DISCUSSION
Here we reported two interstitial Xq28-duplication (involvingMECP2 ) females (daughter and mother) with an asymptomatic phenotype. By HUMARA and RP2 assays, the chromosome with Xq28 duplication was inactive in female II2 (daughter) but was active in female I2 (mother). There should have been a related clinical phenotype in I2, and upregulated MECP2 gene expression, but I2 was an asymptomatic carrier. While by combing RNA-seq and RT-PCR, we found thatXIST only expressed in the Xq28-duplication chromosomes for I2 and II2. It indicated that the Xq28-duplication chromosomes were inactive. We also found that the XCI genes MECP2 and IRAK1on two females’ Xq28-duplicated alleles were not transcriptionally expressed nor upregulated by RNA-seq and qPCR. This can explain why the two females, especially I2, were asymptomatic carriers.
We summarized duplicated regions in Xq28, XCI states and the clinical features of the two asymptomatic females in this study and other seven symptomatic females, inheriting their duplication from their symptomatic mothers (Table 1) (Bijlsma et al., 2012; Novara et al., 2014; Reardon et al., 2010; Scott Schwoerer et al., 2014; Shimada et al., 2013). As similar with I2 in this study, only by HUMARA assay, some females’ clinical phenotype did not necessarily correlate with the XCI pattern, such as the mothers of case 2, 3, 4, 6 and the daughter of case 7. If XCI is really random, transcriptions of MECP2 on the duplicated allele and normal allele are random. Mothers of case 4, 6 with MECP 2 duplication should have abnormal phenotypes, during to overexpression of MECP2, but they were asymptomatic. We thought it was because HUMARA assays could not always refect the real XCI states.
HUMARA/RP2 assays revealed methylation of the X-chromosome at the genomic DNA level (Allen et al., 1992; Machado et al., 2014), but some reports asserted that the ratio of DNA methylation between alleles did not always reflect each allele’s ratio of RNA expression. DNA methylation assays are not always representative of XCI (Swierczek et al., 2012). Clara Xiol et al. reported differences between the XCI pattern detected by HUMARA assay and final RNA levels of eachMECP2 allele in Rett syndrome (RTT) patients caused by mutations in the MECP2 gene. This finding suggested that HUMARAassay did not directly determine the levels of MECP2, and there could be more factors than DNA methylation involved in the regulation ofMECP2 transcript levels (Xiol et al., 2019). Ehrhart F. et al. also reported that there might be other factors involved in regulatingMECP2 transcription and/or RNA degradation that would cause changes in the overall levels of functional MECP2 in RTT (Ehrhart et al., 2016). Therefore, we speculated that DNA methylation detected byHUMARA /RP2 assay might not necessarily be correlated with the severity of patients’ clinical presentation with MECP2 gene defects.
XCI status is maintained by more than one factor. In a homozygous mouse, knockout of one of the XCI factors (XCIF) stanniocalcin 1 (STC1), was expected to have an XCI defect but was phenotypically normal. Remarkably, MECP2 was not overexpressed in female Stc1(-/-) mice (Bhatnagar et al., 2014), and animal experiment also confirmed that genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice can bear a homozygous XCIF deletion (Przanowski et al., 2018). It revealed the existence of a mechanism(s) that could compensate for a persistent XCI deficiency, so that X-linked gene MECP2expression is not upregulated. Meanwhile, many studies showed that duplication dosage inhibition of X-chromosome was also observed in 47,XXX females and many species (Meyer, 2005; Meyer, McDonel, Csankovszki, & Ralston, 2004; Nielsen et al., 2020; L. Sun et al., 2013). These lower transcript levels suggested that a inhibition regulation at a higher level, for example, through mRNA degradation or other epigenetic mechanisms, such as histone modifications, maintaining the XCI status in addition to DNA methylation (Lee, 2011; Prestel, Feller, & Becker, 2010). We speculated a potential inhibition mechanism might occurr at the transcriptional level in the unmethylated X-chromosome with MECP2 duplication, which resulted in a lack of up-regulation of duplicated MECP2 gene expressions in I2.
As a high-throughput RNA expression assay, RNA sequencing can measure the ratio of duplicated to normal alleles that have been activated directly for the MECP2 gene at the transcript level. Direct measurement of the allele expression may provide a better estimate of each inherited chromosome copy’s true cellular activity, and it also increases our power to accurately estimate XCI, thus reflecting a greater influence of XCI on clinical manifestations.
The discovery of molecular mechanisms by DNA and RNA-seq in a patient’s peripheral blood that may be correlated with phenotype in the central nervous system would provide potential benefits in clinical diagnostic cases that remain unresolved. But this finding is supported by some studies that have discovered a strong correlation in the gene expression profile of blood with the affected status of many neurological diseases, such as Parkinson’s disease and Huntington’s disease (Borovecki et al., 2005; Scherzer et al., 2007). Meanwhile, based on the analysis of ASE patterns by RNA-seq, the skewed XCI states of MECP2 were the same across 29 human tissues (Tukiainen et al., 2017), so we speculated that the allelic expression of MECP2 in the blood might reflect the state of the nervous system in Xq28-duplication patients.
In conclusion, we explained why transcriptions of MECP2 andIRAK1 genes were not upregulated in the two Xq28-duplication females with opposite skewed XCI. We showed that XCI detected byHUMARA and RP2 assays did not always reflect the transcriptional level of Xq28 duplication. ASE assay by RNA sequencing, which reflects the transcription of the MECP2 alleles, is more directly correlated with the clinical phenotype. Meanwhile, we speculated there were other factors maintaining the XCI status in addition to DNA methylation, an additional inhibition mechanism might occur at the transcriptional level in the unmethylated X-chromosome to counter balance the detrimental phenotype effects of MECP2duplication.
ACKNOWLEDGMENTS: We thank all the participants in the present study. We also thank Dr. Jiong Gao (BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China) for his assistance in the preparation of this manuscript.
CONFLICT OF INTEREST: The authors have declared no conflicts of interest.
DATA AVAILABILITY STATEMENT: The data that support the findings of this study have been deposited in SRA database with the accession code PRJNA702822
FUNDING: This study was supported by the National Natural Science Foundation of China (Grant No. 81801441), the Key Research and Development Program of the Zhejiang Province (Grant No. 2019C03025), the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (Grant No. 2021KY772), the Public Welfare Technology Research Program of Zhejiang Province (Grant No. LGC20H200003).
AUTHOR CONTRIBUTIONS: MD designed the study. YL and YS performed FISH and SNP array. qPCR detection was carried out by YY1. RNA seq and WES were analyzed by YS, LW, and YH. MC and YY2 contributed theHUMARA and RP2 assays. YS and MD wrote the draft manuscript. All co-authors provided feedback on the estimates and contributed to the subsequent versions of the manuscript. All authors read and approved the final version of the manuscript.