Use of the FMR1 Gene Methylation Status to Assess the X-Chromosome Inactivation Pattern: A Stepwise Analysis.

Bárbara Rodrigues,Nuno Maia,Rosário Santos,António J A Nogueira,Vanessa Sousa,Isabel Marques,Ana Gonçalves,Emídio Vale-Fernandes,Paula Jorge

doi:10.3390/genes13030419

Abstract

X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.

Highlights

Females are usually less susceptible to recessive pathogenic variants in X-chromosome genes, being asymptomatic with a complete absence of clinical features or showing a phenotype less severe than those males presenting the same pathogenic variants [1]
The method described by Allen et al, to analyse the androgen receptor (AR) gene methylation status, designated Human Androgen-Receptor Assay (HUMARA), is considered the gold standard assay to determine the X-chromosome inactivation (XCI) pattern [20]
Retrospective Analysis of Samples Tested by HUMARA

Summary

Introduction

Females are usually less susceptible to recessive pathogenic variants in X-chromosome genes, being asymptomatic with a complete absence of clinical features or showing a phenotype less severe than those males presenting the same pathogenic variants [1]. This is assumed to be due to the X-chromosome inactivation (XCI) phenomena, where preferential inactivation of the X-chromosome with the normal allele results in an incomplete penetrance or in variable clinical presentations [2,3,4,5,6,7,8]. The absence of a complete overlap between the results of both assays led us to propose a set of key points and a stepwise analytical procedure for accurate XCI pattern result assessment, while minimizing the effect of the pitfalls underlying the use of this locus

Objectives

Methods

Results

Conclusion