predilection for affecting MELAS phenotypic expression [40,99]; however, another study reported MELAS low representation on haplogroup J in French patients [100]. In contrast to this, the Spanish population did not show any haplogroup preference [40]. Concerning the geographical variations effect, we can notice that the native American haplogroup B2 was exhibited in Mexican MELAS females [101]; likewise, Eastern Asian haplogroups were detected in Chinese MELAS pedigrees [102] and in Indian MELAS patients [103].
The Leigh disease proband exhibited a preference for haplogroup H, subclade H34 corroborated by detection of non-synonymous substitution at m.15519T>C and m.16291C>T. Further, full sequence analysis detected other polymorphic variants m.152T>C, 315insC and 16093T>C not reported on PhyloTree build 17, but published before on Mitomaster haplogroup H34 (GenBank ID JQ702662.1 and KY797254.2). Furthermore, analysis revealed two variants m.508A>G and m.15948 A>G which is not reported on either the PhyloTree build 17 or Mitomaster for that group; the m.15948 in MT-TT is a conventional tRNA in the acceptor stem domain of threonine and the transition of A to G is considered possibly benign (29.90%) by MitoTIP. Our results are in line with other previous reported Leigh pedigrees that have showed more preference for subclades of haplogroup H; for example, H1r1 in Spanish pedigree harbouring the LS m.1555A>G Mutation in MT-RNR1 [104], and an Indian Leigh case study harbouring m.8993T>C mutation in the MT-ATP6 gene, which was found defining SNP for haplogroup H [105]. Haplogroup heterogeneity for Leigh syndrome has been reported in association with different Leigh-causing mutations; for instance, H13 has been found in association with MILS ATP6 mutant cell lines. Otherwise, our reporting is not in accordance with other studies that have observed susceptibility of Leigh disease on other haplogroups rather than haplogroup H, such as N9a, B5 and Y in Chinese patients [106,107], and haplogroup U5b on mutant cell lines [108]; all are descended from the macro-haplogroup N. Asian haplogroup M is also reported in another Indian Leigh case as harbouring m.8936T>A in the MT-ATP6 gene [109].
Herein, our suggestion that haplogroup H may increase risk to Leigh disease is due to the early onset of severe symptoms in our proband according to Hong et al.’s classification, presented with delayed development under 1 year of age, followed by up to 30 epileptic seizure attacks per day and motor weakness [110]; our prospect is also supported by other literatures which indicated that haplogroup H increases the tendency for other neurodegenerative disorders such as Alzheimer’s disease [111,112], Parkinson’s disease [113], Huntington’s disease [114], amyotrophic lateral sclerosis [115] and multiple sclerosis [116], and is also involved in other non-neurological degenerative disorders such as aortic stenosis [117], diabetes mellitus [118] and osteoarthritis [119,120].

5. Conclusions

Mitochondrial encephalomyopathies in Serbian children presented with specific phenotypes according to the age of onset should be taken into consideration for molecular genetic screening. Our results underscore the importance of recognizing the pathogenic mtDNA mutations and their related mitochondrial haplogroup background, aiming for better definitive diagnosis and assisting in the development of pathogenicity-based therapeutic approaches.

Author Contributions

Conceptualization, P.G.A.D., J.J. and D.N.; Methodology, P.G.A.D., A.M., M.B., M.J. and I.N.; Software, P.G.A.D. and A.M.; Validation, A.G.A.D. and J.S.; Formal Analysis, A.G.A.D. and V.R.; Investigation, P.G.A.D., J.J., A.M., M.B. and I.N.; Resources, J.J., F.I.A.M., V.S.K. and D.N.; Data Curation, J.S., A.G.A.D. and V.R.; Writing—Original Draft Preparation, P.G.A.D., J.J. and I.N.; Writing—Review and Editing, F.I.A.M., V.S.K. and D.N.; Visualization, P.G.A.D. and D.N.; Supervision, F.I.A.M., V.S.K. and D.N.; Project Administration, A.G.A.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This research is approved by the ethical committee (Number: 2650/VI-1) of the Faculty of Medicine, University of Belgrade.

Informed Consent Statement

Conveniently informed consent was obtained from probands’ parents.

Data Availability Statement

Not applicable.

Acknowledgments

This research is supported by Serbian Ministry of Education, Science and Technological Development; grants ON175090 and ON175091 (now contract No. 200110).

Conflicts of Interest

Authors declare no conflict of interest.

