A novel heterozygous pathogenic variation in the MECP2 gene causing typical Rett syndrome: a case report

Abstract

Background

Rett syndrome (RTT) is an X-linked neurodevelopmental disease with clear diagnostic criteria and mainly affects females. Mutations of the methyl-CpG-binding protein 2 (MECP2) gene cause most RTT cases. While numerous MECP2 mutations have been reported, the pathogenicity of novel identified variants often requires functional validation. To obtain a definite genetic diagnosis result and determine the specific pathogenesis, it is essential to conduct functional validation of the novel mutation.

Case Description

We reported one clinical case of a female child diagnosed with typical RTT. The patient presented with major clinical manifestations, including hand dyspraxia, loss of language ability, stereotypical hand movements, and an abnormal gait. Whole-exome sequencing (WES) was performed on this Chinese trio, which confirmed a novel heterozygous nonsense mutation in exon 1 of the MECP2 gene in the proband. Sanger sequencing confirmed that neither parent carried this variant. Functional validation experiments demonstrated that cells transfected with the mutant recombinant plasmid showed significantly reduced levels of both MECP2 messenger RNA (mRNA) and protein compared to those transfected with the wild-type plasmid.

Conclusions

These functional findings confirm the pathogenicity of this de novo MECP2 nonsense mutation and demonstrate that it leads to a loss of function, a mechanism consistent with nonsense-mediated mRNA decay (NMD). Our study elucidates the genotype-phenotype correlation in this case and provides experimental insight into the underlying molecular mechanism.

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Key findings

• We report a novel, de novo heterozygous nonsense mutation (c.44-45insCGAGG; p.G16Efs*30) in exon 1 of the methyl-CpG-binding protein 2 (MECP2) gene in a Chinese patient with typical Rett syndrome (RTT). Functional assays confirm the mutation’s pathogenicity, demonstrating a significant reduction in mutant messenger RNA (mRNA) and a complete absence of the MeCP2 protein. The molecular phenotype is consistent with the nonsense-mediated mRNA decay (NMD) pathway, establishing a clear loss-of-function mechanism.

What is known and what is new?

• RTT is primarily caused by mutations in the MECP2 gene, with the vast majority located in exons 3 and 4. While exon 1 mutations are rare, they are increasingly recognized but often require functional validation to confirm their disease-causing role.

• This study provides conclusive functional evidence for a pathogenic exon 1 mutation. We show that the mutation leads to a complete loss of protein via a mechanism highly consistent with NMD. Our work underscores the critical role of the MeCP2_e1 isoform and exemplifies the necessity of functional studies to bridge genetic findings with definitive diagnosis, especially for variants in non-canonical gene regions.

What is the implication, and what should change now?

• This case expands the mutational spectrum of MECP2 and reinforces that diagnostic genetic screening for RTT must comprehensively include exon 1 to prevent missed diagnoses.

Introduction

Rett syndrome (RTT; OMIM#312750) is a serious neurodevelopmental disorder that mainly affects children’s psychomotor development and is characterized by a series of clinical manifestations, including loss of acquired hand and language skills, stereotyped actions of hands, microcephaly, and abnormal gait (1). The disease came to the attention of an Austrian medical practitioner, Andreas Rett, when he first observed almost identical stereotyped hand movements in two patients in his waiting room in 1966 (2). Seventeen years later, Bengt Hagberg provided a more detailed description of the syndrome based on his observations of many patients (3). To date, RTT is the second most common cause of female intellectual disability, with the morbidity being approximately one in 10 thousand (4).

