- Email: [email protected]
Two novel mutations in the MECP2 gene in patients with Rett syndrome
Journal Pre-proofs Research paper Two novel mutations in the MECP2 gene in patients with Rett syndrome Shayan Khalili Alashti, Jafar Fallahi, Sanaz Mohammadi, Fatemeh Dehghanian, Zahra Farbood, Marjan Masoudi, Shiva Poorang, Arezoo Jokar, Majid Fardaei PII: DOI: Reference:
S0378-1119(20)30006-8 https://doi.org/10.1016/j.gene.2020.144337 GENE 144337
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
27 September 2019 4 December 2019 6 January 2020
Please cite this article as: S. Khalili Alashti, J. Fallahi, S. Mohammadi, F. Dehghanian, Z. Farbood, M. Masoudi, S. Poorang, A. Jokar, M. Fardaei, Two novel mutations in the MECP2 gene in patients with Rett syndrome, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144337
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
Two novel mutations in the MECP2 gene in patients with Rett syndrome Authors: Shayan Khalili Alashti Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Jafar Fallahi Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Sanaz Mohammadi Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Fatemeh Dehghanian Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Zahra Farbood Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Marjan Masoudi Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Shiva Poorang Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected]
Arezoo Jokar Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected]
Majid Fardaei * Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Location of work and address: Zand Avenue, Shiraz University of Medical Sciences, Fars province, Shiraz, Iran Corresponding author: Majid Fardaei Tel: +989173054863 Fax: (+98 71) 32 35 69 96 Email: [email protected]
Two novel mutations in the MECP2 gene in patients with Rett syndrome Shayan Khalili Alashtia, Jafar Fallahib, Sanaz Mohammadic, Fatemeh Dehghanianc, Zahra Farboodc , Marjan Masoudic, Shiva Poorangc, Arezoo Jokard, Majid Fardaeia,* a) Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran b) Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran c) Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran d) Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
_____________________________________________________________________________________
Abstract Rett syndrome (RTT) is an X-linked severe neurological disorder. Mutations in Methyl-CpGBinding Protein2 (MECP2) gene are the main cause of RTT disease. In this study, we report the results of screening the MECP2 gene for mutations in 7 Iranian patients with RTT syndrome. MECP2 sequencing identified two novel mutations in the heterozygous state, a splice mutation, c.354G>T, p.Gly119Gly, resulting in a premature splice-donor site and a 20-bp deletion, c.11671186del20 (p.P390Rfs), leading to modifying the c-terminal parts of the protein and it also changes the reading frames of all coding sequence downstream of the mutation. Multiple sequence alignment showed that amino acid changes occurred in the well conserved protein regions across species. Based on the results of this study and literature reviews, about 70% of mutations are found in exon 3 and 4 of the MECP2 gene, and mutations in exon 4 are more common than other exons. Therefore, it is recommended that exon 4 to be a priority for screening the genetic analysis of RTT patients.
Highlights ►The analysis of MECP2 mutations was performed in 7 Iranian patients with Rett syndrome. ►Two novel mutations such as c.354G>T, p.Gly119Gly (NM_004992.3) and c.1167-1186del20 (p.P390Rfs) (NM_004992) were identified. ►Novel mutations are predicted to be pathogenic and responsible for the phenotype.
Abbreviations RTT, Rett syndrome; MECP2, methyl CpG-binding protein 2; CDKL5, cyclin-dependent kinaselike 5; FOXG1, Forkhead box protein G1; MEF2C, Myocyte-specific enhancer factor 2C; TCF4, Transcription factor 4; MBD, methyl-CpG binding domain; TRD, transcriptional repression domain; CTD, C-terminal domain; NLS, nuclear localization signal; ID, intervening domain; PCR, polymerase chain reaction; DPR, Deletion Prone Region; E, exon;
Keywords Rett syndrome; RTT; MECP2 gene; Novel mutation; DNA sequencing
1. Introduction Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder that was first reported by the Austrian physician Andreas Rett (1). RTT has an incidence rate of 1 in 10000 (2). This disorder is one of the most common causes of mental retardation, which represents up to 10% of cerebral deficiency with hereditary origin in women and the second reason of extreme intellectual inability in females after Down syndrome (3, 4). Although 90% of RTT cases are caused by de novo mutations in the X-linked gene located on Xq28 encoding methyl CpG binding protein 2 (MECP2), mutations in other genes can also cause Rett syndrome, such as cyclin-dependent kinase-like 5 (CDKL5), Forkhead box protein G1 (FOXG1), Myocyte-specific enhancer factor 2C (MEF2C), and Transcription factor 4 (TCF4) (5, 6). The most common RTT cases are called classic form, which described by a duration of normal development up to around 6-18 months of age followed by brain deficiency, impaired speech, hand-wringing, microcephaly, ataxia, scoliosis, and breathing disorder such as alternative ventilation (7). Furthermore, at a cellular level, other indications are observed in patients with RTT including smaller and more compactly neurons that are decremented in length, leading to complications (8). In spite of “classical RTT”, another type of this disorder is called “atypical RTT” or “forme fruste” (9). The two splice variants are produced by the MECP2 gene and each of them encode a protein isoform (10). The primary splicing form of MECP2 gene, now termed MeCP2-e2, has four exons and the start codon is located in exon 2. In the second splicing form, known as MeCP2-e1, exon 2 is missing and translation starts in exon 1. MeCP2-e1 is the main isoform in brain tissues (11, 12). Four protein domains have been identified within MeCP2 protein; however, most of them are highly conserved. The first recognized domain is the methyl-CpG binding domain (MBD) which binds to a DNA sequence at methylated cytosine in the CpG. Subsequently, other functional domains, known as transcriptional repression domain (TRD) and nuclear localization signal (NLS) were also identified. Also, C-terminal domain (CTD) and intervening domain (ID) are less described domains that bind to histones and unmethylated DNA (11). Various researches have postulated that MeCP2 plays several important roles in transcriptional regulation as well as
modifying chromatin organization via binding to methylated CpG and the recruitment of transcription corepressor factors including the NCoR/SMRT complex (13). Currently, over 900 unique variations are found in the MECP2 gene, and up to 80% of them are in exon 3 or 4. Presumably, the intensity of RTT is related to different mutations in diverse domains (11). In this study, we identified 7 Iranian RTT patients of which 2 had a novel mutation and the rest had previously reported mutations in the MECP2 gene.
