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X-Chromosome Inactivation Analysis

2006352

X-Chromosome Inactivation Analysis 2006352

Method

Restriction Enzyme Digestion/Polymerase Chain Reaction (PCR)/Fragment Analysis

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.

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