Currently, the diagnosis of RTT is mainly established on clinical phenotype and genetic testing. The current clinical diagnosis of RTT relies primarily on the guidelines published in 2010 (5). RTT includes two types: typical RTT and atypical RTT. In the typical form, after a period of apparently normal prenatal and postnatal development, patients begin to show different behaviors distinct from their normal peers when they are 6–18 months old. The initial presentation is developmental regression, featuring a decline in purposeful hand function and spoken language. A subsequent phase involves the development of gait abnormalities and stereotyped hand movements. Clinicians should be suspicious of the possibility of RTT when postnatal head circumference growth slows. The core features are frequently accompanied by a constellation of other manifestations—such as dysregulated breathing, awake bruxism, sleep rhythm disorder, dystonia, scoliosis, or kyphosis—all of which constitute supportive diagnostic criteria.

In massive previous studies, it has been found that the main pathogenic gene of RTT is the methyl-CpG-binding protein 2 (MECP2) gene (6). The MECP2 is situated between the interleukin-1 receptor-associated kinase gene (IRAK1) and the red cone pigment (RCP). The MECP2 contains four important exons (exons 1–4) and three introns (introns 1–3) in humans and mice. The MECP2 gene can produce two kinds of isoforms by alternative splicing of the four exons. Isoform e1 includes exons 1, 3, and 4, and the translation start codon is in exon 1. Isoform e2 contains all four exons at the RNA level, however, there is a translation start codon in exon 2, while exon 1 remains a non-coding state (7). MeCP2 protein structure mainly consists of five parts, the first is the terminal sequence at both ends, N-terminal domain (NTD) and C-terminal domain (CTD), and the middle is methyl binding domain (MBD), inter-domain (ID), and transcription repression domain (TRD) (8).

Given the multi-domain structural complexity of the MeCP2 protein, it is understandable that mutations affecting different functional domains result in diverse clinical outcomes. This genetic heterogeneity largely explains the wide spectrum of clinical phenotypes and severity observed among patients diagnosed with RTT (9). By 2,017, approximately 4,688 variants in MECP2 have been found, among which the most common mutation sites are R106W, R133C, T158M, R168X, R255X, R270X, R294X, R306C, and CTD variants (10). According to published reports, the vast majority of RTT-related mutations (approximately 95%) occur in exons 3 and 4, and only a small proportion of mutations are reported to occur in exon 1 (11). Nonsense and missense mutations are the two most common types of mutations. Missense mutations are mainly present at MBD, while nonsense mutations are mostly located at TRD and ID (4,11). The ongoing discovery of novel variants underscores the necessity of functional verification to confirm their pathogenicity.

In vitro functional validation confirmed the pathogenicity of a novel heterozygous nonsense mutation in MECP2, which was initially discovered by whole-exome sequencing (WES) in a case of RTT. Our findings demonstrate that this mutation, likely acting through the nonsense-mediated messenger RNA decay (NMD), is causative for RTT (12). We present this article in accordance with the CARE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-647/rc).

Case presentation

The proband was a 4-year-old girl born at 40 weeks following an uneventful pregnancy and delivery, with no history of asphyxia. Her birth weight, height, and head circumference were all within normal limits. She acquired head control at 3 months old and could sit without support at 8 months. She could walk alone at 18 months and say a few simple words, such as “mama” at 2 years old. After then, her development became stagnant, followed by a period of retrogression. After 2 years of age, she experienced a loss of her speaking and hand-grasping abilities. After some months, stereotypic hand movements such as rubbing automatisms appeared indeterminately. She developed gait abnormalities and could only walk on a broad, wobbly foundation. She could not go up and down the stairs alone. Her head circumference was 46.5 cm [−1 standard deviation (SD) to −2SD] at 33 months old. Diagnostic investigations revealed abnormal epileptiform activity on electroencephalography (EEG; Figure 1) and non-specific white matter changes on magnetic resonance imaging (MRI) of the brain. Hearing and blood system examination showed no obvious abnormality. None of her family members had similar clinical manifestations. Despite intermittent rehabilitation, the patient’s language and cognitive function did not improve significantly. Considering the classic phenotype, specialized pediatricians suspected RTT and advised WES to obtain a definitive molecular diagnosis. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient’s parents for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.

Figure 1.

