Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects brain development and function in females, with a prevalence of one in 10,000 worldwide1. Typical RTT is caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2)2. A database of a large collection of MECP2 variants was established in 2002 (RettBASE: http://mecp2.chw.edu.au/index.shtml)3. To date, associations between clinical phenotypes and related genetic variants for MECP2 as well as other RTT-associated genes, including CDKL5 and FOXG1, are available.

Here, we report a total of 49 RTT patients with 10 novel insertion/deletion variants, eight known insertion/deletion variants and 31 known pathological variants.

All patients were diagnosed with typical RTT by Japanese child neurology experts according to the international diagnostic criteria for RTT. Clinical information and samples from the patient and parents were obtained with written informed consent. The study was approved by the ethical committee of NCNP. Genomic DNA was extracted from peripheral blood using a standard protocol. We first used the MLPA method (MRC-Holland, DL Amsterdam, The Netherlands) to identify the structural abnormalities in the MECP2 locus. In the patients excluded for structural abnormalities, we amplified all coding exons of MECP2 and their exon-intron boundaries by PCR and directly sequenced the PCR products using the Applied Biosystems 3730 DNA analyzer (Thermo Fisher, USA).

The insertion/deletion mutations were detected in 18 (36.7%) of 49 patients with RTT (Table 1). Among the 18 patients, 10 (55.6%) were considered novel by a comparison of our data with the known insertions/deletions deposited in the public databases, including RettBASE, gnomAD, Human Genome Mutation Database Professional 2019.2 and ClinVar. Representative data of pedigrees and sequences of the recombination breakpoints from three families are shown in Fig. 1. Patient 470 showed the insertion/deletion variant at c.1158_1258delinsCCGAGGGTGGCTCC. Patient 488 showed the deletion at c.1168_*539del. Patient 587 showed the insertion/deletion at c.1367_*791delinsCGC. Five (Patients 187, 470, 488, 559, and 587) had lost only exon 4. In addition, Patient 269 has a complex rearrangement with a 2609 bp deletion, including exon 3 and flanking introns, accompanied by two nucleotide substitution and a 25 bp deletion in exon4, c. [27-1707_c.378-206del; 1159_1160CC>AG; 1164_1188del], occurred in cis. The other five showed intragenic deletion involving exon 4 in the MECP2 locus. Hardwick et al. (2007) reported that 12 out of 21 patients (57%)4 experienced breakpoints within the “deletion-prone region (DPR)”, which is characterized by short direct repeat elements and is also known as the hotspot for the smaller deletions5,6. In this study, the breakpoints in seven novel insertion/deletion variants (7/10: 70%) were located within the DPR. In the breakpoints of Patients 269, 289 and 451, no repetitive sequences were found adjacent to the breakpoint. These findings suggest that the de novo deletion events involving MECP2 can be unique to families and that homology-mediated mechanisms are unlikely to be associated with these events.

Table 1 Insertion/deletion variants in MECP2 in Japanese patients with RTT
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Fig. 1: Sequence analysis of the breakpoints of MECP2 structural variants in three RTT patients.
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a–c Pedigrees of each family (left). Nucleotide sequences of MECP2 in patient 470, 488, 587, and controls spanning the breakpoint of each structural variant. All samples were amplified by PCR and then cloned into a plasmid vector, followed by direct sequencing of the junction fragments

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In addition, we identified known pathogenic variants in 31 patients (Supplementary Table 1). No novel change was identified, suggesting that the molecular basis for recurrent de novo nucleotide substitutions in MECP2 is common among the different populations.

The list of MECP2 variants found in 49 Japanese patients with RTT should provide a useful resource to further examine the correlation between genotypes and disease phenotypes.