Two Novel Mutations in the Mecp2 Gene Associated With Typical Rett Syndrome

Background and aim: rett syndrome (rtt, mim 312750) is a progressive neurological disorder with an incidence rate of 1 in 10,000 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 90% of typical rtt patients and 50-60% of atypical cases have mutations in the methyl-cpg binding protein 2 (mecp2; mim 300005) gene as the main pathogenic gene. additionally, mutations in other genes may also induce rett syndrome, such as cycline-dependent kinase-like 5 (cdkl5), forkhead protein g1 (foxg1), myocyte-specific enhancer factor 2c (mef2c), and transcription factor 4 (tcf4). mecp2 gene is located on the chromosome xq28 and encodes the methyl cpg binding protein 2 (mecp2). this gene contains four exon and is a member of the dna binding proteins, which plays an important role in repressing transcription. mecp2 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, mecp2 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 mecp2 database (http://mecp2.chw.edu.au), about 900 pathogenic mutations in the mecp2 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.354g>t, p.gly119gly, resulting in a premature splice-donor site and a 20-bp deletion, c.1167-1186del20 (p.p390rfs), 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 mecp2 gene for patients and their parents. then, pcr amplification was done in a final volume of 20 μl comprising of 10 μl taq dna polymerase, 2x master mix red (amplicon, odense m, denmark), 8 μl dh2o, 0.5 μl of each primer (10 pmol/μl) and 1 μl dna template (50–100 ng). pcr reactions were performed in an abi 96-well thermocycler (applied biosystems instruments, foster city, ca, usa) (table 1). 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_007107.2 for mecp2}. the dna mutations nomenclature was based on hgvs guidelines (http://www.hgvs.org), using genbank ncbi reference sequences.results: patients with novel mutations: patient 1: a 12-year-old girl with rett syndrome, birth tests revealed the possibility of microcephaly in this case (birth weight, 3100 g, and head circumference, 31 cm). symptoms of a delay in gross motor activity emerged at 13 months. moreover, hand wringing started at age 2, and seizure disorders appeared at age 3. 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 p6 were observed including poor sleep patterns, abnormal eeg and poor eye gaze. test results for dtr (deep tendon reflexes) were 2/2, and mp (muscle power) was 5/5, indicating normal dtr and a lack of hypotonia. in this patient, mecp2 sequence analysis identified a novel heterozygous mutation (c.354g>t, p.gly119gly, nm_004992.3) (figure 1) 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 2: a 7-year-old girl with normal birth measures (birth weight, 3700 g, and head circumference, 36 cm), she was born full term. clinical examination at age 2, 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 4. in addition, stereotypic motions occurred at age 4, which was later than other typical patients with rtt. in this patient a novel frameshift deletion mutation in the mecp2 gene (c.1167-1186del20, p.p390rfs, nm_004992) (figure 2) was found, causing typical rett syndrome. this variant was not detected in any of the parents. also, this mutation located inside exon 4 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 werepathogenic (table2). furthermore, according to human splicing finder (hsf; www.umd.be/hsf/), change ggg to ggt in patient 1 can cause an exonic cryptic donor site of splicing within the exon 3 of mecp2 gene.