The electroencephalogram of the proband. The electroencephalogram showed that in the awake stage, the slow wave activities of bilateral occipital regions were the main background. Many stages of sleep and waking up, a large number of low-high amplitude spike, spike and ware waves, and poly spike wave complex are distributed in the left parietal lobe, occipital lobe, middle and posterior temporal area, and the right frontal area, central area, and middle and posterior temporal area, and sometimes they could spread to the ipsilateral hemisphere. Abnormal wave discharge was more in sleep period than in awake period. This figure shows the abnormal brain waves during sleep.

WES identified a heterozygous insertion, c.44-45insCGAGG, in exon 1 of the MECP2 gene (NM_001110792.2). This frameshift variant is predicted to result in a premature termination codon (p.Gly16Glufs*30) located in the NTD (Figure 2). Family verification showed that neither the patient’s father nor mother carried the mutation. Sanger sequencing indicated that it was a de novo mutation (Figure 3). The mutation was a novel site, confirmed through an extensive search on RettBASE, ClinVar, and Human Gene Mutation Database (HGMD). This mutation causes the change in gene open reading frame and protein function (PVS1). This variation is a novel variation verified in parents using family samples (PS2). The mutation is not included in the Exome Aggregation Consortium (ExAC), 1,000 Genomes (1,000G), and Genome Aggregation Database (gnomAD) (PM2-supporting). According to the present evidence, this mutation is defined as a pathogenic mutation.

Figure 2.

The position of the mutation on MECP2 gene. (A) The novel mutation site is located on exon 1 of the MECP2 gene. (B) The mutation site is situated within the NTD of the MeCP2_e1 isoform. ID, inter-domain; MBD, methyl binding domain; MECP2, methyl-CpG-binding protein 2; NTD, N-terminal domain; TRD, transcription repression domain.

Figure 3.

Sanger sequencing diagram of the MECP2 mutation site in the proband. MECP2, methyl-CpG-binding protein 2.

The detailed timeline of the clinical episode and the corresponding research workflow are provided in Figure 4. At meantime, to functionally validate the pathogenicity of the c.44-45insCGAGG mutation in the MECP2 gene, we performed a series of molecular experiments. First, the full-length coding sequence (CDS) of the human MECP2 e1 isoform was synthesized. We then constructed two independent sets of eukaryotic expression vectors to ensure the robustness of our findings: (I) a custom plasmid (designated pHA-MECP2-Flag) for expressing MECP2 with N-terminal hemagglutinin (HA) and C-terminal Flag tags, generated by cloning the CDS using SalI and NotI sites; and (II) a pEGFP-C1-MECP2 vector for expressing an N-terminal enhanced green fluorescent protein (EGFP)-MECP2 fusion protein, created via XhoI and BamHI cloning. Corresponding mutant constructs for both vectors (pHA-MECP2-Flag-mut and pEGFP-C1-MECP2-mut) harboring the specific insertion were generated by site-directed mutagenesis (Figure 5A). Crucially, to preclude any potential amplification of endogenous MECP2 transcripts (including the e1 and e2 isoforms) and to specifically quantify only the expression from our transfected constructs, we designed exon-external real-time quantitative polymerase chain reaction (RT-qPCR) assays. The forward primers were placed within the vector-specific tag sequences (EGFP or HA), while the reverse primer was designed downstream of the mutation site within the MECP2 CDS. This strategy ensures that the assay is exclusively capable of amplifying the recombinant “tag-MECP2” fusion messenger RNA (mRNA). Therefore, the observed mRNA signals are unequivocally attributable to the exogenous plasmid-derived expression, directly confirming the mutation’s impact on the MECP2 e1 transcript. All primer sequences used for plasmid construction and RT-qPCR are listed in Table S1.

Figure 4.

Timeline of clinical episode and corresponding research workflow. EEG, electroencephalography; MRI, magnetic resonance imaging.

Figure 5.

