X-Chromosome Inactivation Analysis

Content Review: November 2020 Last Update:
  • Use to determine XCI pattern for female carriers of X-linked disorders and assess pathogenicity of genetic variant in an X-linked gene
  • XCI ratio reported for the tissue type tested (ranges from 50:50 to 100:0)
  • Does not detect clonality
  • Testing limited to XX females
  • When nonrandom XCI is detected, parent of origin of the active X cannot be determined without testing parental samples
  • Fetal specimens are not accepted

Females typically have two copies of the X chromosome, with one copy randomly inactivated by lyonization early in embryonic development, which allows females to produce the same amount of gene products from X-linked genes as males. In each somatic cell, either the paternally inherited X chromosome or the maternally inherited X chromosome is inactivated. The majority of genes on the inactivated chromosome are silenced, and many of the CpG islands are methylated. Identification of nonrandom, or skewed, X-chromosome inactivation (XCI) patterns in females may assist in evaluation of X-linked disorders. XCI analysis may be useful to help determine pathogenicity of variants detected in X-linked genes. Nonrandom XCI is defined as a ratio of active to inactive X chromosome greater than 85:15. 

 

DISEASE OVERVIEW

Physiology

  • Preferential inactivation of either the paternally or maternally derived X chromosome produces a nonrandom pattern of XCI, which can result from:
    • Secondary cell selection in women who are heterozygous for X-chromosome rearrangements
    • Cell selection bias in females carrying a variant for an X-linked disorder
    • Neoplasia

Diagnostic Issues

  • Nonrandom XCI may influence expression of X-linked disorders.
    • Female carriers may be symptomatic in X-linked recessive disorders if the affected X chromosome is preferentially activated.
    • Female carriers may be asymptomatic in X-linked dominant disorders if the affected X chromosome is preferentially inactivated.
    • In some X-linked diseases, there is a strong selection bias for XCI in favor of cells with the variant.
  • Assessing XCI in a carrier mother may help to determine the pathogenicity of a genetic variant in an X-linked gene detected in her offspring.

GENETICS

The highly polymorphic CAG repeat in exon 1 of the androgen receptor (AR) gene on the X chromosome can be used to distinguish the maternally inherited from the paternally inherited X chromosome.

  • At least 80% of women are heterozygous at the analyzed AR locus, allowing for differentiation between maternal and paternal X chromosomes.
  • Restriction sites near the AR gene are methylated on the inactive X chromosome and unmethylated on the active X chromosome.
  • Methylation-sensitive restriction enzymes are able to digest DNA only on the active X chromosome.
  • Methylation is correlated with XCI.

TEST INTERPRETATION

Sensitivity/Specificity

Clinical sensitivity: 90%

  • 10-15% of females have nonrandom XCI by chance.
    • Increases with age

Results

  • Nonrandom XCI ratio: 86:14 to 100:0
    • Suggests nonrandom pattern of XCI in tissue type tested
  • Random XCI ratio: 50:50 to 74:26
    • Suggests random pattern of XCI in tissue type tested
  • Caution is advised when interpreting XCI ratios between 75:25 and 85:15.
  • Uninformative result: XCI ratio cannot be determined.
    • Maternally and paternally derived X chromosomes could not be distinguished.

Limitations

  • Testing is limited to XX females only.
  • The assay will be uninformative in up to 20% of females due to homozygosity for the polymorphic AR gene locus analyzed.
  • XCI patterns may differ among tissues.
  • XCI ratio reported is for the tissue type tested, with a standard deviation (SD) of 0.08 for XCI ratios of 50:50-79:50, and an SD of 0.05 for XCI ratios of 80:20 or greater.
  • Test will not determine if the X-inactivation pattern is associated with rearrangements of the X chromosome, pathogenic variants in X-linked genes, or neoplastic disease.
  • If nonrandom XCI pattern is present, the parent of origin of the active X cannot be determined without testing parental samples.
  • XCI ratios should not be used to predict prognosis for female carriers of X-linked disorders; variable expressivity may result due to other genetic or environmental modifiers.
  • Test is not recommended for prenatal diagnosis because XCI levels may differ in prenatal specimens and whole blood.
  • Diagnostic errors can occur due to rare sequence variations.