2. Materials and methods 2.1. Patients The 7 cases of RTT from southern Iran and their parents were called for genetics examination in Comprehensive Medical Genetics Center affiliated with Shiraz University of Medical Sciences. Moreover, before any analysis, they completed the informed consent based on the ethics committee of Shiraz University of Medical Sciences.
2.2 DNA extraction The peripheral blood samples were collected from the patients and parents, then DNA extraction was performed using the Cinnagen DNA Extraction Kit (Cinnacolon, Iran) according to the manufacturer's protocol. The concentration of DNA was measured by a NanoDrop (ND1000, NanoDrop Technologies, Wilmington, DE, USA).
2.3 PCR amplification The PCR amplification was done for the coding sequence and the flanking intronic sequence of the MECP2 gene, using the primers that are described in table 1. Primers were designed and evaluated by NCBI BLAST (https://blast.ncbi.nlm.nih.gov), ENSEMBLE BLAST/BLAT (https:// www.ensembl.org), and Allele ID 7.5 (primers and probes design software). The PCR reactions were carried out 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). The conditions for PCR reactions are described in table 1. Table 1. Primers sequences and PCR conditions related to amplification of 4 exons of MECP2 gene.
EXON
Forward primer
Reverse primer
PCR condition
Product size
E1
CCTCTTTTCCCCAAACG ACG
CCCAAAGCCCCGAGA AGG
Anneling:61 95:15min 95:30sec 61:30 sec 72:1 min
751bp
E2
TGTGTTTATCTTCAAAA TGT
GTTATGTCTTTAGTCT TTGG
Anneling:55 95:15min 95:30sec 55:30 sec 72:15 sec
365bp
E3
GTGCTAGGAGACTTGT GG
CACATACATTTTCCTG CTCC
Anneling:60 95:15min 95:30sec 60:30 sec 72:30 sec
686bp
E4
TATCTCTGACATTGCTA T
CTTTATTCTTGTTGGT TTG
Anneling:64 95:15min 95:30sec 64:30 sec
1191bp
72:70 sec
2.4 Mutation and sequence analysis Finally, the Sanger sequencing was done, and the results were analyzed by the Gene tool program and Codon Code Aligner program. Moreover, the results were compared with the reference sequences (NG_007107.2) for MECP2. Nomenclature for mutations was confirmed with HGVS guidelines (http://www.hgvs.org) using GenBank NCBI reference sequences.
3. Results In this study, we have studied 7 female patients (Patients 1–7) with Rett syndrome for presence of mutations in the MECP2 gene. Based on the clinical symptoms, all patients in this study had typical or classic Rett syndrome with mutation in the MECP2 gene. All 4 exons of MECP2 gene were amplified using gene specific primers. As a result, sequence analysis in patients (table2) revealed 2 novel mutations in the MECP2 gene (Patients 6 and patient 7). No pathogenic mutation was observed in the intronic regions.
Table 2. MECP2 gene mutations in this study. Patient 6 and patient 7 had novel mutations, and 5 out of 7 patients had a mutation in exon 4.
Patients 1
Sex F
Exon 4
2
F
4
3
F
4
4
F
3
5
F
4
6
F
3
7
F
4
Mutation c.820C>T p.Q274X c.398C>T p.R133C c.473C>T p.T158M c.352C>T p.R118W c.799C>T p.R267Ter c.354G>T p.G119G c.11671186del20 (p.P390Rfs)
rs rs267608525
Refs. Milunski et al., 2001 (14)
rs61748389
Li et al., 2006 (15)
rs28934906
Ehrhart et al., 2016 (16)
rs28934907
Ehrhart et al., 2016 (16)
rs61749721
Ehrhart et al., 2016 (16)
This study This study
3.1 Genetic analysis of patients with novel mutations 3.1.1 Patient (P6) A 12-year-old girl with Rett syndrome, Measurements at birth indicated the probability of microcephaly in this patient (birth weight, 3,100 g, and head circumference, 31 cm). At 13 months, symptoms of gross motor function delay appeared. Furthermore, the Stereotypic hand movements began at age 2, and seizure disorders appeared after the age of 3. MRI scan revealed Cavum septum pellucidum, and an increase in the signal in the white matter around the ventricles was considered
to be in the terminal zone. Other symptoms of P6 including poor sleep patterns, abnormal EEG, and poor eye-gaze were observed. The DTR (Deep Tendon Reflexes) test result was 2/2, and MP (muscle power) was 5/5, indicating normal DTR and a lack of hypotonia. The sequencing of 4 exons of MECP2 gene showed that she carried a novel mutation c.354G>T, p.Gly119Gly (NM_004992.3) in heterozygous state in exon 3 of MECP2 gene (Fig. 1a). Since the patient’s mother and father did not have this mutation (Fig. 1b, and c); hence, this patient has a de novo mutation. Moreover, this mutation does not alter the amino acid sequence. To evaluate the effect of this mutation, predictive tools (mutation taster and VARSOME) were used. Also, the result showed that p.Gly119Gly variant is disease-causing (table 3).
a)
b)
c) Fig. 1. The sequencing results of patient 6 and parents a) Sanger sequence chromatogram of P6 showing a novel heterozygous missense mutation in exon 3 of the MECP2 gene. The location of the mutation is indicated by the arrow. (Nucleotide substitution from G→T at nucleotide position 354). b and c) The Sanger sequencing of the father (b) and mother (c) indicated the absence of this mutation in parents.
3.1.2 Patient (P7) We carried out mutation analysis of the MECP2 gene for a 7-year-old-girl with RTT. She was born full term with normal birth measurements (birth weight, 3,700 g, and head circumference, 36 cm). At age 2, clinical examination showed typical RTT-related symptoms such as abnormal neurological signs, poor eye-contact, psychomotor delay, poor speech, gait apraxia, and abnormal EEG. Gradually head growth decelerated, and seizures were observed at 4 years of age. Additionally, at age 4, stereotypic movements occurred, which was later than other typical RTT patients.