Expression levels of MECP2 mRNA and protein in the two types of recombinant plasmids. (A) Sequencing results showed that the mutation c.44-45insCGAGG was successfully introduced. (B,C) The expression of MECP2 mRNA by RT-qPCR. (D,E) Western blot analysis of protein lysates from HEK293T cells transfected with the indicated constructs. Note that the WT bands migrate at different apparent molecular weights due to the distinct tags: the pEGFP-C1 construct produces an EGFP-MECP2 fusion protein (~82 kDa), while the pHA-MECP2-Flag construct produces HA/Flag-tagged MECP2 near its native size (~60 kDa). β-actin served as a loading control. **, P<0.01; ***, P<0.001 vs. respective wild-type control (Student’s t-test). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; MECP2, methyl-CpG-binding protein 2; mRNA, messenger RNA; MUT, mutant; qPCR, quantitative polymerase chain reaction; RT-qPCR, real-time quantitative polymerase chain reaction; WT, wild-type.

Following transient transfection of HEK293T cells with the wild-type and mutant plasmids, RT-qPCR analysis of samples collected at 48 hours revealed a significant reduction in mRNA levels for both mutant constructs. The pEGFP-C1-MECP2-mut and pHA-MECP2-Flag-mut plasmids exhibited mRNA levels that were only 65% and 45% of their wild-type counterparts, respectively (Figure 5B,5C; P<0.01). These data confirm that the c.44-45insCGAGG mutation severely impairs the steady-state level of the exogenous MECP2 e1 transcript. Western blot analysis using antibodies against the respective tags (anti-GFP and anti-HA) confirmed the functional impact of the mutation at the protein level. As shown in Figure 5D,5E, cells transfected with the wild-type constructs (pEGFP-C1-MECP2-wt and pHA-MECP2-Flag-wt) exhibited prominent protein bands at the expected molecular weights (approximately 82 kDa for the EGFP fusion and 60 kDa for the HA/Flag-tagged protein). In stark contrast, lysates from cells expressing the corresponding mutant constructs showed a complete absence of any specific band at these positions. This indicates that the c.44-45insCGAGG mutation prevents the synthesis or causes the immediate degradation of the MeCP2 protein. The pattern of mRNA reduction and complete loss of protein suggests that the mutant transcript is likely subjected to NMD.

Discussion

This study reports a case of typical RTT in a Chinese patient, characterized by regression of hand skills and language, gait ataxia, and hand stereotypies. WES identified a novel, de novo heterozygous nonsense mutation (c.44-45insCGAGG; p.G16Efs*30) in exon 1 of the MECP2 gene. Functional assays subsequently confirmed the pathogenicity of this variant. The observed reduction in mutant mRNA levels, coupled with the complete absence of the protein product, is consistent with the action of NMD pathway, thereby elucidating its role in disease etiology.

MECP2 gene is known as a primary causative gene in RTT patients. Furthermore, more than 95% of RTT cases are caused by mutations in MECP2, most of which are de novo mutations (13). MECP2 gene is involved in regulating various biological functions of the human body, which not only influences the methylation process by regulating the DNA methylation pathway but also acts as a transcription inhibitor to regulate gene transcription. Furthermore, more and more evidence has shown that it manifests genome-wide functions by influencing the landscape of coding and non-coding transcription and the expression of wrong elements (14,15). Therefore, if the mutation of the MECP2 gene leads to the dysfunction of its encoded protein, it may cause damage to the growth and development of the nervous system (16). In people’s previous cognition, mutations occur primarily in exons 3 and 4. Among the four exons, exon 2 is the beginning of translation, and most of exon 1 and exon 2 constitute the 5' untranslated region. In 2004, Mnatzakanian et al. mentioned for the first time that two transcripts were produced during the translation of MECP2, MeCP2A, and MeCP2B, which were later known as MeCP2E2 and MeCP2E1 (14). Currently, people are not only limited to exons 3 and 4 in gene testing of RTT patients but have also begun to pay attention to the mutations in exon 1 and exon 2, thereby further reducing the missed diagnosis in the gene testing of RTT patients. To date, more than 20 cases have occurred on exon 1, according to the RettBASE database (17).