References

  1. Nyhan, W.L.; Hoffmann, G.F.; Hoffmann, G.F. Atlas of Inherited Metabolic Diseases, 4th ed.; CRC Press: Boca Raton, FL, USA, 2019. [Google Scholar]
  2. Sperl, W. [Diagnosis and therapy of mitochondriopathies]. Wien. Klin. Wochenschr. 1997, 109, 93–99. [Google Scholar] [PubMed]
  3. Bugiardini, E.; Mitchell, A.L.; Rosa, I.D.; Horning-Do, H.-T.; Pitmann, A.; Poole, O.V.; Holton, J.L.; Shah, S.; Woodward, C.; Hargreaves, I.; et al. MRPS25 Mutations Impair Mitochondrial Translation and Cause Encephalomyopathy. Hum. Mol. Genet. 2019, 28, 2711–2719. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Colina-Tenorio, L.; Horten, P.; Pfanner, N.; Rampelt, H. Shaping the Mitochondrial Inner Membrane in Health and Disease. J. Intern. Med. 2020, 287, 645–664. [Google Scholar] [CrossRef]
  5. Rahman, S.; Wolf, N.I. Diagnostic Workup of Patients with Mitochondrial Diseases. In Inherited Metabolic Diseases; Hoffmann, G., Zschocke, J., Nyhan, W., Eds.; Springer: Berlin/Heidelberg, Germany, 2017; pp. 521–535. [Google Scholar]
  6. DiMauro, S.; Moraes, C.T. Mitochondrial Encephalomyopathies. Arch. Neurol. 1993, 50, 1197–1208. [Google Scholar] [CrossRef] [PubMed]
  7. Fiuza-Luces, C.; Valenzuela, P.L.; Laine-Menéndez, S.; Fernández-de la Torre, M.; Bermejo-Gómez, V.; Rufián-Vázquez, L.; Arenas, J.; Martín, M.A.; Lucia, A.; Morán, M. Physical Exercise and Mitochondrial Disease: Insights from a Mouse Model. Front. Neurol. 2019, 10, 790. [Google Scholar] [CrossRef] [PubMed]
  8. Roberta, C.; Falchetti, A. Mitochondriopathies and Bone Health. Tre. Bio. Res. 2018, 1, 1–7. [Google Scholar] [CrossRef]
  9. Swerdlow, R.H. The Neurodegenerative Mitochondriopathies. J. Alzheimers Dis. 2009, 17, 737–751. [Google Scholar] [CrossRef] [Green Version]
  10. McWilliams, T.G.; Suomalainen, A. Mitochondrial DNA Can Be Inherited from Fathers, Not Just Mothers. Nature 2019, 565, 296. [Google Scholar] [CrossRef] [PubMed]
  11. Chinnery, P.F. Primary Mitochondrial Disorders Overview. In GeneReviews®; Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J., Mirzaa, G., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 1993. [Google Scholar]
  12. de Laat, P.; Janssen, M.C.H.; Alston, C.L.; Taylor, R.W.; Rodenburg, R.J.T.; Smeitink, J.A.M. Three Families with ‘de Novo’ m.3243A > G Mutation. BBA Clin. 2016, 6, 19–24. [Google Scholar] [CrossRef] [Green Version]
  13. Poulton, J.; Finsterer, J.; Yu-Wai-Man, P. Genetic Counselling for Maternally Inherited Mitochondrial Disorders. Mol. Diagn. Ther. 2017, 21, 419–429. [Google Scholar] [CrossRef] [Green Version]
  14. Inak, G.; Rybak-Wolf, A.; Lisowski, P.; Juettner, R.; Zink, A.; Mlody, B.; Glazar, P.; Secker, C.; Ciptasari, U.H.; Stenzel, W.; et al. SURF1 Mutations Causative of Leigh Syndrome Impair Human Neurogenesis. bioRxiv 2019. [Google Scholar] [CrossRef] [Green Version]
  15. Kimura, S.; Shiraishi, H.; Egawa, K.; Uchida, M.; Ueno, M. Efficacy of Perampanel for Epileptic Seizures and Daily Behavior in a Patient with Leigh Syndrome: A Case Report. Brain Dev. 2021, 43, 157–159. [Google Scholar] [CrossRef] [PubMed]
  16. Lake, N.J.; Compton, A.G.; Rahman, S.; Thorburn, D.R. Leigh Syndrome: One Disorder, More than 75 Monogenic Causes. Ann. Neurol. 2016, 79, 190–203. [Google Scholar] [CrossRef] [PubMed]
  17. Sofou, K.; Kollberg, G.; Hedberg-Oldfors, C.; Oldfors, A. The Phenotypic Variability and Natural History of NARS2 Associated Disease. Eur. J. Paediatr. Neurol. 2021, 31, 31–37. [Google Scholar] [CrossRef]
  18. Torraco, A.; Nasca, A.; Verrigni, D.; Pennisi, A.; Zaki, M.S.; Olivieri, G.; Assouline, Z.; Martinelli, D.; Maroofian, R.; Rizza, T.; et al. Novel NDUFA12 Variants Are Associated with Isolated Complex I Defect and Variable Clinical Manifestation. Hum. Mutat. 2021, 42, 699–710. [Google Scholar] [CrossRef] [PubMed]
  19. Holt, I.J.; Harding, A.E.; Petty, R.K.; Morgan-Hughes, J.A. A New Mitochondrial Disease Associated with Mitochondrial DNA Heteroplasmy. Am. J. Hum. Genet. 1990, 46, 428–433. [Google Scholar]
  20. Saneto, R.P.; Patrick, K.E.; Perez, F.A. Homoplasmy of the m. 8993 T>G Variant in a Patient without MRI Findings of Leigh Syndrome, Ataxia or Retinal Abnormalities. Mitochondrion 2021, 59, 58–62. [Google Scholar] [CrossRef]
  21. de Vries, D.D.; van Engelen, B.G.; Gabreëls, F.J.; Ruitenbeek, W.; van Oost, B.A. A Second Missense Mutation in the Mitochondrial ATPase 6 Gene in Leigh’s Syndrome. Ann. Neurol. 1993, 34, 410–412. [Google Scholar] [CrossRef]
  22. Sgarbi, G.; Baracca, A.; Lenaz, G.; Valentino, L.M.; Carelli, V.; Solaini, G. Inefficient Coupling between Proton Transport and ATP Synthesis May Be the Pathogenic Mechanism for NARP and Leigh Syndrome Resulting from the T8993G Mutation in MtDNA. Biochem. J. 2006, 395, 493–500. [Google Scholar] [CrossRef] [Green Version]
  23. Ruhoy, I.S.; Saneto, R.P. The Genetics of Leigh Syndrome and Its Implications for Clinical Practice and Risk Management. Appl. Clin. Genet. 2014, 7, 221–234. [Google Scholar]
  24. Uziel, G.; Ghezzi, D.; Zeviani, M. Infantile Mitochondrial Encephalopathy. Semin. Fetal Neonatal Med. 2011, 16, 205–215. [Google Scholar] [CrossRef] [PubMed]
  25. Schon, E.A.; Bonilla, E.; DiMauro, S. Mitochondrial DNA Mutations and Pathogenesis. J. Bioenerg. Biomembr. 1997, 29, 131–149. [Google Scholar] [CrossRef] [PubMed]
  26. Yasukawa, T.; Suzuki, T.; Ishii, N.; Ohta, S.; Watanabe, K. Wobble Modification Defect in TRNA Disturbs Codon–Anticodon Interaction in a Mitochondrial Disease. EMBO J. 2001, 20, 4794–4802. [Google Scholar] [CrossRef] [PubMed]
