Two novel mutations in the MECP۲ gene associated with typical Rett syndrome

Background and Aim: Rett syndrome (RTT, MIM ۳۱۲۷۵۰) is a progressive neurological disorder with an incidence rate of ۱ in ۱۰,۰۰۰ that was first reported by the Austrian physician Andreas Rett. Patients with Rett syndrome have similar characteristics to other autism spectrum disorders including developmental delay, hand wringing, microcephaly, ataxia, hypotonia, seizure, poor-eye contact, and gait abnormalities. The RTT disease is classified into two forms based on clinical aspects; classic or typical Rett syndrome and variant or atypical Rett syndrome. Over ۹۰% of typical RTT patients and ۵۰-۶۰% of atypical cases have mutations in the Methyl-CpG binding protein ۲ (MECP۲; MIM ۳۰۰۰۰۵) gene as the main pathogenic gene. Additionally, Mutations in other genes may also induce Rett syndrome, such as Cycline-dependent kinase-like ۵ (CDKL۵), Forkhead protein G۱ (FOXG۱), Myocyte-specific enhancer factor ۲C (MEF۲C), and Transcription factor ۴ (TCF۴). MECP۲ gene is located on the chromosome Xq۲۸ and encodes the methyl CpG binding protein ۲ (MECP۲). This gene contains four exon and is a member of the DNA Binding proteins, which plays an important role in repressing transcription. Mecp۲ protein domains include methyl-CpG binding domain (MBD), transcriptional repression domain (TRD), nuclear localization signal (NLS), C-terminal, domain (CTD), and intervening domain (ID). In terms of transcriptional regulation, Mecp۲ protein plays important role in altering the organization of chromatin by binding to methylated CpG and recruiting corepressor transcription factors including the NCoR / SMRT complex. According to MECP۲ database (http://mecp۲.chw.edu.au), about ۹۰۰ pathogenic mutations in the MECP۲ gene have been reported, most of them in the MBD domain. In this study, we genetically analyzed seven patients with the classic form of Rett syndrome of which two patients had novel mutations such as; a splice mutation, c.۳۵۴G>T, p.Gly۱۱۹Gly, resulting in a premature splice-donor site and a ۲۰-bp deletion, c.۱۱۶۷-۱۱۸۶del۲۰ (p.P۳۹۰Rfs), leading to modifying the c-terminal parts of the protein.Methods: DNA extraction: Peripheral blood samples were taken from patients and their parents, and Genomic DNA was extracted, using the Cinnagen DNA Extraction Kit (Cinnacolon, Iran) based on the manufacturer's method. PCR amplification and Sanger sequencing: Primers were designed to amplify the coding sequence and the flanking intronic sequence of the MECP۲ gene for patients and their parents. Then, PCR amplification was done in a final volume of ۲۰ μl comprising of ۱۰ μl Taq DNA Polymerase, ۲x Master Mix Red (Amplicon, Odense M, Denmark), ۸ μl dH۲O, ۰.۵ μl of each primer (۱۰ pmol/μl) and ۱ μl DNA template (۵۰–۱۰۰ ng). PCR reactions were performed in an ABI ۹۶-well thermocycler (Applied Biosystems Instruments, Foster City, CA, USA) (Table ۱). Finally, Sanger Sequencing was carried out, and the Codon Code Aligner software was used to analyze the results and the results were compared with the reference sequences {NG_۰۰۷۱۰۷.۲ for MECP۲}. The DNA mutations nomenclature was based on HGVS guidelines (http://www.hgvs.org), using GenBank NCBI reference sequences.Results: Patients with novel mutations: Patient ۱: A ۱۲-year-old girl with Rett syndrome, Birth tests revealed the possibility of microcephaly in this case (birth weight, ۳۱۰۰ g, and head circumference, ۳۱ cm). Symptoms of a delay in gross motor activity emerged at ۱۳ months. Moreover, hand wringing started at age ۲, and seizure disorders appeared at age ۳. MRI scan showed Cavum pellucidum septum, and an increase in the white matter signal around the ventricles was considered to be in the terminal zone. Other symptoms of P۶ were observed including poor sleep patterns, abnormal EEG and poor eye gaze. Test results for DTR (Deep Tendon Reflexes) were ۲/۲, and MP (muscle power) was ۵/۵, indicating normal DTR and a lack of hypotonia. In this patient, MECP۲ sequence analysis identified a novel heterozygous mutation (c.۳۵۴G>T, p.Gly۱۱۹Gly, NM_۰۰۴۹۹۲.۳) (figure ۱) that was not observed in the parents. This mutation resulted in a change in glycine codon's third (wobble)position (changes the codon GGG to GGT) but does not change the coded amino acid (i.e. a synonymous change). The probable pathogenicity of this wobble position mutation is related to the creation of an aberrant splice site. Patient ۲: A ۷-year-old girl with normal birth measures (birth weight, ۳۷۰۰ g, and head circumference, ۳۶ cm), she was born full term. Clinical examination at age ۲, showed typical RTT-related symptoms such as abnormal neurological signs, poor eye contact, delay of the psychomotor, poor speech, gait apraxia, and abnormal EEG. Gradually, growth in the head decelerated, and seizures were observed at age ۴. In addition, stereotypic motions occurred at age ۴, which was later than other typical patients with RTT. In this patient a novel frameshift deletion mutation in the MECP۲ gene (c.۱۱۶۷-۱۱۸۶del۲۰, p.P۳۹۰Rfs, NM_۰۰۴۹۹۲) (figure ۲) was found, causing typical Rett syndrome. This variant was not detected in any of the parents. Also, this mutation located inside exon ۴ and modifying the c-terminal section of protein. Bioinformatics analysis: Bioinformatics analysis was conducted to investigate the effects of these variants on the structure and function of genes through online tools, including, Varsome, and Mutation Taster. The results showed that these mutations were pathogenic (table۲). Furthermore, according to Human Splicing Finder (HSF; www.umd.be/HSF/), change GGG to GGT in patient ۱ can cause an exonic cryptic donor site of splicing within the exon ۳ of MECP۲ gene.Conclusion: In conclusion, we described genetic analysis for two patients with symptoms related to typical Rett syndrome and found two novel disease-causing variants in the MECP۲ gene. Our findings expand information about patients with MECP۲ mutations. Further, the results of this study and similar studies have shown that most mutations occur in the MECP۲ gene's third and fourth exons. The molecular methods of diagnosis described in this study could serve as the basis for further study. Additional studies are also warranted to examine other possible outcomes of the mutations at cellular level.

کلیدواژه ها:

Rett syndrome ، MECP۲ gene ، DNA sequencing ، novel mutation