Gene detection plays a vital role in diagnosing RTT. With the continuous development of gene detection methods, more and more unexplained diseases have been diagnosed clearly, and more and more mutation sites have been discovered. However, the goal of this method is still limited; therefore, some unresolved problems in the exon sequencing process may need to be clarified using the whole-genome sequencing (WGS) in the future (18). Moreover, in many cases, genome sequencing results contain multiple mutation sites, which may lead to diseases. However, establishing a definitive diagnosis from sequencing data alone remains challenging. The mere presence of a rare variant in a disease-associated gene is insufficient. To be considered pathogenic, a variant must not only have a predicted deleterious effect on the gene product, but its functional consequence must also be biologically plausible for explaining the patient’s specific phenotype. This often necessitates functional validation, as performed in this study, to move from a genetic finding to a causal diagnosis. Therefore, how to verify whether the mutation site is the root cause of the disease has become a new hot topic.

This study identifies and functionally validates a novel de novo nonsense mutation (c.44-45insCGAGG) in MECP2 exon 1 in a Chinese patient with typical RTT.

The mutation causes a frameshift and introduces a premature termination codon (p.G16Efs*30) within the NTD specific to the MeCP2_e1 isoform. Functional assays demonstrated a significant reduction in exogenous e1 transcript levels and a complete absence of the corresponding protein. This “mRNA reduction-protein loss” phenotype is highly consistent with the NMD pathway, constituting a clear loss-of-function mechanism. Notably, the loss of protein was more complete than the reduction in mRNA. This may indicate that the mutant transcript is a highly efficient substrate for NMD, or that any trace amounts of truncated protein produced are structurally unstable and rapidly degraded by cellular quality control systems. As the mutation resides in the e1-specific N-terminal sequence and does not affect the e2 isoform, our results highlight the critical role of the MeCP2_e1 isoform in RTT pathogenesis, aligning with the recognized functional distinction between the two major transcripts (MeCP2_e1 and MeCP2_e2).

Our study exemplifies the essential role of functional validation as a critical bridge in the genomics era. While sequencing technologies continue to uncover an increasing number of rare variants in patients, the mere presence of a variant in a disease-associated gene or its predicted deleterious effect is insufficient to confirm pathogenicity. As demonstrated here, experimental confirmation that a variant disrupts gene function—such as causing a complete loss of protein expression—is required to reliably establish a causal link between a genetic finding and the patient’s specific clinical phenotype, thereby moving from a variant of uncertain significance to a definitive etiological diagnosis.

A limitation of our study is that the RT-qPCR assay, while specific for the exogenous transcript, did not separately quantify the stability of different transcript isoforms (e1 vs. e2). Furthermore, although the data strongly support the NMD mechanism, the precise efficiency of decay and potential concurrent impairment of translation initiation warrant further investigation using tools such as NMD inhibitors. In conclusion, our integrated clinical and functional approach definitively establishes that this novel mutation causes RTT through a loss-of-function mechanism. This expands the mutational spectrum of MECP2 and reinforces the clinical importance of comprehensive functional assessment of coding exons in the diagnostic pipeline.

Conclusions

In conclusion, we have identified and characterized a novel de novo frameshift mutation in MECP2 exon 1. Our functional validation definitively establishes this variant as pathogenic, primarily through a loss-of-function mechanism, as evidenced by the absence of the encoded protein. This case expands the mutational spectrum of MECP2 and underscores the importance of including exon 1 in diagnostic screening panels. Future studies are needed to fully elucidate the precise molecular cascade leading from this specific mutation to the complete loss of protein expression.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study were in accordance with the ethical standards of the institutional and/or national research committee(s) and with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from the patient’s parents for the publication of this case report and accompanying images. A copy of the written consent is available for review by the editorial office of this journal.