Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial ß-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls. Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

Vitamin B1 deficiency leads to high oxidative stress and mtDNA depletion caused by SLC19A3 mutation in consanguineous family with Leigh syndrome.

Leigh syndrome (LS) and Leigh-like spectrum are the most common infantile mitochondrial disorders characterized by heterogeneous neurologic and metabolic manifestations. Pathogenic variants in SLC carriers are frequently reported in LS given their important role in transporting various solutes across the blood-brain barrier. SLC19A3 (THTR2) is one of these carriers transporting vitamin-B1 (vitB1, thiamine) into the cell. Targeted NGS of nuclear genes involved in mitochondrial diseases was performed in a patient belonging to a consanguineous Tunisian family with LS and revealed a homozygous c.1264 A > G (p.T422A) variant in SLC19A3. Molecular docking revealed that the p.T422A aa change is located at a key position interacting with vitB1 and causes conformational changes compromising vitB1 import. We further disclosed decreased plasma antioxidant activities of CAT, SOD and GSH enzymes, and a 42% decrease of the mtDNA copy number in patient blood.Altogether, our results disclose that the c.1264 A > G (p.T422A) variant in SLC19A3 affects vitB1 transport, induces a mtDNA depletion and reduces the expression level of oxidative stress enzymes, altogether contributing to the LS phenotype of the patient.

First description of the MEGDEHL syndrome in the Tunisian population via whole-exome sequencing: Novel nonsense mutation in SERAC1 gene.

INTRODUCTION: MEGDEL syndrome is a rare recessive disorder, with about 100 cases reported worldwide, which is defined by 3-methylglutaconic aciduria (MEG), deafness (D), encephalopathy (E) and Leigh-like syndrome (L). When these manifestations were added to hepatopathy (H), the syndrome was labelled as MEGD(H)EL. Mutations in SERAC1 gene encoding a serine active site containing 1 protein were described in patients affected by this syndrome. PATIENTS AND METHODS: The present study reports the Whole Exome Sequencing (WES) of the first case of MEGDEHL syndrome in Tunisia in a consanguineous family with three affected children. Bioinformatic analysis was also performed in addition to mtDNA deletion screening and mtDNA copy number quantification in the blood of the indexed case, carried out, respectively by Long-Range PCR and qPCR. RESULTS: The WES revealed a novel homozygous nonsense mutation (c.1379G > A; p.W460X) in the SERAC1 gene, which was confirmed by Sanger sequencing. This nonsense mutation was present at a homozygous state in the three affected children and was heterozygous in the parents. In silico analysis using various softwares was performed, and the predictive results supported the pathogenic effect of the identified mutation. Further, long-range PCR and qPCR analyses of the patient's blood excluded any mtDNA deletions or depletions. CONCLUSION: Sequencing results and bioinformatic tools confirmed that the novel mutation (p.W460X) in the SERAC1 gene causes the severe phenotype in the studied family with MEGDEHL syndrome.

A novel thymidine phosphorylase mutation in a family with Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Molecular docking, dynamic simulation and computational investigations.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/ß domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity.

Mitochondrial disease patients with novel ND4 12058A > C and ND1 m.3911A > G variations: implications for a role in the phenotype following a bioinformatic investigation.

Mitochondrial diseases include a wide group of clinically heterogeneous disorders caused by a dysfunction of the mitochondrial respiratory chain and can be related to mutations in nuclear or mitochondrial DNA genes. In the present report, we performed a whole mitochondrial genome screening in two patients with clinical features of mitochondrial diseases. Mutational analysis revealed the presence of two undescribed heteroplasmic mitochondrial variations, the m.3911A > G (E202G) variant in the MT-ND1 gene found in two patients (P1 and P2) and the m.12058A > C (E433D) pathogenic variant in the MT-ND4 gene present only in patient P2 who had a more severe phenotype. These two substitutions were predicted to be damaging by several bioinformatics tools and lead to amino acid changes in two conserved residues localized in two important functional domains of the mitochondrial subunits of complex I. Furthermore, the 3D modeling suggested that the two amino acid changes could therefore alter the structure of the two subunits and may decrease the stability and the function of complex I. The two described pathogenic variants found in patient P2 could act synergically and alter the complex I function by affecting the proton pumping processes and the energy production and then could explain the severe phenotype compared to patient P1 presenting only the E202G substitution in ND1.