We found a novel mutation that had not been previously reported. Parent’s analysis was negative for this mutation (Fig. 2b, and c), indicating that this was a de novo mutation. Accordingly, this novel frameshift mutation is c.1167-1186del20 (p.P390Rfs) (NM_004992) (Fig. 2a), located inside the exon 4 and modifying the c-terminal section of protein and based on the online software this mutation is pathogenic (table3).
a)
b)
c) Fig. 2. The sequencing results of patient 7 and parents a) Sequencing (reverse strand) of the P7 demonstrated a 20-bp deletion (c.1167-1186del20) in exon 4 of MECP2 gene, which resulted in a frameshift leading to premature termination of the 486 amino acid protein at the 390th codon. The deletion location is indicated by a cursor. b and c) The Sanger sequencing of the father (b) and mother (c) indicated the absence of this mutation in parents.
Table 3. Results of online software show that the MECP2 gene variants in patient 6 and 7 are pathogenic.
3.2 Sequence and structure analysis Multiple sequence alignment was accomplished for these novel mutations using Clustal Omega program with its homologs retrieved from NCBI database and those are amino acid sequences of Homo sapiens (Accession No NP_001104262.1), Nomascus leucogenys (XP_030663420.1), Pan Troglodytes (XP_521333.3), Pongo abelii (PNJ09457.1), Mus musculus (NP_001075448.1). Sequences alignment showed that the mutation in patient 7 (p.390fs) is well conserved across species (Figs. 3a and b)
a)
b)
Fig. 3. Comparison of MECP2 gene conservation rates to its homologs. (a) Multiple sequence alignment of p.390fs with homologous sequences from different species. (b) Weblogo revealing the well conservation of this mutation across species.
4. Discussion In more than 95% of patients with Rett syndrome, MECP2 gene is mutated. According to a study by Wendi et al. about 70% of the pathogenic mutations in the MECP2 gene are located in MBD and TRD domains, which are in the third and fourth exons. Also, there are eight mutations in these domains, accounting for about 50% of all the mutations in the MECP2 gene (p.R106W, p.R133C, p.T158M, p.R168X, p.R255X, p.R270X, p.R294X, and p.R306C) (6, 17). In this study, we carried
out mutation analysis of this gene for 7 patient with symptoms related to Rett syndrome and their parents. Two novel mutations were found in these patients, which were not reported in any previous study. Patient 6 carried a novel mutation c.354G>T, p.Gly119Gly in heterozygous state in exon 3 of MECP2 gene. This substitution causes a change in the third (wobble) position of glycine codon but does not change in coded amino acid (i.e. a synonymous change). The probable pathogenicity of the mutation in wobble position is related to creating an aberrant splice site (18). In this study, we described the pathogenic consequences of a synonymous mutation in MECP2 exon 3. This SNP changes the codon GGG to GGT, coding for the same amino acid (p.Gly119Gly). This mutation can cause premature splice-donor site (Fig. 4); hence, it can cause frameshift and alterations in gene expression. Furthermore, in silico analysis with Human Splicing Finder (HSFVersion 3.1) showed that this mutation can activate an exonic cryptic donor site.
Fig. 4. Activation of cryptic splice site at c.354G>T in MECP2 exon 3. The splicing system uses the GT sequence within the 5' end of the intron as the donor site and the AG sequence within the 3' end of the intron as the acceptor site. Based on in-silico results, change GGG to GGT in patient 6 can cause an exonic cryptic donor site of splicing within the exon 3 of MECP2 gene. Graphic representation, offering the alternative pre-mRNA splicing of the mutant transcript of MECP2 gene.
We carried out mutation analysis of MECP2 gene for patient 7 (7-year-old-girl with RTT) and her healthy parents and found a novel mutation that had not been reported previously in RettBase (http://mecp2.chw.edu.au) and other mutation databases. Accordingly, this novel frameshift mutation is c.1167-1186del20 (p.P390Rfs), located inside exon 4. This area of exon 4 is a hotspot for deletion occurrence, known as Deletion Prone Region (DPR) of MECP2 (19). Additionally, some deletions occurred in nearby areas which were described in other studies (Fig. 5).
C.1167-1186del20
Fig. 5. Some of deletion mutations around the deleted 20-bp in the P7 (the novel mutation of patient 7 is shown with arrow).
There are particular characteristics around the c-terminal area of MECP2 gene such as, small direct/inverted and short tandem repeats, making this region prone to deletion/insertion mutagenesis (20) Based on this, about 10% of all Rett syndrome cases are caused by small deletions in exon 4 of MECP2 gene (21). P7 deletion (c.1167-1186del20) is located between direct repeats ACCCC and CCCCACC…CCTGAGCCC (20) as well as the inverted repeats ACCCC/CCCCA (Figs. 6a, b, and c). Therefore, based on previous studies, the “polymerase slippage” is the most likely mechanisms responsible for this deletion.
a
b
c Fig. 6. There are particular repeated Sequences around the c-terminal area of MECP2 gene such as small direct/inverted and short tandem repeats. (a) There are many repeated sequences such as, small direct/inverted and short tandem repeats in the Deletion Prone Region (DPR) of MECP2 gene (almost in between c.1000-1200) that are susceptible to deletion mutation in this area. (b and c) since there are repeated sequences such as directed repeats (ACCCC and CCCCACC….CCTGAGCCC) and inverted repeats (ACCCC/CCCCA) around the 20-bp deleted sequence in P7, as a result, polymerase slippage probably causes deletion in this region.
In a study by Telethon Institute for Child Health Research, Center for Child Health Research, University of Western Australia, A Bebbington et al. Genotype-phenotype correlations studies showed that patients with deletions in c-terminal of MECP2 gene have mild symptoms, including conserved hand function, lack feeding difficulties, tend to have the latest onset of regression as well as later onset of stereotypies, seizure, and scoliosis. Also, in these patients, head circumference at birth is usually in the normal range (about 35cm) (22, 23). In conclusion, two novel mutations were reported in the MECP2 gene related to RTT syndrome. Based on the results of bioinformatics analysis, mutation in the patient 6 may possibly causes splice defect and the patient 7 had a c-terminal deletion mutation. In addition, the results of this study and similar studies have shown that most mutations occur in the third and fourth exons of the MECP2 gene. The molecular diagnostic methods described in this study could be the basis for further studies. Also, further studies are warranted to examine other possible outcomes of the mutations at cellular level. Competing interests
The authors declare no conflict of interest. Acknowledgment The authors thank the families for participating in this study and wish to thank Mr. H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.
