First report of an unusual novel double mutation affecting the transcription repression domain of MeCP2 and causing a severe phenotype of Rett syndrome: Molecular analyses and computational investigation.

Rett syndrome is an X-linked neurodevelopmental disorder that develops a profound intellectual and motor disability and affects 1 from 10 000 to 15 000 live female births. This disease is characterized by a period of apparently normal development until 6-18 months of age when motor and communication abilities regress which is caused by mutations occurred in the X-linked MECP2 gene, encoding the methyl-CpG binding protein 2. This research study reports a molecular analysis via an exhaustive gene sequencing which reveals an unusual novel double mutation (c.695 G > T; c.880C > T) located in a highly conserved region in MECP2 gene affecting the transcription repression domain (TRD) of MeCP2 protein and leading for the first time to a severe phenotype of Rett syndrome. Moreover, a computational investigation of MECP2 mutations demonstrates that the novel mutation c.695 G > T is highly deleterious which affects the MeCP2 protein showing also an adverse impact on MECP2 gene expression and resulting in an affected folding and decreased stability of MECP2 structures. Thus, the altered TRD domain engenders a disrupted process of MECP2 functions. Therefore, this is the first study which highlights a novel double mutation among the transcription repression domain (TRD) of MeCP2 protein in Rett patient with a severe clinical phenotype in North Africa region.

Unusual double mutation in MECP2 and CDKL5 genes in Rett-like syndrome: Correlation with phenotype and genes expression.

INTRODUCTION: Rett syndrome (RTT) is a neuro-developmental disorder affecting almost exclusively females and it divided into classical and atypical forms of the disease. RTT-like syndrome was also described and presents an overlapping phenotype of RTT. RTT-like syndrome has been associated with several genes including MECP2 and CDKL5 having common biological pathways and regulatory interactions especially during neural maturation and synaptogenesis. METHODS: We report patient with Rett-like syndrome for whom clinical features and their progression guided toward the screening of two candidate genes MECP2 and CDKL5 by sequencing. Severity score was evaluated by "Rett Assessment Rating Scale" (R.A.R.S.). Predictions of pahogenicity and functional effects used several bioinformatic tools and qRT-PCR was conducted to evaluate gene expression. RESULTS: Mutational screening revealed two mutations c.1065 C > A (p.S355R) in MECP2 gene and c.616 G > A (p.D206N) mutation in CDKL5 gene in the patient with a high R.A.R.S. Bioinformatic investigations predicted a moderate effect of p.S355R in MECP2 gene but a more pathogenic one of p.D206N mutation in CDKL5. Effect of c.616 G > A mutation on structure and stability of CDKL5 mRNA was confirmed by qRT-PCR. Additionally, analysis of gene expression revealed a drastic effect of CDKL5 mutant on its MeCP2 and Dnmt1 substrates and also on its MYCN regulator. CONCLUSIONS: The co-existence of the two mutations in CDKL5 and MECP2 genes could explain the severe phenotype in our patient with RTT-Like and is consistent with the data related to the interactions of CDKL5 with MeCP2 and Dnmt1 proteins.

Do GSTM1 and GSTT1 polymorphisms influence the risk of developing mitochondrial diseases in a Tunisian population?

Mitochondria play an essential role to supply the cell with metabolic energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). As a consequence, they are also the primary source of cellular reactive oxygen species (ROS) which can cause oxidative damage of individual respiratory chain complexes. Indeed, affected OXPHOS subunits result in decreases in ATP production and increases in ROS formation which generate oxidative phosphorylation deficiency leading to mitochondrial dysfunctions. It has been suggested that ROS play a vital role in the pathogenesis of mitochondrial diseases. To the best of our knowledge, this is the first study which aimed to investigate the genetic variant effect of the antioxidant enzymes GSTM1 and GSTT1 on mitochondrial disease among a Tunisian population. In this report, 109 patients with mitochondrial disease and 154 healthy controls were genotyped by multiplex PCR amplification, and data were analyzed by SPSS v20 software. The results showed that GSTM1 null genotype was found to be associated with mitochondrial disease with a protective effect; however, no significant association of GSTT1 polymorphism with mitochondrial disease risk was revealed. But, interestingly, our findings highlight that GSTM1 active and GSTT1 null genotype combination increased by three fold the risk of developing mitochondrial disease with p c = 0.020, notably mitochondrial myopathy with p c = 0.046 and Leigh syndrome with p c = 0.042. In conclusion, this study suggests that GSTM1 active and GSTT1 null genotype combination might be a risk factor in developing mitochondrial disease.

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 Novel Mutation p.A59P in N-Terminal Domain of Methyl-CpG-Binding Protein 2 Confers Phenotypic Variability in 3 Cases of Tunisian Rett Patients: Clinical Evaluations and In Silico Investigations.

Rett syndrome is a monogenic X-linked dominant neurodevelopmental disorder related to mutation in MECP2, which encodes the methyl-CpG-binding protein MeCP2. The aim of this study was to search for mutations of MECP2 gene in Tunisian Rett patients and to evaluate the impact of the found variants on structural and functional features of MeCP2. The result of mutation analysis revealed that 3 Rett patients shared the same novel heterozygous point mutation c.175G>C (p.A59P). The p.A59P mutation was located in a conserved amino acid in the N-terminal segment of MeCP2. This novel mutation confers a phenotypic variability with different clinical severity scores (3, 8, and 9) and predicted by Sift and PolyPhen to be damaging. Modeling results showed that p.A59P adds 2 hydrogen bonds and changes the structural conformation of MeCP2 with a significant root mean square deviation value (9.66 Å), suggesting that this mutation could probably affect the conformation, function and stability of MeCP2.

Novel double deletions in the MECP2 gene in Tunisian Rett patient.

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively girls. Rett patients present an apparently normal psychomotor development during the first 6-18 months of life. Thereafter, they show a short period of developmental stagnation followed by a rapid regression in language and motor development. RTT is currently considered as monogenic X-linked dominant disorder due to mutations in the MECP2 gene, encoding the methyl-CpG binding protein 2. The aim of this study was to perform a mutational analysis of the MECP2 gene in a classical Rett patient.The results showed the presence of a novel point mutation c.C1142T (p.P381L) and two deletions at the heterozygous state: a novel deletion c.1075delTTC (p.S359) and a known one c.1157del44 (p.L386Q fs X2) in the C-terminal region of MeCP2.

Novel mutations in the C-terminal region of the MECP2 gene in Tunisian Rett syndrome patients.

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder in females, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Rett patients present an apparently normal psychomotor development during the first 6 to 18 months of life. Thereafter, they show a short period of developmental stagnation followed by a rapid regression in language and motor development. In the present study, we performed a mutational analysis of the MECP2 gene in 2 typical Rett syndrome patients and in 1 atypical Rett syndrome girl. The results showed the presence of 3 de novo point mutations in the C-terminal region: 2 novel mutations: c.1065C>A (p.S355R) and c.1030C>G (p.R344G) in the 2 typical Rett syndrome girls, but also the c.996C>T (p.S332S) mutation first described in the atypical Rett syndrome patient.