INTRODUCTION
Xq28 (involving MECP2 ) duplication syndrome (MDS) is a severe
neurodevelopmental disorder. Signs and symptoms include infantile
hypotonia, delayed psychomotor development, severe intellectual
disability, poor speech development, progressive spasticity, recurrent
respiratory infections, and seizures (Van Esch, 1993). MDS is caused by
a duplication (or triplication) of the gene methyl CpG binding protein 2
(MECP2 ), which increases the expression of transcription
regulator MeCP2 (Makrythanasis et al., 2010; Ramocki et al., 2009).
MeCP2 protein overexpression could have detrimental effects on brain
development and function, as shown in mouse models and humans (Collins
et al., 2004; Ramocki & Zoghbi, 2008; Van Esch et al., 2005).
Initially, MDS was considered fully penetrant in males, and most females
with duplications were asymptomatic carriers. However, later reports
revealed a mild to severe clinical phenotype in more than 20 affected
females with MECP2 duplication, including larger cytogenetic
changes resulting in MECP2 duplication and intrachromosomal
(intraC) Xq28 duplication (El Chehadeh et al., 2017; Van Esch, 1993).
X-chromosome inactivation (XCI) is a transcriptional silencing mechanism
of the X-chromosome initiated by the transcription of XIST , which
encodes a long non-coding RNA, transcribes mono-allelically, and spreads
in cis along the inactive X (Xi) chromosome. XCI induces progressive
epigenetic silencing by recruiting chromatin remodeling enzymatic
complexes, thereby imposing repressive histone and DNA changes to the Xi
chromosome. One X-chromosome is inactivated at random in females, but
some females show preferential inactivation for one of the two
X-chromosomes (skewed XCI) (Lee, 2011). Skewed XCI can cause phenotypic
heterogeneity of X-linked disorders in females (Horga et al., 2019;
Orstavik, 2009; Y. Sun et al., 2019). XCI seems to be essential forMECP2 expression in Xq28 duplication females, which affects the
phenotype.
XCI pattern analysis has generally been conducted by human androgen
receptor (HUMARA ) assay, a polymerase chain reaction (PCR)-based
X-chromosome inactivation assay that uses a methylation-sensitive
restriction enzyme. However, using the classic mean, some reports did
not correlate XCI and the severity of the phenotype in females affected
by Xq28 duplication (Bijlsma et al., 2012; El Chehadeh et al., 2017;
Fieremans et al., 2014; Scott Schwoerer et al., 2014). We speculated it
was because the HUMARA assay did not directly reflect the
transcriptional level of the genes in Xq28. Recently, quantification of
allelic read counts and allele-specific expression (ASE) by RNA
sequencing has been used as a direct XCI assay (Tukiainen et al., 2017;
Zito et al., 2019). We investigated two females in a daughter-mother
relationship, each possessing interstitial Xq28 duplication (involvingMECP2 ) opposite skewed XCI at the methylated level, as detected
by HUMARA assay and RP2 assay. Through RNA sequencing
(RNA-seq), we revealed that chromosomes with Xq28 duplication were both
silent at the transcriptional level, and the dosages of genesMECP2 and IRAK1 spanning the Xq28-duplicated region were
not upregulated. This finding explains why both females were
asymptomatic, especially the mother.