X-Inactivation Studies

Clinical Information

Determination of maternal and paternal X chromosome activation status is useful in the diagnostic analysis of nonrandom patterns. Skewed patterns of inactivation in female carriers of a number of X-linked recessive disorders and asymptomatic female carriers of some X-linked dominant diseases have been observed. By using the methylation-sensitive restriction enzyme HpaII and the polymerase chain reaction, the methylation status of both the maternal and paternal X chromosome is determined. Methylation of the HpaII restriction endonuclease site in the human androgen-receptor gene, HUMARA, correlates with X-inactivation. In approximately 10% of cases, the testing is uninformative due to the presence of the same allele size on both X chromosomes.

Technical Information

X-inactivation analysis of the Androgen Receptor (AR) locus is performed on genomic DNA. The X-inactivation pattern is determined by polymerase chain reaction (PCR) analysis of a polymorphic CAG repeat in the first exon of the AR gene. Methylation of sites close to this short tandem repeat has been demonstrated to correlate with X chromosome inactivation (Allen et al. (1992) Am J Hum Genet. 51, 1229-1239). In this assay, amplification of the AR gene both before and after digestion with the methylation-sensitive HpaII restriction enzyme is used to determine the methylation status of the maternal and paternal X chromosome. The reliability of the analysis depends on the assumption that the methylation status of the AR locus reflects that of the X chromosomes in the submitted sample type.

Inactivation ratios of less than 80:20 are considered random patterns. Ratios between 80:20 and 90:10 are reported as moderately skewed and ratios greater than 90:10 are considered highly skewed and may be clinically significant. In approximately 10% of cases, the testing is uninformative due to the presence of the same allele size on both X chromosomes (homozygous).

Specimen Requirements

• The preferred sample type is 3-4 ml of peripheral blood collected in an EDTA (purple top) tube. Extracted DNA and saliva are also accepted for this test. Blood and saliva kits are available by request.
• Send approximately 5µg of extracted DNA at a requested concentration of 90-130 ng/µl.
• Saliva samples must be submitted in an approved saliva kit, and sample kits are available upon provider request.

Transport Instructions

• The blood specimen should be kept at room temperature and delivered via overnight shipping. If shipment is delayed by one or two days, the specimen should be refrigerated and shipped at room temperature. Do not freeze the specimen. Samples collected on Friday can be safely designated for Monday delivery.
• Extracted DNA should be sent at room temperature via overnight delivery.
• Saliva is stable at room temperature and can be delivered via overnight or ground shipping.