The first concurrent detection of mitochondrial DNA m.3243A>G mutation, deletion, and depletion in a family with mitochondrial diabetes.

BACKGROUND: Mitochondrial diabetes (MD) is a rare monogenic form of diabetes and divided into type l and type 2. It is characterized by a strong familial clustering of diabetes with the presence of maternal transmission in conjunction with bilateral hearing impairment in most of the carriers. The most common form of MD is associated with the m.3243A>G mutation in the mitochondrial MT-TL1, but there are also association with a range of other point mutations, deletion, and depletion in mtDNA. METHODS: The mitochondrial genome anomalies were investigated in a family with clinical features of MD, which includes a proband presenting severe MD conditions including cardiomyopathy, retinopathy, and psychomotor retardation. RESULTS: By investigating the patient's blood leukocytes and skeletal muscle, we identified the m.3243A>G mutation in heteroplasmic state. This mutation was absent in the rest of the family members. In addition, our analysis revealed in the proband a large mtDNA heteroplasmic deletion (~1 kb) and a reduction in mtDNA copy number. CONCLUSION: Our study points out, for the first time, a severe phenotypic expression of the m.3243A>G point mutation in association with mtDNA deletion and depletion in MD.

Mutations in aARS genes revealed by targeted next-generation sequencing in patients with mitochondrial diseases.

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.

Next generation sequencing in family with MNGIE syndrome associated to optic atrophy: Novel homozygous POLG mutation in the C-terminal sub-domain leading to mtDNA depletion.

INTRODUCTION: Mitochondrial diseases are a group of disorders caused mainly by the impairment of the mitochondrial oxidative phosphorylation process, due to mutations either in the mitochondrial or nuclear genome. Among them, the mitochondrial neuro-gastrointestinal encephalo-myopathy (MNGIE) syndrome affects adolescents or young adults, and is mostly caused by TYMP mutations encoding a cytosolic thymidine phosphorylase (TP). PATIENTS AND METHODS: The present study reports the molecular investigation by next-generation re-sequencing of 281 nuclear genes, encoding mitochondrial proteins, of consanguineous family including two individuals with MNGIE syndrome associated to optic atrophy. Bioinformatic analysis was also performed in addition to mtDNA deletion screening and mtDNA copy number quantification in blood of the two patients which were carried out by solf clipping program and qPCR respectively. RESULTS: Next-generation re-sequencing revealed a novel homozygous c.2391G > T POLG mutation (p.M797I) co-occurring with the hypomorphic c.1311A > G OPA1 variant (p.I437M). Analysis of the mitochondrial genome in the two patients disclosed mtDNA depletion in blood, but no deletion. Bio-informatics investigations supported the pathogenicity of the novel POLG mutation that is located in the C-terminal subdomain and might change POLG 3D structure, stability and function. CONCLUSION: The novel homozygous p.M797I POLG mutation is responsible for MNGIE combined to optic atrophy and mtDNA depletion in the two patients.

Cytochrome C oxydase deficiency: SURF1 gene investigation in patients with Leigh syndrome.

Leigh syndrome (LS) is a rare progressive neurodegenerative disorder occurring in infancy. The most common clinical signs reported in LS are growth retardation, optic atrophy, ataxia, psychomotor retardation, dystonia, hypotonia, seizures and respiratory disorders. The paper reported a manifestation of 3 Tunisian patients presented with LS syndrome. The aim of this study is the MT[HYPHEN]ATP6 and SURF1 gene screening in Tunisian patients affected with classical Leigh syndrome and the computational investigation of the effect of detected mutations on its structure and functions by clinical and bioinformatics analyses. After clinical investigations, three Tunisian patients were tested for mutations in both MT-ATP6 and SURF1 genes by direct sequencing followed by in silico analyses to predict the effects of sequence variation. The result of mutational analysis revealed the absence of mitochondrial mutations in MT-ATP6 gene and the presence of a known homozygous splice site mutation c.516-517delAG in sibling patients added to the presence of a novel double het mutations in LS patient (c.752-18 A > C/c. c.751 + 16G > A). In silico analyses of theses intronic variations showed that it could alters splicing processes as well as SURF1 protein translation. Leigh syndrome (LS) is a rare progressive neurodegenerative disorder occurring in infancy. The most common clinical signs reported in LS are growth retardation, optic atrophy, ataxia, psychomotor retardation, dystonia, hypotonia, seizures and respiratory disorders. The paper reported a manifestation of 3 Tunisian patients presented with LS syndrome. The aim of this study is MT-ATP6 and SURF1 genes screening in Tunisian patients affected with classical Leigh syndrome and the computational investigation of the effect of detected mutations on its structure and functions. After clinical investigations, three Tunisian patients were tested for mutations in both MT-ATP6 and SURF1 genes by direct sequencing followed by in silico analysis to predict the effects of sequence variation. The result of mutational analysis revealed the absence of mitochondrial mutations in MT-ATP6 gene and the presence of a known homozygous splice site mutation c.516-517delAG in sibling patients added to the presence of a novel double het mutations in LS patient (c.752-18 A>C/ c.751+16G>A). In silico analysis of theses intronic vaiations showed that it could alters splicing processes as well as SURF1 protein translation.

