Mitochondrial genes modulate the phenotypic expression of congenital scoliosis syndrome caused by mutations in the TBXT gene.

BACKGROUND: Congenital scoliosis (CS) is a spinal disorder caused by genetic-congenital vertebral malformations and may be associated with other congenital defects or may occur alone. It is genetically heterogeneous and numerous genes contributing to this disease have been identified. In addition, CS has a wide range of phenotypic and genotypic variability, which has been explained by the intervention of genetic factors like modifiers and environment genes. The aim of the present study was to determine the possible cause of CS in a Tunisian patient and to examine the association between mtDNA mutations and mtDNA content and CS. METHODS: Here we performed Whole-Exome Sequencing (WES) in a patient presenting clinical features suggestive of severe congenital scoliosis syndrome. Direct sequencing of the whole mitochondrial DNA (mtDNA) was also performed in addition to copy number quantification in the blood of the indexed case. In silico prediction tools, 3D modeling and molecular docking approaches were used. RESULTS: The WES revealed the homozygous missense mutation c.512A > G (p.H171R) in the TBXT gene. Bioinformatic analysis demonstrated that the p.H171R variant was highly deleterious and caused the TBXT structure instability. Molecular docking revealed that the p.H171R mutation disrupted the monomer stability which seemed to be crucial for maintaining the stability of the homodimer and consequently to the destabilization of the homodimer-DNA complex. On the other hand, we hypothesized that mtDNA can be a modifier factor, so, the screening of the whole mtDNA showed a novel heteroplasmic m.10150T > A (p.M31K) variation in the MT-ND3 gene. Further, qPCR analyses of the patient's blood excluded mtDNA depletion. Bioinformatic investigation revealed that the p.M31K mutation in the ND3 protein was highly deleterious and may cause the ND3 protein structure destabilization and could disturb the interaction between complex I subunits. CONCLUSION: We described the possible role of mtDNA genetics on the pathogenesis of congenital scoliosis by hypothesizing that the presence of the homozygous variant in TBXT accounts for the CS phenotype in our patient and the MT-ND3 gene may act as a modifier gene.

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.

A Novel Mutation in the MAP7D3 Gene in Two Siblings with Severe Intellectual Disability and Autistic Traits: Concurrent Assessment of BDNF Functional Polymorphism, X-Inactivation and Oxidative Stress to Explain Disease Severity.

Intellectual disabilities (ID) and autism spectrum disorders (ASD) are characterized by extreme genetic and phenotypic heterogeneity. However, understanding this heterogeneity is difficult due to the intricate interplay among multiple interconnected genes, epigenetic factors, oxidative stress, and environmental factors. Employing next-generation sequencing (NGS), we revealed the genetic cause of ID and autistic traits in two patients from a consanguineous family followed by segregation analysis. Furthermore, in silico prediction methods and 3D modeling were conducted to predict the effect of the variants. To establish genotype-phenotype correlation, X-chromosome inactivation using Methylation-specific PCR and oxidative stress markers were also investigated. By analyzing the NGS data of the two patients, we identified a novel frameshift mutation c.2174_2177del (p.Thr725MetfsTer2) in the MAP7D3 gene inherited from their mother along with the functional BDNF Val66Met polymorphism inherited from their father. The 3D modeling demonstrated that the p.Thr725MetfsTer2 variant led to the loss of the C-terminal tail of the MAP7D3 protein. This change could destabilize its structure and impact kinesin-1's binding to microtubules via an allosteric effect. Moreover, the analysis of oxidative stress biomarkers revealed an elevated oxidative stress in the two patients compared to the controls. To the best of our knowledge, this is the first report describing severe ID and autistic traits in familial cases with novel frameshift mutation c.2174_2177del in the MAP7D3 gene co-occurring with the functional polymorphism Val66M in the BDNF gene. Besides, our study underlines the importance of investigating combined genetic variations, X-chromosome inactivation (XCI) patterns, and oxidative stress markers for a better understanding of ID and autism etiology.

Identification of a novel homozygous mutation in NAXE gene associated with early-onset progressive encephalopathy by whole-exome sequencing: in silico protein structure characterization, molecular docking, and dynamic simulation.