  27. Goto, Y.; Horai, S.; Matsuoka, T.; Koga, Y.; Nihei, K.; Kobayashi, M.; Nonaka, I. Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS): A Correlative Study of the Clinical Features and Mitochondrial DNA Mutation. Neurology 1992, 42, 545. [Google Scholar] [CrossRef] [PubMed]
  28. Van Haute, L.; Pearce, S.F.; Powell, C.A.; D’Souza, A.R.; Nicholls, T.J.; Minczuk, M. Mitochondrial Transcript Maturation and Its Disorders. J. Inherit. Metab. Dis. 2015, 38, 655–680. [Google Scholar] [CrossRef] [Green Version]
  29. Queen, R.A.; Steyn, J.S.; Lord, P.; Elson, J.L. Mitochondrial DNA Sequence Context in the Penetrance of Mitochondrial T-RNA Mutations: A Study across Multiple Lineages with Diagnostic Implications. PLoS ONE 2017, 12, e0187862. [Google Scholar] [CrossRef] [Green Version]
  30. Al Yazidi, G.; Mulder, J.; Licht, C.; Harvey, E.; Robertson, J.; Sondheimer, N.; Tein, I. Reversal of Stroke-Like Episodes With L-Arginine and Meticulous Perioperative Management of Renal Transplantation in a Patient with Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes (MELAS) Syndrome. Case Report. Neurohospitalist 2021. [Google Scholar] [CrossRef]
  31. Montagna, P.; Gallassi, R.; Medori, R.; Govoni, E.; Zeviani, M.; Mauro, S.D.; Lugaresi, E.; Andermann, F. MELAS Syndrome: Characteristic Migrainous and Epileptic Features and Maternal Transmission. Neurology 1988, 38, 751. [Google Scholar] [CrossRef]
  32. Pavlakis, S.G.; Phillips, P.C.; DiMauro, S.; De Vivo, D.C.; Rowland, L.P. Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Strokelike Episodes: A Distinctive Clinical Syndrome. Ann. Neurol. 1984, 16, 481–488. [Google Scholar] [CrossRef]
  33. Caporali, L.; Maresca, A.; Capristo, M.; Del Dotto, V.; Tagliavini, F.; Valentino, M.L.; La Morgia, C.; Carelli, V. Incomplete Penetrance in Mitochondrial Optic Neuropathies. Mitochondrion 2017, 36, 130–137. [Google Scholar] [CrossRef]
  34. Carelli, V.; Achilli, A.; Valentino, M.L.; Rengo, C.; Semino, O.; Pala, M.; Olivieri, A.; Mattiazzi, M.; Pallotti, F.; Carrara, F.; et al. Haplogroup Effects and Recombination of Mitochondrial DNA: Novel Clues from the Analysis of Leber Hereditary Optic Neuropathy Pedigrees. Am. J. Hum. Genet. 2006, 78, 564–574. [Google Scholar] [CrossRef] [Green Version]
  35. Georgiou, A.; Demetriou, C.A.; Heraclides, A.; Christou, Y.P.; Leonidou, E.; Loukaides, P.; Yiasoumi, E.; Panagiotou, D.; Manoli, P.; Thomson, P.; et al. Mitochondrial Superclusters Influence Age of Onset of Parkinson’s Disease in a Gender Specific Manner in the Cypriot Population: A Case-Control Study. PLoS ONE 2017, 12, e0183444. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Ridge, P.G.; Maxwell, T.J.; Corcoran, C.D.; Norton, M.C.; Tschanz, J.T.; O’Brien, E.; Kerber, R.A.; Cawthon, R.M.; Munger, R.G.; Kauwe, J.S.K. Mitochondrial Genomic Analysis of Late Onset Alzheimer’s Disease Reveals Protective Haplogroups H6A1A/H6A1B: The Cache County Study on Memory in Aging. PLoS ONE 2012, 7, e45134. [Google Scholar] [CrossRef] [PubMed]
  37. Wei, W.; Chinnery, P.F. Inheritance of Mitochondrial DNA in Humans: Implications for Rare and Common Diseases. J. Intern. Med. 2020, 287, 634–644. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Leung, G.P.H. Iatrogenic Mitochondriopathies: A Recent Lesson from Nucleoside/Nucleotide Reverse Transcriptase Inhibitors. In: Scatena R., Bottoni P., Giardina B. (eds) Advances in Mitochondrial Medicine. Adv. Exp. Med. Biol. 2012, 942, 347–369. [Google Scholar]
  39. Lorenzo, C.D.; Pierelli, F.; Coppola, G.; Grieco, G.S.; Rengo, C.; Ciccolella, M.; Magis, D.; Bolla, M.; Casali, C.; Santorelli, F.M.; et al. Mitochondrial DNA Haplogroups Influence the Therapeutic Response to Riboflavin in Migraineurs. Neurology 2009, 72, 1588–1594. [Google Scholar] [CrossRef]
  40. Torroni, A.; Campos, Y.; Rengo, C.; Sellitto, D.; Achilli, A.; Magri, C.; Semino, O.; García, A.; Jara, P.; Arenas, J.; et al. Mitochondrial DNA Haplogroups Do Not Play a Role in the Variable Phenotypic Presentation of the A3243G Mutation. Am. J. Hum. Genet. 2003, 72, 1005–1012. [Google Scholar] [CrossRef] [Green Version]
  41. Wang, C.; Kong, Q.; Zhang, Y. Application of the Phylogenetic Analysis in Mitochondrial Disease Study. Sci. Bull. 2008, 53, 2733–2738. [Google Scholar] [CrossRef] [Green Version]
  42. Makino, M.; Horai, S.; Goto, Y.; Nonaka, I. Mitochondrial DNA Mutations in Leigh Syndrome and Their Phylogenetic Implications. J. Hum. Genet. 2000, 45, 69–75. [Google Scholar] [CrossRef]
  43. Taylor, R.W.; Taylor, G.A.; Durham, S.E.; Turnbull, D.M. The Determination of Complete Human Mitochondrial DNA Sequences in Single Cells: Implications for the Study of Somatic Mitochondrial DNA Point Mutations. Nucleic Acids Res. 2001, 29, e74. [Google Scholar] [CrossRef]
  44. Dawod, P.G.A.; Jancic, J.; Marjanovic, A.; Brankovic, M.; Jankovic, M.; Samardzic, J.; Potkonjak, D.; Djuric, V.; Mesaros, S.; Novakovic, I.; et al. Whole Mitochondrial Genome Analysis in Serbian Cases of Leber’s Hereditary Optic Neuropathy. Genes 2020, 11, 1037. [Google Scholar] [CrossRef]
  45. Brandon, M.C.; Ruiz-Pesini, E.; Mishmar, D.; Procaccio, V.; Lott, M.T.; Nguyen, K.C.; Spolim, S.; Patil, U.; Baldi, P.; Wallace, D.C. MITOMASTER—A Bioinformatics Tool for the Analysis of Mitochondrial DNA Sequences. Hum. Mutat. 2009, 30, 1–6. [Google Scholar] [CrossRef] [Green Version]