Clinical, Molecular, and Computational Analysis in two cases with mitochondrial encephalomyopathy associated with SUCLG1 mutation in a consanguineous family.

Deficiency of the mitochondrial enzyme succinyl COA ligase (SUCL) is associated with encephalomyopathic mtDNA depletion syndrome and methylmalonic aciduria. This disorder is caused by mutations in both SUCL subunits genes: SUCLG1 (α subnit) and SUCLA2 (ß subnit). We report here, two Tunisian patients belonging to a consanguineous family with mitochondrial encephalomyopathy, hearing loss, lactic acidosis, hypotonia, psychomotor retardation and methylmalonic aciduria. Mutational analysis of SUCLG1 gene showed, for the first time, the presence of c.41T > C in the exon 1 at homozygous state. In-silico analysis revealed that this mutation substitutes a conserved methionine residue to a threonine at position 14 (p.M14T) located at the SUCLG1 protein mitochondrial targeting sequence. Moreover, these analysis predicted that this mutation alter stability structure and mitochondrial translocation of the protein. In Addition, a decrease in mtDNA copy number was revealed by real time PCR in the peripheral blood leukocytes in the two patients compared with controls.

Splicing Defects in the AAAS Gene Leading to both Exon Skipping and Partial Intron Retention in a Tunisian Patient with Allgrove Syndrome.

BACKGROUND/AIMS: Allgrove syndrome is a rare autosomal recessive disorder characterized by the triad of adrenal insufficiency, achalasia, and alacrima. This syndrome is caused by mutations in the AAAS gene. A major splice site mutation c.1331+1G>A was found previously in North African families affected by Allgrove syndrome. In this study, we analyzed in vivo and in silico the effect of this mutation on the splicing process. METHODS: Using reverse transcriptase-polymerase chain reaction, sequencing and bioinformatics tools, we analyzed all transcripts produced by the AAAS gene containing this splice site mutation. RESULTS: The altered splicing of mRNA produces two aberrant transcripts: one with exon 14 skipping, the other with concurrent exon 14 skipping and retention of 99 bp of intron 14, both outcomes resulting in frameshifts with a new stop codon generation in the untranslated region of the last exon. Using in silico bioinformatics tools, we demonstrated that this mutation abolishes the splice donor site of exon 14 and activates a new intronic cryptic splice site in intron 14. CONCLUSION: This study demonstrated that a single splicing mutation affects the AAAS transcripts and consequently the ALADIN protein structure and function.

A de-novo large deletion of 2.8 kb produced in the ABCD1 gene causing adrenoleukodystrophy disease.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long chain fatty acids (VLCFAs) in plasma, adrenal, testicular, and nerve tissues. For this study, our objective was to conduct clinical, molecular, and genetic studies of a Tunisian patient with X-ALD. The diagnosis was based on clinical indications, biochemical analyses, typical brain-scan patterns, and molecular biology; the molecular analyses were based on PCR, long-range PCR, and sequencing. The molecular analysis by long-range PCR and direct sequencing of the ABCD1 gene showed the presence of a de-novo 2794 bp deletion covering the whole of exon 2. Using bioinformatics tools, we demonstrate that the large deletion is located in a region rich with Alu sequences. Furthermore, we suggest that the AluJb sequence could be the cause of the large deletion of intron 1, exon 2, and intron 2, and the creation of a premature stop codon within exon 3. This report is the first report in which we demonstrate the breakpoints and the size of a large deletion in a Tunisian with X-ALD.