Progressive encephalopathy with brain edema and/or leukoencephalopathy, PEBEL1, is a severe neurometabolic disorder characterized by rapidly progressive neurologic deterioration associated with a febrile illness. PEBEL1 is a lethal encephalopathy caused by NAXE gene mutations. Here we report a 6-month-old boy with mitochondrial encephalomyopathy from a consanguineous family. Molecular analysis was performed using whole-exome sequencing followed by segregation analysis. In addition, in silico prediction tools and molecular dynamic approaches were used to predict the structural effect of the mutation. Furthermore, molecular docking of the substrate NADP in both wild-type and mutated NAXE protein was carried out. Molecular analysis revealed the presence of the novel homozygous mutation c.641 T > A (p. Ile214Asn) in the NAXE gene, located at the NAD (P)H hydrate epimerase domain. In addition, bioinformatics analyses and molecular dynamics revealed that p. Ile214Asn mutation could affect the structure, stability, and compactness of the NAXE protein. Moreover, the result of the molecular docking showed that the p. Ile214Asn mutation leads to conformational changes in the catalytic cavity, thus modifying interaction with the substrate and restricting its access. We also compared the phenotype of our patient with those of previously reported cases with PEBEL syndrome. All bioinformatics findings provide evidence that the NAXE variant Asn214 disrupts NAXE protein functionality leading to an insufficient NAD (P)HX repair system and the development of clinical features of PEBEL1 syndrome in our patient. To our knowledge, our case is the 21st case of PEBEL1 patient worldwide and the first case in North Africa.

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.

Combined in Silico Prediction Methods, Molecular Dynamic Simulation, and Molecular Docking of FOXG1 Missense Mutations: Effect on FoxG1 Structure and Its Interactions with DNA and Bmi-1 Protein.

FoxG1 encoded by FOXG1 gene is a transcriptional factor interacting with the DNA of targeted genes as well as with several proteins to regulate the forebrain development. Mutations in the FOXG1 gene have been shown to cause a wide spectrum of brain disorders, including the congenital variant of Rett syndrome. In this study, the direct sequencing of FOXG1 gene revealed a novel c.645C > A (F215L) variant in the patient P1 and a de novo known one c.755G > A (G252D) in the patient P2. To investigate the putative impact of FOXG1 missense variants, a computational pipeline by the application of in silico prediction methods, molecular dynamic simulation, and molecular docking approaches was used. Bioinformatics analysis and molecular dynamics simulation have demonstrated that F215L and G252D variants found in the DNA binding domain are highly deleterious mutations that may cause the protein structure destabilization. On the other hand, molecular docking revealed that F215L mutant is likely to have a great impact on destabilizing the protein structure and the disruption of the Bmi-1 binding site quite significantly. Regarding G252D mutation, it seems to abolish the ability of FoxG1 to bind DNA target, affecting the transcriptional regulation of targeted genes. Our study highlights the usefulness of combined computational approaches, molecular dynamic simulation, and molecular docking for a better understanding of the dysfunctional effects of FOXG1 missense mutations and their role in the etiopathogenesis as well as in the genotype-phenotype correlation.

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.

A first description of ataxia with vitamin E deficiency associated with MT-TG gene mutation.

Ataxia with isolated vitamin E deficiency (AVED) is a rare autosomal recessive cerebellar ataxia disorder that is caused by a mutation in the alpha-tocopherol transfer protein gene TTPA, leading to a lower level of serum vitamin E. Although it is almost clinically similar to Friedreich's ataxia, its devastating neurological features can be prevented with appropriate treatment. In this study, we present a patient who was initially diagnosed with Friedreich's ataxia, but was later found to have AVED. Frataxin gene screening revealed the absence of GAA expansion in homozygous or heterozygous state. However, TTPAgene sequencing showed the presence of the c.744delA mutation, leading to a premature stop codon (p.E249fx). In addition, the result of mutational analysis of MT-DNA genes revealed the presence of several variants, including the m.10044A>G mutation in MT-TG gene. Here, we report for the first time the coexistence of both mitochondrial and nuclear genes mutations in AVED.