  46. van Oven, M. PhyloTree Build 17: Growing the Human Mitochondrial DNA Tree. Forensic Sci. Int. Genet. Suppl. Ser. 2015, 5, e392–e394. [Google Scholar] [CrossRef] [Green Version]
  47. Andrews, R.M.; Kubacka, I.; Chinnery, P.F.; Lightowlers, R.N.; Turnbull, D.M.; Howell, N. Reanalysis and Revision of the Cambridge Reference Sequence for Human Mitochondrial DNA. Nat. Genet. 1999, 23, 147. [Google Scholar] [CrossRef]
  48. Benson, D.A.; Cavanaugh, M.; Clark, K.; Karsch-Mizrachi, I.; Lipman, D.J.; Ostell, J.; Sayers, E.W. GenBank. Nucleic Acids Res. 2013, 41, D36–D42. [Google Scholar] [CrossRef] [Green Version]
  49. Ruiz-Pesini, E.; Lott, M.T.; Procaccio, V.; Poole, J.C.; Brandon, M.C.; Mishmar, D.; Yi, C.; Kreuziger, J.; Baldi, P.; Wallace, D.C. An Enhanced MITOMAP with a Global MtDNA Mutational Phylogeny. Nucleic Acids Res. 2007, 35, D823–D828. [Google Scholar] [CrossRef] [Green Version]
  50. Adzhubei, I.A.; Schmidt, S.; Peshkin, L.; Ramensky, V.E.; Gerasimova, A.; Bork, P.; Kondrashov, A.S.; Sunyaev, S.R. A Method and Server for Predicting Damaging Missense Mutations. Nat. Methods 2010, 7, 248–249. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Choi, Y.; Chan, A.P. PROVEAN Web Server: A Tool to Predict the Functional Effect of Amino Acid Substitutions and Indels. Bioinformatics 2015, 31, 2745–2747. [Google Scholar] [CrossRef] [Green Version]
  52. Mi, H.; Huang, X.; Muruganujan, A.; Tang, H.; Mills, C.; Kang, D.; Thomas, P.D. PANTHER Version 11: Expanded Annotation Data from Gene Ontology and Reactome Pathways, and Data Analysis Tool Enhancements. Nucleic Acids Res. 2017, 45, D183–D189. [Google Scholar] [CrossRef] [Green Version]
  53. Sonney, S.; Leipzig, J.; Lott, M.T.; Zhang, S.; Procaccio, V.; Wallace, D.C.; Sondheimer, N. Predicting the Pathogenicity of Novel Variants in Mitochondrial TRNA with MitoTIP. PLoS Comput. Biol. 2017, 13, e1005867. [Google Scholar] [CrossRef] [PubMed]
  54. Pütz, J.; Dupuis, B.; Sissler, M.; Florentz, C. Mamit-TRNA, a Database of Mammalian Mitochondrial TRNA Primary and Secondary Structures. RNA 2007, 13, 1184–1190. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Gorman, G.S.; Schaefer, A.M.; Ng, Y.; Gomez, N.; Blakely, E.L.; Alston, C.L.; Feeney, C.; Horvath, R.; Yu-Wai-Man, P.; Chinnery, P.F.; et al. Prevalence of Nuclear and Mitochondrial DNA Mutations Related to Adult Mitochondrial Disease. Ann. Neurol. 2015, 77, 753–759. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Ng, Y.S.; DiMauro, S.; Turnbull, D.M. Mitochondrial Medicine: A Historical Point of View. In Diagnosis and Management of Mitochondrial Disorders; Mancuso, M., Klopstock, T., Eds.; Springer International Publishing: Cham, Switzerland, 2019; pp. 1–18. [Google Scholar]
  57. Sharma, R.; Reinstadler, B.; Engelstad, K.; Skinner, O.S.; Stackowitz, E.; Haller, R.G.; Clish, C.B.; Pierce, K.; Walker, M.A.; Fryer, R.; et al. Circulating Markers of NADH-Reductive Stress Correlate with Mitochondrial Disease Severity. J. Clin. Investig. 2021, 131, e136055. [Google Scholar] [CrossRef] [PubMed]
  58. Rahman, S.; Thorburn, D. Nuclear Gene-Encoded Leigh Syndrome Spectrum Overview. In GeneReviews® [Internet]; Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Mirzaa, G., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 2015; [Updated 2020 July 16]. Available online: https://www.ncbi.nlm.nih.gov/books/NBK320989/ (accessed on 31 August 2021).
  59. Thorburn, D.R.; Rahman, J.; Rahman, S. Mitochondrial DNA-Associated Leigh Syndrome and NARP. In GeneReviews®; Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J., Stephens, K., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 1993. [Google Scholar]
  60. Zhang, H.; Burr, S.P.; Chinnery, P.F. The Mitochondrial DNA Genetic Bottleneck: Inheritance and Beyond. Essays Biochem. 2018, 62, 225–234. [Google Scholar]
  61. Fujii, T.; Hattori, H.; Higuchi, Y.; Tsuji, M.; Mitsuyoshi, I. Phenotypic Differences between T-->C and T-->G Mutations at Nt 8993 of Mitochondrial DNA in Leigh Syndrome. Pediatr. Neurol. 1998, 18, 275–277. [Google Scholar] [CrossRef]
  62. Weerasinghe, C.A.L.; Bui, B.-H.T.; Vu, T.T.; Nguyen, H.-L.T.; Phung, B.-K.; Nguyen, V.-M.; Pham, V.-A.; Cao, V.-H.; Phan, T.-N. Leigh Syndrome T8993C Mitochondrial DNA Mutation: Heteroplasmy and the First Clinical Presentation in a Vietnamese Family. Mol. Med. Rep. 2018, 17, 6919–6925. [Google Scholar] [CrossRef] [Green Version]
  63. Balasubramaniam, S.; Lewis, B.; Mock, D.M.; Said, H.M.; Tarailo-Graovac, M.; Mattman, A.; van Karnebeek, C.D.; Thorburn, D.R.; Rodenburg, R.J.; Christodoulou, J. Leigh-Like Syndrome Due to Homoplasmic m.8993T>G Variant with Hypocitrullinemia and Unusual Biochemical Features Suggestive of Multiple Carboxylase Deficiency (MCD). JIMD Rep. 2016, 33, 99–107. [Google Scholar]
  64. Blok, R.B.; Gook, D.A.; Thorburn, D.R.; Dahl, H.H. Skewed Segregation of the MtDNA Nt 8993 (T-->G) Mutation in Human Oocytes. Am. J. Hum. Genet. 1997, 60, 1495–1501. [Google Scholar] [CrossRef] [Green Version]
  65. Burr, S.P.; Pezet, M.; Chinnery, P.F. Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line. Dev. Growth Differ. 2018, 60, 21–32. [Google Scholar] [CrossRef] [Green Version]
  66. White, S.L.; Shanske, S.; McGill, J.J.; Mountain, H.; Geraghty, M.T.; DiMauro, S.; Dahl, H.H.; Thorburn, D.R. Mitochondrial DNA Mutations at Nucleotide 8993 Show a Lack of Tissue- or Age-Related Variation. J. Inherit. Metab. Dis. 1999, 22, 899–914. [Google Scholar] [CrossRef]
  67. Fassone, E.; Wedatilake, Y.; DeVile, C.J.; Chong, W.K.; Carr, L.J.; Rahman, S. Treatable Leigh-like Encephalopathy Presenting in Adolescence. BMJ Case Rep. 2013, 2013. [Google Scholar] [CrossRef]