References 1. Townend GS, Ehrhart F, van Kranen HJ, Wilkinson M, Jacobsen A, Roos M, et al. MECP2 variation in Rett syndrome—An overview of current coverage of genetic and phenotype data within existing databases. Human mutation. 2018;39(7):914-24. 2. Lyst MJ, Bird A. Rett syndrome: a complex disorder with simple roots. Nature Reviews Genetics. 2015;16(5):261. 3. Villard L, Kpebe A, Cardoso C, Chelly J, Tardieu M, Fontes M. Two affected boys in a Rett syndrome family: clinical and molecular findings. Neurology. 2000;55(8):1188-93. 4. Ehinger Y, Matagne V, Villard L, Roux J-C. Rett syndrome from bench to bedside: Recent advances. F1000Research. 2018;7. 5. Dajani R, Koo SE, Sullivan GJ, Park IH. Investigation of Rett syndrome using pluripotent stem cells. Journal of cellular biochemistry. 2013;114(11):2446-53. 6. Gold WA, Krishnarajy R, Ellaway C, Christodoulou J. Rett syndrome: a genetic update and clinical review focusing on comorbidities. ACS chemical neuroscience. 2017;9(2):167-76. 7. Ip JP, Mellios N, Sur M. Rett syndrome: insights into genetic, molecular and circuit mechanisms. Nature Reviews Neuroscience. 2018;19(6):368. 8. Sheikh TI, Mittal K, Willis MJ, Vincent JB. A synonymous change, p. Gly16Gly in MECP2 Exon 1, causes a cryptic splice event in a Rett syndrome patient. Orphanet journal of rare diseases. 2013;8(1):108. 9. Hadzsiev K, Polgar N, Bene J, Komlosi K, Karteszi J, Hollody K, et al. Analysis of Hungarian patients with Rett syndrome phenotype for MECP2, CDKL5 and FOXG1 gene mutations. Journal of human genetics. 2011;56(3):183. 10. Gianakopoulos PJ, Zhang Y, Pencea N, Orlic‐Milacic M, Mittal K, Windpassinger C, et al. Mutations in MECP2 exon 1 in classical Rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2012;159(2):210-6. 11. Xiang F, Buervenich S, Nicolao P, Bailey ME, Zhang Z, Anvret M. Mutation screening in Rett syndrome patients. Journal of medical genetics. 2000;37(4):250-5.
12. Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, Klein F, et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell. 1992;69(6):905-14. 13. Lebo R, Ikuta T, Milunsky J, Milunsky A. Rett syndrome from quintuple and triple deletions within the MECP2 deletion hotspot region. Clinical genetics. 2001;59(6):406-17. 14. Milunsky JM, Lebo RV, Ikuta T, Maher TA, Haverty CE, Milunsky A. Mutation analysis in Rett syndrome. Genetic testing. 2001;5(4):321-5. 15. Li M-r, Pan H, Bao X-H, Zhang Y-Z, Wu X-R. MECP2 and CDKL5 gene mutation analysis in Chinese patients with Rett syndrome. Journal of human genetics. 2007;52(1):38. 16. Ehrhart F, Coort SL, Cirillo E, Smeets E, Evelo CT, Curfs LM. Rett syndrome–biological pathways leading from MECP2 to disorder phenotypes. Orphanet journal of rare diseases. 2016;11(1):158. 17. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature genetics. 1999;23(2):185. 18. Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature reviews genetics. 2002;3(4):285. 19. Kammoun F, De Roux N, Boespflug-Tanguy O, Vallee L, Seng R, Tardieu M, et al. Screening of MECP2 coding sequence in patients with phenotypes of decreasing likelihood for Rett syndrome: a cohort of 171 cases. Journal of medical genetics. 2004;41(6):e85-e. 20. Philippe C, Villard L, De Roux N, Raynaud M, Bonnefond J, Pasquier L, et al. Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. European journal of medical genetics. 2006;49(1):9-18. 21. Fichou Y, Nectoux J, Bahi-Buisson N, Rosas-Vargas H, Girard B, Chelly J, et al. The first missense mutation causing Rett syndrome specifically affecting the MeCP2_e1 isoform. Neurogenetics. 2009;10(2):127. 22. Kriaucionis S, Bird A. The major form of MeCP2 has a novel N‐terminus generated by alternative splicing. Nucleic acids research. 2004;32(5):1818-23. 23. Bebbington A, Anderson A, Ravine D, Fyfe S, Pineda M, De Klerk N, et al. Investigating genotype–phenotype relationships in Rett syndrome using an international data set. Neurology. 2008;70(11):868-75.
Abbreviations
RTT, Rett syndrome; MECP2, methyl CpG-binding protein 2; CDKL5, cyclin-dependent kinaselike 5; FOXG1, Forkhead box protein G1; MEF2C, Myocyte-specific enhancer factor 2C; TCF4, Transcription factor 4; MBD, methyl-CpG binding domain; TRD, transcriptional repression domain; CTD, C-terminal domain; NLS, nuclear localization signal; ID, intervening domain; PCR, polymerase chain reaction; DPR, Deletion Prone Region; E, exon;
Authors
Contribution
Shayan Khalili Alashti
Fatemeh Dehghanian
Conceptualization, Writing - Original Draft, Writing - Review & Editing, Investigation, Methodology Project administration, Writing - Review & Editing, Project administration Writing - Original Draft, Data Curation, Visualization Data Curation, Formal analysis, Investigation
Zahra Farbood
Writing - Original Draft, Resources, Software
Marjan Masoudi
Data Curation, Resources
Shiva Poorang
Investigation, Methodology
Arezoo Jokar
Software, Validation
Majid Fardaei
Correspond
Jafar Fallahi Sanaz Mohammadi
Highlights ►The analysis of MECP2 mutations was performed in 7 Iranian patients with Rett syndrome. ►Two novel mutations such as c.354G>T, p.Gly119Gly (NM_004992.3) and c.1167-1186del20 (p.P390Rfs) (NM_004992) were identified. ►Novel mutations are predicted to be pathogenic and responsible for the phenotype.
