Aims-To examine persistent CD3-large granular lymphocytosis (LGL) cases for clonality, both by lineage specific (T cell receptor) and lineage independent (X-inactivation) molecular methods; and to find out whether X-inactivation studies are more appropriate than gene rearrangement studies for this subset of LGL disorders. An alternative method of clonal analysis is provided by the study of differential methylation in certain X chromosome genes in females.10 The inactivation of one X chromosome with concomitant methylation of the 5' end of genes, such as PGK and hypoxanthine phosphoribosyl transferase (HPRT) during early embryogenesis, provides a stably inherited genetic marker. A normal population of cells in an adult woman will comprise a random mixture of paternally and maternally derived X-inactivated cells. A clonal population of cells, having originated from a single cell, will feature inactivation of the same X chromosome in each cell. The PGK gene can be used for clonal analysis by a polymerase chain reaction (PCR) method where amplification of DNA around the BstXI polymorphic restriction site and a methylation site permits analysis of heterozygous females." The BstXI site is polymorphic in about 33% of females.'0To examine the nature of persistent increases in CD3 -LGIJnatural killer cells, we analysed X-inactivation at the PGK locus in six informative women from 17 female CD3 -cases identified in the YLG survey.5 Methods To date the YLG survey has identified 97 people with persistently increased LGL and/or lymphocytes expressing natural killer cell associated membrane determinants (defined as lasting more than six months).5 Of these, 23 were found with a primary increase in CD3 -natural killer cell associated + cells. Seventeen were women and were screened for heterozygosity at the polymorphic BstXI site in the PGK gene (fig 1). LGL lymphocytes were identified by conventional morphologi-399 on 11 May 2018 by guest. Protected by copyright.