  68. Licchetta, L.; Ferri, L.; Morgia, C.L.; Zenesini, C.; Caporali, L.; Valentino, M.L.; Minardi, R.; Fulitano, D.; Vito, L.D.; Mostacci, B.; et al. Epilepsy in MT-ATP6 - Related Mils/NARP: Correlation of Elettroclinical Features with Heteroplasmy. Ann. Clin. Transl. Neurol. 2021, 8, 704–710. [Google Scholar] [CrossRef]
  69. Distelmaier, F.; Huppke, P.; Pieperhoff, P.; Amunts, K.; Schaper, J.; Morava, E.; Ma yatepek, E.; Kohlhase, J.; Karenfort, M. Biotin-Responsive Basal Ganglia Disease: A Treatable Differential Diagnosis of Leigh Syndrome. JIMD Rep. 2014, 13, 53–57. [Google Scholar]
  70. Yatsuga, S.; Povalko, N.; Nishioka, J.; Katayama, K.; Kakimoto, N.; Matsuishi, T.; Kakuma, T.; Koga, Y. MELAS: A Nationwide Prospective Cohort Study of 96 Patients in Japan. Biochim. Biophys. Acta 2012, 1820, 619–624. [Google Scholar] [CrossRef] [PubMed]
  71. Hirano, M.; Ricci, E.; Richard Koenigsberger, M.; Defendini, R.; Pavlakis, S.G.; DeVivo, D.C.; DiMauro, S.; Rowland, L.P. MELAS: An Original Case and Clinical Criteria for Diagnosis. Neuromuscul. Disord. 1992, 2, 125–135. [Google Scholar] [CrossRef]
  72. Kimata, K.G.; Gordan, L.; Ajax, E.T.; Davis, P.H.; Grabowski, T. A Case of Late-Onset MELAS. Arch. Neurol. 1998, 55, 722. [Google Scholar] [CrossRef] [Green Version]
  73. Sinnecker, T.; Andelova, M.; Mayr, M.; Rüegg, S.; Sinnreich, M.; Hench, J.; Frank, S.; Schaller, A.; Stippich, C.; Wuerfel, J.; et al. Diagnosis of Adult-Onset MELAS Syndrome in a 63-Year-Old Patient with Suspected Recurrent Strokes—A Case Report. BMC Neurol. 2019, 19, 91. [Google Scholar] [CrossRef] [Green Version]
  74. Parsons, T.; Weimer, L.; Engelstad, K.; Linker, A.; Battista, V.; Wei, Y.; Hirano, M.; DiMauro, S. Autonomic Symptoms in Carriers of the m.3243AϾG Mitochondrial DNA Mutation. Arch. Neurol. 2010, 67, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Robinson, R. The Wide Phenotypic Spectrum of the Common MELAS-Causing Mutation. Neurol. Today 2007, 7, 26. [Google Scholar] [CrossRef]
  76. Liang, C.; Ahmad, K.; Sue, C.M. The Broadening Spectrum of Mitochondrial Disease: Shifts in the Diagnostic Paradigm. Biochim. Biophys. Acta 2014, 1840, 1360–1367. [Google Scholar] [CrossRef] [Green Version]
  77. Kaufmann, P.; Engelstad, K.; Wei, Y.; Kulikova, R.; Oskoui, M.; Sproule, D.M.; Battista, V.; Koenigsberger, D.Y.; Pascual, J.M.; Shanske, S.; et al. Natural History of MELAS Associated with Mitochondrial DNA m.3243A>G Genotype. Neurology 2011, 77, 1965–1971. [Google Scholar] [CrossRef] [Green Version]
  78. Weiduschat, N.; Kaufmann, P.; Mao, X.; Engelstad, K.M.; Hinton, V.; DiMauro, S.; Vivo, D.D.; Shungu, D. Cerebral Metabolic Abnormalities in A3243G Mitochondrial DNA Mutation Carriers. Neurology 2014, 82, 798–805. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Finsterer, J. Lifestyle Changes Normalize Serum Lactate Levels in an m.3243A>G Carrier. Am. J. Case Rep. 2021, 22, e930175. [Google Scholar] [CrossRef] [PubMed]
  80. Sofou, K.; de Coo, I.F.M.; Ostergaard, E.; Isohanni, P.; Naess, K.; De Meirleir, L.; Tzoulis, C.; Uusimaa, J.; Lönnqvist, T.; Bindoff, L.A.; et al. Phenotype-Genotype Correlations in Leigh Syndrome: New Insights from a Multicentre Study of 96 Patients. J. Med. Genet. 2018, 55, 21–27. [Google Scholar] [CrossRef] [PubMed]
  81. Yiş, U.; Seneca, S.; Dırık, E.; Kurul, S.H.; Özer, E.; Çakmakçi, H.; De Meırleır, L. Unusual Findings in Leigh Syndrome Caused by T8993C Mutation. Eur. J. Paediatr. Neurol. 2009, 13, 550–552. [Google Scholar] [CrossRef] [PubMed]
  82. Levy, R.J.; Ríos, P.G.; Akman, H.O.; Sciacco, M.; De Vivo, D.C.; DiMauro, S. Long Survival in Patients with Leigh Syndrome and the m.10191T>C Mutation in MT-ND3: A Case Report and Review of the Literature. J. Child. Neurol. 2014, 29, NP105–NP110. [Google Scholar] [CrossRef] [Green Version]
  83. Han, J.; Lee, Y.-M.; Kim, S.M.; Han, S.Y.; Lee, J.B.; Han, S.-H. Ophthalmological Manifestations in Patients with Leigh Syndrome. Br. J. Ophthalmol. 2015, 99, 528–535. [Google Scholar] [CrossRef]
  84. Loeffen, J.; Elpeleg, O.; Smeitink, J.; Smeets, R.; Stöckler-Ipsiroglu, S.; Mandel, H.; Sengers, R.; Trijbels, F.; van den Heuvel, L. Mutations in the Complex I NDUFS2 Gene of Patients with Cardiomyopathy and Encephalomyopathy. Ann. Neurol. 2001, 49, 195–201. [Google Scholar] [CrossRef]
  85. Bénit, P.; Beugnot, R.; Chretien, D.; Giurgea, I.; De Lonlay-Debeney, P.; Issartel, J.-P.; Corral-Debrinski, M.; Kerscher, S.; Rustin, P.; Rötig, A.; et al. Mutant NDUFV2 Subunit of Mitochondrial Complex I Causes Early Onset Hypertrophic Cardiomyopathy and Encephalopathy. Hum. Mutat. 2003, 21, 582–586. [Google Scholar] [CrossRef]
  86. Tay, S.K.H.; Sacconi, S.; Akman, H.O.; Morales, J.F.; Morales, A.; De Vivo, D.C.; Shanske, S.; Bonilla, E.; DiMauro, S. Unusual Clinical Presentations in Four Cases of Leigh Disease, Cytochrome C Oxidase Deficiency, and SURF1 Gene Mutations. J. Child. Neurol. 2005, 20, 670–674. [Google Scholar] [CrossRef]
  87. Leslie, N.; Wang, X.; Peng, Y.; Valencia, C.A.; Khuchua, Z.; Hata, J.; Witte, D.; Huang, T.; Bove, K.E. Neonatal Multiorgan Failure Due to ACAD9 Mutation and Complex I Deficiency with Mitochondrial Hyperplasia in Liver, Cardiac Myocytes, Skeletal Muscle, and Renal Tubules. Hum. Pathol. 2016, 49, 27–32. [Google Scholar] [CrossRef]
  88. Finsterer, J. Rare Phenotypic Manifestations of MELAS. Yonsei Med. J. 2020, 61, 904–906. [Google Scholar] [CrossRef]