Competing interests The authors declare no conflict of interest.
S0378-1119(20)30006-8 https://doi.org/10.1016/j.gene.2020.144337 GENE 144337
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
27 September 2019 4 December 2019 6 January 2020
Please cite this article as: S. Khalili Alashti, J. Fallahi, S. Mohammadi, F. Dehghanian, Z. Farbood, M. Masoudi, S. Poorang, A. Jokar, M. Fardaei, Two novel mutations in the MECP2 gene in patients with Rett syndrome, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144337
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.
Two novel mutations in the MECP2 gene in patients with Rett syndrome Authors: Shayan Khalili Alashti Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Jafar Fallahi Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Sanaz Mohammadi Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Fatemeh Dehghanian Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Zahra Farbood Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Marjan Masoudi Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Shiva Poorang Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected]
Arezoo Jokar Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected]
Majid Fardaei * Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran Email: [email protected] Location of work and address: Zand Avenue, Shiraz University of Medical Sciences, Fars province, Shiraz, Iran Corresponding author: Majid Fardaei Tel: +989173054863 Fax: (+98 71) 32 35 69 96 Email: [email protected]
Two novel mutations in the MECP2 gene in patients with Rett syndrome Shayan Khalili Alashtia, Jafar Fallahib, Sanaz Mohammadic, Fatemeh Dehghanianc, Zahra Farboodc , Marjan Masoudic, Shiva Poorangc, Arezoo Jokard, Majid Fardaeia,* a) Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran b) Department of Molecular Medicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran c) Comprehensive Medical Genetic Center, Shiraz University of Medical Sciences, Shiraz, Iran d) Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
_____________________________________________________________________________________
Abstract Rett syndrome (RTT) is an X-linked severe neurological disorder. Mutations in Methyl-CpGBinding Protein2 (MECP2) gene are the main cause of RTT disease. In this study, we report the results of screening the MECP2 gene for mutations in 7 Iranian patients with RTT syndrome. MECP2 sequencing identified two novel mutations in the heterozygous state, a splice mutation, c.354G>T, p.Gly119Gly, resulting in a premature splice-donor site and a 20-bp deletion, c.11671186del20 (p.P390Rfs), leading to modifying the c-terminal parts of the protein and it also changes the reading frames of all coding sequence downstream of the mutation. Multiple sequence alignment showed that amino acid changes occurred in the well conserved protein regions across species. Based on the results of this study and literature reviews, about 70% of mutations are found in exon 3 and 4 of the MECP2 gene, and mutations in exon 4 are more common than other exons. Therefore, it is recommended that exon 4 to be a priority for screening the genetic analysis of RTT patients.
Highlights ►The analysis of MECP2 mutations was performed in 7 Iranian patients with Rett syndrome. ►Two novel mutations such as c.354G>T, p.Gly119Gly (NM_004992.3) and c.1167-1186del20 (p.P390Rfs) (NM_004992) were identified. ►Novel mutations are predicted to be pathogenic and responsible for the phenotype.
Abbreviations RTT, Rett syndrome; MECP2, methyl CpG-binding protein 2; CDKL5, cyclin-dependent kinaselike 5; FOXG1, Forkhead box protein G1; MEF2C, Myocyte-specific enhancer factor 2C; TCF4, Transcription factor 4; MBD, methyl-CpG binding domain; TRD, transcriptional repression domain; CTD, C-terminal domain; NLS, nuclear localization signal; ID, intervening domain; PCR, polymerase chain reaction; DPR, Deletion Prone Region; E, exon;
Keywords Rett syndrome; RTT; MECP2 gene; Novel mutation; DNA sequencing
1. Introduction Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder that was first reported by the Austrian physician Andreas Rett (1). RTT has an incidence rate of 1 in 10000 (2). This disorder is one of the most common causes of mental retardation, which represents up to 10% of cerebral deficiency with hereditary origin in women and the second reason of extreme intellectual inability in females after Down syndrome (3, 4). Although 90% of RTT cases are caused by de novo mutations in the X-linked gene located on Xq28 encoding methyl CpG binding protein 2 (MECP2), mutations in other genes can also cause Rett syndrome, such as cyclin-dependent kinase-like 5 (CDKL5), Forkhead box protein G1 (FOXG1), Myocyte-specific enhancer factor 2C (MEF2C), and Transcription factor 4 (TCF4) (5, 6). The most common RTT cases are called classic form, which described by a duration of normal development up to around 6-18 months of age followed by brain deficiency, impaired speech, hand-wringing, microcephaly, ataxia, scoliosis, and breathing disorder such as alternative ventilation (7). Furthermore, at a cellular level, other indications are observed in patients with RTT including smaller and more compactly neurons that are decremented in length, leading to complications (8). In spite of “classical RTT”, another type of this disorder is called “atypical RTT” or “forme fruste” (9). The two splice variants are produced by the MECP2 gene and each of them encode a protein isoform (10). The primary splicing form of MECP2 gene, now termed MeCP2-e2, has four exons and the start codon is located in exon 2. In the second splicing form, known as MeCP2-e1, exon 2 is missing and translation starts in exon 1. MeCP2-e1 is the main isoform in brain tissues (11, 12). Four protein domains have been identified within MeCP2 protein; however, most of them are highly conserved. The first recognized domain is the methyl-CpG binding domain (MBD) which binds to a DNA sequence at methylated cytosine in the CpG. Subsequently, other functional domains, known as transcriptional repression domain (TRD) and nuclear localization signal (NLS) were also identified. Also, C-terminal domain (CTD) and intervening domain (ID) are less described domains that bind to histones and unmethylated DNA (11). Various researches have postulated that MeCP2 plays several important roles in transcriptional regulation as well as
modifying chromatin organization via binding to methylated CpG and the recruitment of transcription corepressor factors including the NCoR/SMRT complex (13). Currently, over 900 unique variations are found in the MECP2 gene, and up to 80% of them are in exon 3 or 4. Presumably, the intensity of RTT is related to different mutations in diverse domains (11). In this study, we identified 7 Iranian RTT patients of which 2 had a novel mutation and the rest had previously reported mutations in the MECP2 gene.