First description of an unusual novel double mutation in MECP2 co-occurring with the m.827A>G mutation in the MT-RNR1 gene associated with angelman-like syndrome.

Mutations in Methyl-CpG-Binding protein 2 (MECP2), located on Xq28 and encoding a methyl CpG binding protein, are commonly related to Rett syndrome. However, MECP2 mutations have already been reported in patients with neurodevelopmental abnormalities such as X-linked mental retardation, severe neonatal encephalopathy and Angelman-like syndrome (AS-like). Accordingly, we report the clinical, molecular and bioinformatic analyses in a Tunisian patient with AS-like phenotype. In fact, the direct sequencing of MECP2 and cloning essay reveals the emergence of an unusual novel double mutation, including a de novo mutation c.397C > T (p.R133C) and an inherited one c.608C > T (p.T203 M) co-occurring in Trans. We also provide the molecular evidence of the c.608C > T transmission to the patient which was present in her father at somatic mosaicism state. To gain insight into the molecular basis of this disorder, we undertook, for the first time, a whole mitochondrial genome mutational analysis. Thus, the results showed the presence of several variations and a homoplasmic mutation m.827A > G in the MT-RNR1 gene, leading to the disruption of the 12S rRNA secondary structure. Our report is considered as the first to describe an unusual novel double mutation (c.397C > T in trans with c.608C > T) in MECP2 co-occurring with the mitochondrial m.827A > G mutation in the MT-RNR1 gene in a Tunisian patient with AS-like. Besides, our results highlight the importance of studying MECP2 and the significance of mDNA screening in AS-like disorder for a better understanding of its etiopathogenesis.

First description of a novel mitochondrial mutation in the MT-TI gene associated with multiple mitochondrial DNA deletion and depletion in family with severe dilated mitochondrial cardiomyopathy.

Mitochondria are essential for early cardiac development and impaired mitochondrial function was described associated with heart diseases such as hypertrophic or dilated mitochondrial cardiomyopathy. In this study, we report a family including two individuals with severe dilated mitochondrial cardiomyopathy. The whole mitochondrial genome screening showed the presence of several variations and a novel homoplasmic mutation m.4318-4322delC in the MT-TI gene shared by the two patients and their mother and leading to a disruption of the tRNAIle secondary structure. In addition, a mitochondrial depletion was present in blood leucocyte of the two affected brother whereas a de novo heteroplasmic multiple deletion in the major arc of mtDNA was present in blood leucocyte and mucosa of only one of them. These deletions in the major arc of the mtDNA resulted to the loss of several protein-encoding genes and also some tRNA genes. The mtDNA deletion and depletion could result to an impairment of the oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patients. Our report is the first description of a family with severe lethal dilated mitochondrial cardiomyopathy and presenting several mtDNA abnormalities including punctual mutation, deletion and depletion.

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.

Clinical, Molecular, and Computational Analysis in Patients With a Novel Double Mutation and a New Synonymous Variant in MeCP2: Report of the First Missense Mutation Within the AT-hook1 Cluster in Rett Syndrome.

Rett syndrome is an X-linked neurodevelopmental disorder, primarily caused by MECP2 mutations. In this study, clinical, molecular and bioinformatics analyses were performed in Rett patients to understand the relationship between MECP2 mutation type and the clinical severity. Two double MeCP2 mutations were detected: a novel one (p.G185 V in cis with p.R255X) in P1 and a known one (p.P179 S in cis with p.R255X) in P2. Besides, a novel synonymous mutation (c.807C>T; p.G269G), which could affect mRNA splicing, was identified in P3. The results from clinical severity analysis have shown that P1 was more severely affected than P2 with CSS being 35 and 14, respectively. Therefore, the phenotypic variability in P1 and P2 could be explained by the potential pathogenic effect of the RTT-causing missense mutation p.G185 V in the AT-hook1. In conclusion, clinical, molecular, and in silico investigations in the studied patients have been proven to be substantial for the genotype-phenotype correlation.

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.