  89. Keilland, E.; Rupar, C.A.; Prasad, A.N.; Tay, K.Y.; Downie, A.; Prasad, C. The Expanding Phenotype of MELAS Caused by the m.3291T > C Mutation in the MT-TL1 Gene. Mol. Genet. Metab. Rep. 2016, 6, 64–69. [Google Scholar] [CrossRef]
  90. Mochizuki, H.; Joh, K.; Kawame, H.; Imadachi, A.; Nozaki, H.; Ohashi, T.; Usui, N.; Eto, Y.; Kanetsuna, Y.; Aizawa, S. Mitochondrial Encephalomyopathies Preceded by De-Toni-Debré-Fanconi Syndrome or Focal Segmental Glomerulosclerosis. Clin. Nephrol. 1996, 46, 347–352. [Google Scholar] [PubMed]
  91. Motoda, A.; Kurashige, T.; Sugiura, T.; Nakamura, T.; Yamawaki, T.; Arihiro, K.; Matsumoto, M. [A case of MELAS with G13513A mutation presenting with chronic kidney disease long before stroke-like episodes]. Rinsho Shinkeigaku 2013, 53, 446–451. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  92. Seitun, S.; Massobrio, L.; Rubegni, A.; Nesti, C.; Castiglione Morelli, M.; Boccalini, S.; Galletto Pregliasco, A.; Budaj, I.; Deferrari, L.; Rosa, G.M.; et al. MELAS Syndrome with Cardiac Involvement: A Multimodality Imaging Approach. Case Rep. Cardiol. 2016, 2016, 1–4. [Google Scholar] [CrossRef]
  93. Strachan, J.; McLellan, A.; Kirkpatrick, M.; Hume, R.; Mechan, D. Ketoacidosis: An Unusual Presentation of MELAS. J. Inherit. Metab. Dis. 2001, 24, 409–410. [Google Scholar] [CrossRef]
  94. Chaig, M.R.; Zernotti, M.E.; Soria, N.W.; Romero, O.F.; Romero, M.F.; Gerez, N.M. A Mutation in Mitochondrial 12S RRNA, A827G, in Argentinean Family with Hearing Loss after Aminoglycoside Treatment. Biochem. Biophys. Res. Commun. 2008, 368, 631–636. [Google Scholar] [CrossRef]
  95. Dowlati, M.A.; Derakhshandeh-peykar, P.; Houshmand, M.; Farhadi, M.; Shojaei, A.; Fallah, M.; Mohammadi, E.; Tajdini, A.; Arastoo, S.; Tavakkoly-Bazzaz, J. Novel Nucleotide Changes in Mutational Analysis of Mitochondrial 12SrRNA Gene in Patients with Nonsyndromic and Aminoglycoside-Induced Hearing Loss. Mol. Biol. Rep. 2013, 40, 2689–2695. [Google Scholar] [CrossRef]
  96. Häkli, S.; Luotonen, M.; Sorri, M.; Majamaa, K. Mutations in the Two Ribosomal RNA Genes in Mitochondrial DNA among Finnish Children with Hearing Impairment. BMC Med. Genet. 2015, 16, 3. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  97. Pacheu-Grau, D.; Pérez-Delgado, L.; Gómez-Díaz, C.; Fraile-Rodrigo, J.; Montoya, J.; Ruiz-Pesini, E. Mitochondrial Ribosome and Ménière’s Disease: A Pilot Study. Eur. Arch. Otorhinolaryngol. 2012, 269, 2003–2008. [Google Scholar] [CrossRef]
  98. Wallace, D.C. Mitochondrial DNA Variation in Human Radiation and Disease. Cell 2015, 163, 33–38. [Google Scholar] [CrossRef] [Green Version]
  99. Manwaring, N.; Jones, M.M.; Wang, J.J.; Rochtchina, E.; Howard, C.; Mitchell, P.; Sue, C.M. Population Prevalence of the MELAS A3243G Mutation. Mitochondrion 2007, 7, 230–233. [Google Scholar] [CrossRef]
  100. Pierron, D.; Rocher, C.; Amati-Bonneau, P.; Reynier, P.; Martin-Négrier, M.-L.; Allouche, S.; Batandier, C.; de Camaret, B.M.; Godinot, C.; Rotig, A.; et al. New Evidence of a Mitochondrial Genetic Background Paradox: Impact of the J Haplogroup on the A3243G Mutation. BMC Med. Genet. 2008, 9, 41. [Google Scholar] [CrossRef]
  101. Delgado-Sánchez, R.; Zárate-Moysen, A.; Monsalvo-Reyes, A.; Herrero, M.D.; Ruiz-Pesini, E.; López-Pérez, M.; Montoya, J.; Montiel-Sosa, J.F. Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like Episodes (MELAS) with the A3243G Mutation of the TRNALeu (UUR) Gene of MtDNA in Native American Haplogroup B2. Rev. Neurol. 2007, 44, 18–22. [Google Scholar]
  102. Liu, G.; Shen, X.; Sun, Y.; Lv, Q.; Li, Y.; Du, A. Heteroplasmy and Phenotype Spectrum of the Mitochondrial TRNALeu (UUR) Gene m.3243A>G Mutation in Seven Han Chinese Families. J. Neurol. Sci. 2020, 408, 116562. [Google Scholar] [CrossRef] [PubMed]
  103. Chakrabarty, S.; Govindaraj, P.; Sankaran, B.P.; Nagappa, M.; Kabekkodu, S.P.; Jayaram, P.; Mallya, S.; Deepha, S.; Ponmalar, J.N.J.; Arivinda, H.R.; et al. Contribution of Nuclear and Mitochondrial Gene Mutations in Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS) Syndrome. J. Neurol. 2021, 268, 2192–2207. [Google Scholar] [CrossRef] [PubMed]
  104. Habbane, M.; Llobet, L.; Bayona-Bafaluy, M.P.; Bárcena, J.E.; Ceberio, L.; Gómez-Díaz, C.; Gort, L.; Artuch, R.; Montoya, J.; Ruiz-Pesini, E. Leigh Syndrome in a Pedigree Harboring the m.1555A>G Mutation in the Mitochondrial 12S RRNA. Genes 2020, 11, 1007. [Google Scholar] [CrossRef]
  105. Prasad, H.V.R.; Neelima, K.; Shalini, M.; Chandak, G.R. Leigh’s Syndrome: A Case Report. Internet J. Pediatr. Neonatol. 2006, 7, 1–9. [Google Scholar]
  106. Chen, Z.; Zhao, Z.; Ye, Q.; Chen, Y.; Pan, X.; Sun, B.; Huang, H.; Zheng, A. Mild Clinical Manifestation and Unusual Recovery upon Coenzyme Q10 Treatment in the First Chinese Leigh Syndrome Pedigree with Mutation m.10197 G>A. Mol. Med. Rep. 2015, 11, 1956–1962. [Google Scholar] [CrossRef] [Green Version]
  107. Hao, X.-D.; Yang, Y.-L.; Tang, N.L.S.; Kong, Q.-P.; Wu, S.-F.; Zhang, Y.-P. Mitochondrial DNA Haplogroup Y Is Associated to Leigh Syndrome in Chinese Population. Gene 2013, 512, 460–463. [Google Scholar] [CrossRef]
  108. D’Aurelio, M.; Vives-Bauza, C.; Davidson, M.M.; Manfredi, G. Mitochondrial DNA Background Modifies the Bioenergetics of NARP/MILS ATP6 Mutant Cells. Hum. Mol. Genet. 2010, 19, 374–386. [Google Scholar] [CrossRef] [Green Version]