2. Materials and methods 2.1. Patients The 7 cases of RTT from southern Iran and their parents were called for genetics examination in Comprehensive Medical Genetics Center affiliated with Shiraz University of Medical Sciences. Moreover, before any analysis, they completed the informed consent based on the ethics committee of Shiraz University of Medical Sciences.
2.2 DNA extraction The peripheral blood samples were collected from the patients and parents, then DNA extraction was performed using the Cinnagen DNA Extraction Kit (Cinnacolon, Iran) according to the manufacturer's protocol. The concentration of DNA was measured by a NanoDrop (ND1000, NanoDrop Technologies, Wilmington, DE, USA).
2.3 PCR amplification The PCR amplification was done for the coding sequence and the flanking intronic sequence of the MECP2 gene, using the primers that are described in table 1. Primers were designed and evaluated by NCBI BLAST (https://blast.ncbi.nlm.nih.gov), ENSEMBLE BLAST/BLAT (https:// www.ensembl.org), and Allele ID 7.5 (primers and probes design software). The PCR reactions were carried out 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). The conditions for PCR reactions are described in table 1. Table 1. Primers sequences and PCR conditions related to amplification of 4 exons of MECP2 gene.
EXON
Forward primer
Reverse primer
PCR condition
Product size
E1
CCTCTTTTCCCCAAACG ACG
CCCAAAGCCCCGAGA AGG
Anneling:61 95:15min 95:30sec 61:30 sec 72:1 min
751bp
E2
TGTGTTTATCTTCAAAA TGT
GTTATGTCTTTAGTCT TTGG
Anneling:55 95:15min 95:30sec 55:30 sec 72:15 sec
365bp
E3
GTGCTAGGAGACTTGT GG
CACATACATTTTCCTG CTCC
Anneling:60 95:15min 95:30sec 60:30 sec 72:30 sec
686bp
E4
TATCTCTGACATTGCTA T
CTTTATTCTTGTTGGT TTG
Anneling:64 95:15min 95:30sec 64:30 sec
1191bp
72:70 sec
2.4 Mutation and sequence analysis Finally, the Sanger sequencing was done, and the results were analyzed by the Gene tool program and Codon Code Aligner program. Moreover, the results were compared with the reference sequences (NG_007107.2) for MECP2. Nomenclature for mutations was confirmed with HGVS guidelines (http://www.hgvs.org) using GenBank NCBI reference sequences.
3. Results In this study, we have studied 7 female patients (Patients 1–7) with Rett syndrome for presence of mutations in the MECP2 gene. Based on the clinical symptoms, all patients in this study had typical or classic Rett syndrome with mutation in the MECP2 gene. All 4 exons of MECP2 gene were amplified using gene specific primers. As a result, sequence analysis in patients (table2) revealed 2 novel mutations in the MECP2 gene (Patients 6 and patient 7). No pathogenic mutation was observed in the intronic regions.
Table 2. MECP2 gene mutations in this study. Patient 6 and patient 7 had novel mutations, and 5 out of 7 patients had a mutation in exon 4.
Patients 1
Sex F
Exon 4
2
F
4
3
F
4
4
F
3
5
F
4
6
F
3
7
F
4
Mutation c.820C>T p.Q274X c.398C>T p.R133C c.473C>T p.T158M c.352C>T p.R118W c.799C>T p.R267Ter c.354G>T p.G119G c.11671186del20 (p.P390Rfs)
rs rs267608525
Refs. Milunski et al., 2001 (14)
rs61748389
Li et al., 2006 (15)
rs28934906
Ehrhart et al., 2016 (16)
rs28934907
Ehrhart et al., 2016 (16)
rs61749721
Ehrhart et al., 2016 (16)
This study This study
3.1 Genetic analysis of patients with novel mutations 3.1.1 Patient (P6) A 12-year-old girl with Rett syndrome, Measurements at birth indicated the probability of microcephaly in this patient (birth weight, 3,100 g, and head circumference, 31 cm). At 13 months, symptoms of gross motor function delay appeared. Furthermore, the Stereotypic hand movements began at age 2, and seizure disorders appeared after the age of 3. MRI scan revealed Cavum septum pellucidum, and an increase in the signal in the white matter around the ventricles was considered
to be in the terminal zone. Other symptoms of P6 including poor sleep patterns, abnormal EEG, and poor eye-gaze were observed. The DTR (Deep Tendon Reflexes) test result was 2/2, and MP (muscle power) was 5/5, indicating normal DTR and a lack of hypotonia. The sequencing of 4 exons of MECP2 gene showed that she carried a novel mutation c.354G>T, p.Gly119Gly (NM_004992.3) in heterozygous state in exon 3 of MECP2 gene (Fig. 1a). Since the patient’s mother and father did not have this mutation (Fig. 1b, and c); hence, this patient has a de novo mutation. Moreover, this mutation does not alter the amino acid sequence. To evaluate the effect of this mutation, predictive tools (mutation taster and VARSOME) were used. Also, the result showed that p.Gly119Gly variant is disease-causing (table 3).
a)
b)
c) Fig. 1. The sequencing results of patient 6 and parents a) Sanger sequence chromatogram of P6 showing a novel heterozygous missense mutation in exon 3 of the MECP2 gene. The location of the mutation is indicated by the arrow. (Nucleotide substitution from G→T at nucleotide position 354). b and c) The Sanger sequencing of the father (b) and mother (c) indicated the absence of this mutation in parents.
3.1.2 Patient (P7) We carried out mutation analysis of the MECP2 gene for a 7-year-old-girl with RTT. She was born full term with normal birth measurements (birth weight, 3,700 g, and head circumference, 36 cm). At age 2, clinical examination showed typical RTT-related symptoms such as abnormal neurological signs, poor eye-contact, psychomotor delay, poor speech, gait apraxia, and abnormal EEG. Gradually head growth decelerated, and seizures were observed at 4 years of age. Additionally, at age 4, stereotypic movements occurred, which was later than other typical RTT patients.