Mutational screening in patients with profound sensorineural hearing loss and neurodevelopmental delay: Description of a novel m.3861A > C mitochondrial mutation in the MT-ND1 gene.

Mitochondrial diseases caused by mitochondrial dysfunction are a clinically and genetically, heterogeneous group of disorders involving multiple organs, particularly tissues with high-energy demand. Hearing loss is a recognized symptom of a number of mitochondrial diseases and can result from neuronal or cochlear dysfunction. The tissue affected in this pathology is most probably the cochlear hair cells, which are essential for hearing function since they are responsible for maintaining the ionic gradients necessary for sound signal transduction. Several mitochondrial DNA mutations have been associated with hearing loss and since mitochondria are crucial for the cellular energy supply in many tissues, most of these mtDNA mutations affect several tissues and will cause syndromic hearing loss. In the present study, we described 2 patients with sensorineural hearing loss and neurodevelopmental delay in whom we tested mitochondrial genes described to be associated with syndromic hearing loss. One of these patients showed a novel heteroplasmic mitochondrial mutation m.3861A > C (W185C) which lead to a loss of stability of the ND1 protein since it created a new hydrogen bund between the unique created cystein C185 and the A182 residue. In the second patient, we detected two novel heteroplasmic variations m.12350C > A (T5N) and m.14351T > C (E108G) respectively in the MT-ND5 and the MT-ND6 genes. The TopPred II prediction for the E108G variation revealed a decrease of the hydrophobicity in the mutated MT-ND6.

Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders.

Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder.

Mutational analysis in patients with neuromuscular disorders: Detection of mitochondrial deletion and double mutations in the MT-ATP6 gene.

Mitochondrial diseases encompass a wide variety of pathologies characterized by a dysfunction of the mitochondrial respiratory chain resulting in an energy deficiency. The respiratory chain consists of five multi-protein complexes providing coupling between nutrient oxidation and phosphorylation of ADP to ATP. In the present report, we studied mitochondrial genes of complex I, III, IV and V in 2 Tunisian patients with mitochondrial neuromuscular disorders. In the first patient, we detected the m.8392C>T variation (P136S) in the mitochondrial ATPase6 gene and the m.8527A>G transition at the junction MT-ATP6/MT-ATP8 which change the initiation codon AUG to GUG. The presence of these two variations in such an important gene could probably affect the ATP synthesis in the studied patient. In the second patient, we detected several known variations in addition to a mitochondrial deletion in the major arc of the mtDNA eliminating tRNA and respiratory chain protein genes. This deletion could be responsible of an inefficient translation leading to an inefficient mitochondrial protein synthesis in P2.

Molecular Analysis of Libyan Families with Allgrove Syndrome: Geographic Expansion of the Ancestral Mutation c.1331+1G>A in North Africa.

BACKGROUND/AIMS: Allgrove syndrome is a rare autosomal recessive disorder characterized by alacrima, achalasia, and adrenal insufficiency. It is caused by mutations of the AAAS gene located on chromosome 12q13 encoding the WD-repeat protein ALADIN. The c.1331+1G>A mutation is one of the most common mutations described in the literature and was identified in Tunisian and Algerian populations. Herein, we describe the clinical and genetic profile of two families from Libya in North Africa associated with Allgrove syndrome. METHODS: Two unrelated families clinically diagnosed with Allgrove syndrome were evaluated for sequence variations in the AAAS gene. Blood samples were collected, and isolated DNA derived from the subjects was amplified. The entire sequence of the AAAS gene was analyzed by PCR-RFLP and direct sequencing. RESULTS: Molecular analysis revealed the major homozygous mutation (c.1331+1G>A) in all patients. The presence of a major mutation in Tunisia, Algeria and, as discovered in this report, in Libya in patients with Allgrove syndrome suggests the existence of an ancestral mutation and a founder effect in North Africa. CONCLUSIONS: The findings allow for a fast genetic counseling in North African families with Allgrove syndrome. To the best of our knowledge, this is the first report of Allgrove syndrome in Libya.