  109. Angural, A.; Sharma, I.; Pandoh, P.; Sharma, V.; Spolia, A.; Rai, E.; Singh, V.; Razdan, S.; Pandita, K.K.; Sharma, S. A Case Report on a Novel MT-ATP6 Gene Variation in Atypical Mitochondrial Leigh Syndrome Associated with Bilateral Basal Ganglia Calcifications. Mitochondrion 2019, 46, 209–213. [Google Scholar] [CrossRef]
  110. Hong, C.-M.; Na, J.-H.; Park, S.; Lee, Y.-M. Clinical Characteristics of Early-Onset and Late-Onset Leigh Syndrome. Front. Neurol. 2020, 11, 267. [Google Scholar] [CrossRef]
  111. Tapias, V.; Mastroberardino, P.G.; Maio, R.D. Mitochondrial Dysfunction and Neurodegeneration; Frontiers Media SA: Lausanne, Switzerland, 2020. [Google Scholar]
  112. Wang, Y.; Brinton, R.D. Triad of Risk for Late Onset Alzheimer’s: Mitochondrial Haplotype, APOE Genotype and Chromosomal Sex. Front. Aging Neurosci. 2016, 8, 232. [Google Scholar] [CrossRef] [PubMed]
  113. Hudson, G.; Nalls, M.; Evans, J.R.; Breen, D.P.; Winder-Rhodes, S.; Morrison, K.E.; Morris, H.R.; Williams-Gray, C.H.; Barker, R.A.; Singleton, A.B.; et al. Two-Stage Association Study and Meta-Analysis of Mitochondrial DNA Variants in Parkinson Disease. Neurology 2013, 80, 2042–2048. [Google Scholar] [CrossRef]
  114. Arning, L.; Haghikia, A.; Taherzadeh-Fard, E.; Saft, C.; Andrich, J.; Pula, B.; Höxtermann, S.; Wieczorek, S.; Akkad, D.A.; Perrech, M.; et al. Mitochondrial Haplogroup H Correlates with ATP Levels and Age at Onset in Huntington Disease. J. Mol. Med. 2010, 88, 431–436. [Google Scholar] [CrossRef] [PubMed]
  115. Mancuso, M.; Conforti, F.L.; Rocchi, A.; Tessitore, A.; Muglia, M.; Tedeschi, G.; Panza, D.; Monsurrò, M.; Sola, P.; Mandrioli, J.; et al. Could Mitochondrial Haplogroups Play a Role in Sporadic Amyotrophic Lateral Sclerosis? Neurosci. Lett. 2004, 371, 158–162. [Google Scholar] [CrossRef] [PubMed]
  116. Ghabaee, M.; Omranisikaroudi, M.; Amrisaroukolaei, S.; Meysamie, A.; Sahraian, M.A.; Bayati, A.; Sanati, M.H.; Houshman, M.; Sadeghian, H.; Vajihazaman, K. Mitochondrial Mutation in Iranian Patients with Multiple Sclerosis, Correlation between Haplogroups H, A and Clinical Manifestations. Cell. Mol. Neurobiol. 2009, 29, 341–346. [Google Scholar] [CrossRef]
  117. Serrano-Teruel, M.E.; Garcia-Vieites, M.; Rego-Perez, I.; Domenech-Garcia, N.; Blanco-Garcia, F.; Cuenca-Castillo, J.J.; Bautista-Hernandez, V. Mitochondrial DNA Haplogroups Influence the Risk of Aortic Stenosis. Asian Cardiovasc. Thorac. Ann. 2019, 27, 5–10. [Google Scholar] [CrossRef]
  118. Alwehaidah, M.S.; Bakhiet, M.; AlFadhli, S. Mitochondrial Haplogroup Reveals the Genetic Basis of Diabetes Mellitus Type 2 Comorbidity in Psoriasis. Med. Princ. Pract. 2021, 30, 62–68. [Google Scholar] [PubMed]
  119. Soto-Hermida, A.; Fernández-Moreno, M.; Pértega-Díaz, S.; Oreiro, N.; Fernández-López, C.; Blanco, F.J.; Rego-Pérez, I. Mitochondrial DNA Haplogroups Modulate the Radiographic Progression of Spanish Patients with Osteoarthritis. Rheumatol. Int. 2015, 35, 337–344. [Google Scholar] [CrossRef] [PubMed]
  120. Fajardo, R.G.; Fariña, F.O.; Rey, A.M.; Rego-Pérez, I.; García, F.J.B.; García, J.L.F. Relationship between the Dynamics of Telomere Loss in Peripheral Blood Leukocytes from Osteoarthritis Patients and Mitochondrial DNA Haplogroup. J. Rheumatol. 2021, jrheum.201316, (Online ahead of print). [Google Scholar] [CrossRef]
Diagnostics 11 01969 g001 550
Figure 1. Electropherogram is showing m.8993T>G homoplasmic mutation and m.3243A>G heteroplasmic mutation. Reference sequence is on the top.
Figure 1. Electropherogram is showing m.8993T>G homoplasmic mutation and m.3243A>G heteroplasmic mutation. Reference sequence is on the top.
Diagnostics 11 01969 g001
Diagnostics 11 01969 g002 550
Figure 2. Phylogenetic reconstruction is presenting full mtDNA Sanger sequencing of three Serbian probands have mitochondrial encephalomyopathies mutations. The tree was rooted by following the nomenclature of mtDNA tree Build 17. ! Indicates back mutation, () indicates recurrent mutation; rCRS indicates Revised Cambridge Reference Sequence; nc, s, ns, t, r1 and r2 indicate non-coding region, synonymous, nonsynonymous, transfer RNA, ribosomal RNA1, and 2 variants, respectively. Primary mitochondrial encephalomyopathies mutations are shown in bold, 3243 for MELAS and 8993 for Leigh syndrome. Underlining indicates variants specific for haplogroups reported on Mitomaster analysis vs. PhyloTree Build 17.
Figure 2. Phylogenetic reconstruction is presenting full mtDNA Sanger sequencing of three Serbian probands have mitochondrial encephalomyopathies mutations. The tree was rooted by following the nomenclature of mtDNA tree Build 17. ! Indicates back mutation, () indicates recurrent mutation; rCRS indicates Revised Cambridge Reference Sequence; nc, s, ns, t, r1 and r2 indicate non-coding region, synonymous, nonsynonymous, transfer RNA, ribosomal RNA1, and 2 variants, respectively. Primary mitochondrial encephalomyopathies mutations are shown in bold, 3243 for MELAS and 8993 for Leigh syndrome. Underlining indicates variants specific for haplogroups reported on Mitomaster analysis vs. PhyloTree Build 17.
Diagnostics 11 01969 g002
Table
Table 1. Informatics predictors for m.3243A>G and m.8993T>G mutations.
Table 1. Informatics predictors for m.3243A>G and m.8993T>G mutations.
MitochondriopathiesMELASLeigh Disease
Mutationm.3243A>Gm.8993T>G
GeneRNA Gene
MT-TL1		Protein Coding gene
MT-ATP6
Codon number−156
Amino acid changetRNA LeuLeu-Arg
MitomapConfirmed-PathogenicConfirmed-Pathogenic
MitoTIPPathogenic−
Mamit-tRNAPathogenic−
UniProt ID−P00846
Polyphen Prediction−probably damaging
PANTHER−probably damaging