We found a novel mutation that had not been previously reported. Parent’s analysis was negative for this mutation (Fig. 2b, and c), indicating that this was a de novo mutation. Accordingly, this novel frameshift mutation is c.1167-1186del20 (p.P390Rfs) (NM_004992) (Fig. 2a), located inside the exon 4 and modifying the c-terminal section of protein and based on the online software this mutation is pathogenic (table3).
a)
b)
c) Fig. 2. The sequencing results of patient 7 and parents a) Sequencing (reverse strand) of the P7 demonstrated a 20-bp deletion (c.1167-1186del20) in exon 4 of MECP2 gene, which resulted in a frameshift leading to premature termination of the 486 amino acid protein at the 390th codon. The deletion location is indicated by a cursor. b and c) The Sanger sequencing of the father (b) and mother (c) indicated the absence of this mutation in parents.
Table 3. Results of online software show that the MECP2 gene variants in patient 6 and 7 are pathogenic.
3.2 Sequence and structure analysis Multiple sequence alignment was accomplished for these novel mutations using Clustal Omega program with its homologs retrieved from NCBI database and those are amino acid sequences of Homo sapiens (Accession No NP_001104262.1), Nomascus leucogenys (XP_030663420.1), Pan Troglodytes (XP_521333.3), Pongo abelii (PNJ09457.1), Mus musculus (NP_001075448.1). Sequences alignment showed that the mutation in patient 7 (p.390fs) is well conserved across species (Figs. 3a and b)
a)
b)
Fig. 3. Comparison of MECP2 gene conservation rates to its homologs. (a) Multiple sequence alignment of p.390fs with homologous sequences from different species. (b) Weblogo revealing the well conservation of this mutation across species.
4. Discussion In more than 95% of patients with Rett syndrome, MECP2 gene is mutated. According to a study by Wendi et al. about 70% of the pathogenic mutations in the MECP2 gene are located in MBD and TRD domains, which are in the third and fourth exons. Also, there are eight mutations in these domains, accounting for about 50% of all the mutations in the MECP2 gene (p.R106W, p.R133C, p.T158M, p.R168X, p.R255X, p.R270X, p.R294X, and p.R306C) (6, 17). In this study, we carried
out mutation analysis of this gene for 7 patient with symptoms related to Rett syndrome and their parents. Two novel mutations were found in these patients, which were not reported in any previous study. Patient 6 carried a novel mutation c.354G>T, p.Gly119Gly in heterozygous state in exon 3 of MECP2 gene. This substitution causes a change in the third (wobble) position of glycine codon but does not change in coded amino acid (i.e. a synonymous change). The probable pathogenicity of the mutation in wobble position is related to creating an aberrant splice site (18). In this study, we described the pathogenic consequences of a synonymous mutation in MECP2 exon 3. This SNP changes the codon GGG to GGT, coding for the same amino acid (p.Gly119Gly). This mutation can cause premature splice-donor site (Fig. 4); hence, it can cause frameshift and alterations in gene expression. Furthermore, in silico analysis with Human Splicing Finder (HSFVersion 3.1) showed that this mutation can activate an exonic cryptic donor site.
Fig. 4. Activation of cryptic splice site at c.354G>T in MECP2 exon 3. The splicing system uses the GT sequence within the 5' end of the intron as the donor site and the AG sequence within the 3' end of the intron as the acceptor site. Based on in-silico results, change GGG to GGT in patient 6 can cause an exonic cryptic donor site of splicing within the exon 3 of MECP2 gene. Graphic representation, offering the alternative pre-mRNA splicing of the mutant transcript of MECP2 gene.
We carried out mutation analysis of MECP2 gene for patient 7 (7-year-old-girl with RTT) and her healthy parents and found a novel mutation that had not been reported previously in RettBase (http://mecp2.chw.edu.au) and other mutation databases. Accordingly, this novel frameshift mutation is c.1167-1186del20 (p.P390Rfs), located inside exon 4. This area of exon 4 is a hotspot for deletion occurrence, known as Deletion Prone Region (DPR) of MECP2 (19). Additionally, some deletions occurred in nearby areas which were described in other studies (Fig. 5).
C.1167-1186del20
Fig. 5. Some of deletion mutations around the deleted 20-bp in the P7 (the novel mutation of patient 7 is shown with arrow).
There are particular characteristics around the c-terminal area of MECP2 gene such as, small direct/inverted and short tandem repeats, making this region prone to deletion/insertion mutagenesis (20) Based on this, about 10% of all Rett syndrome cases are caused by small deletions in exon 4 of MECP2 gene (21). P7 deletion (c.1167-1186del20) is located between direct repeats ACCCC and CCCCACC…CCTGAGCCC (20) as well as the inverted repeats ACCCC/CCCCA (Figs. 6a, b, and c). Therefore, based on previous studies, the “polymerase slippage” is the most likely mechanisms responsible for this deletion.
a
b
c Fig. 6. There are particular repeated Sequences around the c-terminal area of MECP2 gene such as small direct/inverted and short tandem repeats. (a) There are many repeated sequences such as, small direct/inverted and short tandem repeats in the Deletion Prone Region (DPR) of MECP2 gene (almost in between c.1000-1200) that are susceptible to deletion mutation in this area. (b and c) since there are repeated sequences such as directed repeats (ACCCC and CCCCACC….CCTGAGCCC) and inverted repeats (ACCCC/CCCCA) around the 20-bp deleted sequence in P7, as a result, polymerase slippage probably causes deletion in this region.
In a study by Telethon Institute for Child Health Research, Center for Child Health Research, University of Western Australia, A Bebbington et al. Genotype-phenotype correlations studies showed that patients with deletions in c-terminal of MECP2 gene have mild symptoms, including conserved hand function, lack feeding difficulties, tend to have the latest onset of regression as well as later onset of stereotypies, seizure, and scoliosis. Also, in these patients, head circumference at birth is usually in the normal range (about 35cm) (22, 23). In conclusion, two novel mutations were reported in the MECP2 gene related to RTT syndrome. Based on the results of bioinformatics analysis, mutation in the patient 6 may possibly causes splice defect and the patient 7 had a c-terminal deletion mutation. In addition, the results of this study and similar studies have shown that most mutations occur in the third and fourth exons of the MECP2 gene. The molecular diagnostic methods described in this study could be the basis for further studies. Also, further studies are warranted to examine other possible outcomes of the mutations at cellular level. Competing interests
The authors declare no conflict of interest. Acknowledgment The authors thank the families for participating in this study and wish to thank Mr. H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.