PROVEAN−Deleterious (−5.180)
MT-ATP6: mitochondrially encoded ATP synthase membrane subunit 6; MT-TL1: Mitochondrially Encoded TRNA-Leu (UUA/G) 1; Arg: Arginine; Leu: leucine.
Table
Table 2. Polymorphic mtDNA variants detected in Serbian probands with mitochondrial encephalomyopathies.
Table 2. Polymorphic mtDNA variants detected in Serbian probands with mitochondrial encephalomyopathies.
ProbandsHaplogroupVariantsLocusNucleotide ChangesA.A ChangesPhyloTree Build 17 in HG BranchMitomaster Frequencies in HG Branch
P1U2e73MT-CRA>GCRReported96.46%
(MELAS) 152MT-CRT>CCRReported90.26%
263MT-CRA>GCRReported96.46%
508MT-CRA>GCRReported94.39%
739MT-RNR1C>TrRNANot reported0.29%
750MT-RNR1A>GrRNAReported100.00%
1438MT-RNR1A>GrRNAReported98.82%
1811MT-RNR2A>GrRNAReported94.39%
2706MT-RNR2A>GrRNAReported99.11%
3116MT-RNR2C>TrRNANot reported22.71%
3720MT-ND1A>GQ138QReported98.23%
4769MT-ND2A>GM100MReported98.52%
5390MT-ND2A>GM307MReported98.52%
5426MT-ND3T>CH319HReported98.52%
6045MT-COIC>TL48LReported98.52%
6152MT-COIT>CV83VReported98.23%
7028MT-COIC>TA375AReported98.80%
8860MT-ATP6A>GT112AReported99.41%
10,876MT-ND4A>GL39LReported99.11%
11,197MT-ND4C>TG146GNot reported22.42%
11,467MT-ND4A>GL236LReported98.82%
11,719MT-ND4G>AG320GReported99.40%
12,308MT-TL2A>GtRNAReported98.80%
12,372MT-ND5G>AL12LReported99.70%
13,020MT-ND5T>CG228GReported99.11%
13,359MT-ND5G>AM341MNot reported0.00%
14,766MT-CYBC>TT7IReported99.70%
15,326MT-CYBA>GT194AReported99.70%
15,907MT-TTA>GtRNAReported98.82%
16,051MT-CRA>GCRReported96.75%
16,129MT-CRG>CCRReported95.28%
16,183MT-CRA>CCRNot considered75.52%
16,189MT-CRT>CCRReported84.36%
P2H1555MT-CRT>CCRReported63.33%
(MELAS) 56MT-CRinsCCRNot reported6.67%
143MT-CRG>ACRNot reported0.00%
263MT-RNR1A>GCRReported80.00%
750MT-RNR1A>GrRNAReported100.00%
1438MT-RNR2A>GrRNAReported96.70%
2706MT-ND1A>GrRNAReported95.60%
3847MT-ND2T>CL181LReported96.67%
4769MT-COIA>GM100MReported96.70%
6253MT-COIT>CM117TReported96.67%
7028MT-ATP6C>TA375AReported97.80%
8860MT-CYBA>GT112AReported96.67%
15,326MT-CRA>GT194AReported96.70%
P3H34152MT-CRT>CCRNot reported90.91%
(LS) 263MT-CRA>GCRReported100.00%
315MT-CRinsCCRNot reported45.46%
508MT-RNR1A>GCRNot reported0.00%
750MT-RNR1A>GrRNAReported100.00%
1438MT-RNR1A>GrRNAReported100.00%
4769MT-ND2A>GM100MReported100.00%
8860MT-ATP6A>GT112AReported100.00%
15,326MT-CYBA>GT194AReported100.00%
15,519MT-CYBT>CL258PReported100.00%
15,948MT-TTA>GtRNANot reported0.00%
16,093MT-CRT>CCRNot reported45.46%
16,291MT-CRC>TCRReported90.91%
16,519MT-CRT>CCRReported100.00%
This table shows haplogrouping analysis for two MELAS probands (P1 and P2) and one Leigh syndrome proband (P3). Mitomaster analysis for complete mtDNA sequences detected numerous mtDNA haplotypes for haplogroups (U2e, H15 and H34) in different frequencies, of which almost all are reported on the PhyloTree build 17 in specific haplogroup branches, with the exception of m.16183A>C, which was not considered for phylogenetic reconstruction. A.A: amino acid; HG: haplogroup; LS: Leigh syndrome; MELAS: mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MT-ATP6: mitochondrially encoded ATP synthase membrane subunit 6; MT-CO1: mitochondrially encoded cytochrome c oxidase 1; MT-CR: mitochondrial control region; MT-CYB: mitochondrially encoded cytochrome b; MT-ND1, MT-ND2, MT-ND3, MT-ND4 and MT-ND5: mitochondrially encoded NADH:Ubiquinone Oxidoreductase Core Subunit 1, 2, 3, 4 and 5 respectively; MT-RNR1 and MT-RNR2: Mitochondrially Encoded 12S and 16S, respectively; MT-TL2: mitochondrially encoded tRNA leucine; MT-TT: mitochondrially encoded tRNA threonine; P: proband.
Table
Table 3. Clinical evaluation for MELAS and Leigh probands.
Table 3. Clinical evaluation for MELAS and Leigh probands.
Clinical EvaluationMELASLeigh Disease
ProbandP1P2P3
GenderMaleMaleFemale
Age at onset of the disease14 years old12 years oldFew months after birth
Duration of the disease1 month9 years4 years
Family history of MEMP−––
Epileptic seizures+++
Psychosis++−
Psychomotor retardation–−+
Confusion+−−
Behaviour changes+++
Dementia−+−
Episodes like stroke−−−
Headache+−−
Eye deviation during seizures−−+
Speech delay−−+
Hemiparesis++−
Muscle weakness+++
Muscle twitches+−+
Associated vomiting++−
Preceding infection−++
Lactate acidosis+++
MRI changes+++
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Dawod, P.G.A.; Jancic, J.; Marjanovic, A.; Brankovic, M.; Jankovic, M.; Samardzic, J.; Gamil Anwar Dawod, A.; Novakovic, I.; Abdel Motaleb, F.I.; Radlovic, V.; et al. Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review. Diagnostics 2021, 11, 1969. https://doi.org/10.3390/diagnostics11111969

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Dawod PGA, Jancic J, Marjanovic A, Brankovic M, Jankovic M, Samardzic J, Gamil Anwar Dawod A, Novakovic I, Abdel Motaleb FI, Radlovic V, et al. Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review. Diagnostics. 2021; 11(11):1969. https://doi.org/10.3390/diagnostics11111969

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Dawod, Phepy G. A., Jasna Jancic, Ana Marjanovic, Marija Brankovic, Milena Jankovic, Janko Samardzic, Ayman Gamil Anwar Dawod, Ivana Novakovic, Fayda I. Abdel Motaleb, Vladimir Radlovic, and et al. 2021. "Mutational Analysis and mtDNA Haplogroup Characterization in Three Serbian Cases of Mitochondrial Encephalomyopathies and Literature Review" Diagnostics 11, no. 11: 1969. https://doi.org/10.3390/diagnostics11111969

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