References 1. Townend GS, Ehrhart F, van Kranen HJ, Wilkinson M, Jacobsen A, Roos M, et al. MECP2 variation in Rett syndrome—An overview of current coverage of genetic and phenotype data within existing databases. Human mutation. 2018;39(7):914-24. 2. Lyst MJ, Bird A. Rett syndrome: a complex disorder with simple roots. Nature Reviews Genetics. 2015;16(5):261. 3. Villard L, Kpebe A, Cardoso C, Chelly J, Tardieu M, Fontes M. Two affected boys in a Rett syndrome family: clinical and molecular findings. Neurology. 2000;55(8):1188-93. 4. Ehinger Y, Matagne V, Villard L, Roux J-C. Rett syndrome from bench to bedside: Recent advances. F1000Research. 2018;7. 5. Dajani R, Koo SE, Sullivan GJ, Park IH. Investigation of Rett syndrome using pluripotent stem cells. Journal of cellular biochemistry. 2013;114(11):2446-53. 6. Gold WA, Krishnarajy R, Ellaway C, Christodoulou J. Rett syndrome: a genetic update and clinical review focusing on comorbidities. ACS chemical neuroscience. 2017;9(2):167-76. 7. Ip JP, Mellios N, Sur M. Rett syndrome: insights into genetic, molecular and circuit mechanisms. Nature Reviews Neuroscience. 2018;19(6):368. 8. Sheikh TI, Mittal K, Willis MJ, Vincent JB. A synonymous change, p. Gly16Gly in MECP2 Exon 1, causes a cryptic splice event in a Rett syndrome patient. Orphanet journal of rare diseases. 2013;8(1):108. 9. Hadzsiev K, Polgar N, Bene J, Komlosi K, Karteszi J, Hollody K, et al. Analysis of Hungarian patients with Rett syndrome phenotype for MECP2, CDKL5 and FOXG1 gene mutations. Journal of human genetics. 2011;56(3):183. 10. Gianakopoulos PJ, Zhang Y, Pencea N, Orlic‐Milacic M, Mittal K, Windpassinger C, et al. Mutations in MECP2 exon 1 in classical Rett patients disrupt MECP2_e1 transcription, but not transcription of MECP2_e2. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics. 2012;159(2):210-6. 11. Xiang F, Buervenich S, Nicolao P, Bailey ME, Zhang Z, Anvret M. Mutation screening in Rett syndrome patients. Journal of medical genetics. 2000;37(4):250-5.
12. Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, Klein F, et al. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell. 1992;69(6):905-14. 13. Lebo R, Ikuta T, Milunsky J, Milunsky A. Rett syndrome from quintuple and triple deletions within the MECP2 deletion hotspot region. Clinical genetics. 2001;59(6):406-17. 14. Milunsky JM, Lebo RV, Ikuta T, Maher TA, Haverty CE, Milunsky A. Mutation analysis in Rett syndrome. Genetic testing. 2001;5(4):321-5. 15. Li M-r, Pan H, Bao X-H, Zhang Y-Z, Wu X-R. MECP2 and CDKL5 gene mutation analysis in Chinese patients with Rett syndrome. Journal of human genetics. 2007;52(1):38. 16. Ehrhart F, Coort SL, Cirillo E, Smeets E, Evelo CT, Curfs LM. Rett syndrome–biological pathways leading from MECP2 to disorder phenotypes. Orphanet journal of rare diseases. 2016;11(1):158. 17. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nature genetics. 1999;23(2):185. 18. Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature reviews genetics. 2002;3(4):285. 19. Kammoun F, De Roux N, Boespflug-Tanguy O, Vallee L, Seng R, Tardieu M, et al. Screening of MECP2 coding sequence in patients with phenotypes of decreasing likelihood for Rett syndrome: a cohort of 171 cases. Journal of medical genetics. 2004;41(6):e85-e. 20. Philippe C, Villard L, De Roux N, Raynaud M, Bonnefond J, Pasquier L, et al. Spectrum and distribution of MECP2 mutations in 424 Rett syndrome patients: a molecular update. European journal of medical genetics. 2006;49(1):9-18. 21. Fichou Y, Nectoux J, Bahi-Buisson N, Rosas-Vargas H, Girard B, Chelly J, et al. The first missense mutation causing Rett syndrome specifically affecting the MeCP2_e1 isoform. Neurogenetics. 2009;10(2):127. 22. Kriaucionis S, Bird A. The major form of MeCP2 has a novel N‐terminus generated by alternative splicing. Nucleic acids research. 2004;32(5):1818-23. 23. Bebbington A, Anderson A, Ravine D, Fyfe S, Pineda M, De Klerk N, et al. Investigating genotype–phenotype relationships in Rett syndrome using an international data set. Neurology. 2008;70(11):868-75.
Abbreviations
RTT, Rett syndrome; MECP2, methyl CpG-binding protein 2; CDKL5, cyclin-dependent kinaselike 5; FOXG1, Forkhead box protein G1; MEF2C, Myocyte-specific enhancer factor 2C; TCF4, Transcription factor 4; MBD, methyl-CpG binding domain; TRD, transcriptional repression domain; CTD, C-terminal domain; NLS, nuclear localization signal; ID, intervening domain; PCR, polymerase chain reaction; DPR, Deletion Prone Region; E, exon;
Authors
Contribution
Shayan Khalili Alashti
Fatemeh Dehghanian
Conceptualization, Writing - Original Draft, Writing - Review & Editing, Investigation, Methodology Project administration, Writing - Review & Editing, Project administration Writing - Original Draft, Data Curation, Visualization Data Curation, Formal analysis, Investigation
Zahra Farbood
Writing - Original Draft, Resources, Software
Marjan Masoudi
Data Curation, Resources
Shiva Poorang
Investigation, Methodology
Arezoo Jokar
Software, Validation
Majid Fardaei
Correspond
Jafar Fallahi Sanaz Mohammadi
Highlights ►The analysis of MECP2 mutations was performed in 7 Iranian patients with Rett syndrome. ►Two novel mutations such as c.354G>T, p.Gly119Gly (NM_004992.3) and c.1167-1186del20 (p.P390Rfs) (NM_004992) were identified. ►Novel mutations are predicted to be pathogenic and responsible for the phenotype.
Competing interests The authors declare no conflict of interest.