Rearrangements of immunoglobulinTCR genes in lymphoid neoplasia

The rearrangement of multiple gene segments to form functional immunoglobulin or T-cell receptor genes occur early in lymphocyte development and each lymphocyte has a particular combination of variable (V), diversity (D) and joining (J) segments. The junctional regions V-(D)-J are clonal markers of lymphoid neoplasia and can be used as targets for diagnosis and disease tracking. However the 'uniqueness' of each rearrangement means that it is not possible to use standardized real-time PCR reagents; in most instances individual clonal rearrangements are sequenced and primers and/or probes are designed. As probes are the most costly component in a real-time PCR assay, it would be prohibitively expensive to design patient specific probes. Therefore, several groups have designed assays using one ASO primer and a germline primer and probe (Figure 16.3). This reduces the complexity of the assays as only one forward primer be designed, although in practice at least three versions of the ASO are designed. These approaches are discussed in depth by (van der Velden et al., 2003). The use of ASO primers in lymphoid malignancies is highly specialized and requires experience in design and testing of reagents to produce the highly sensitive and

Variable gene segments Diversity genes Joining genes Constant region

COOH

V-D-J Recombination

ACG CT GAG GGG

GAG GGG

NH, NNNN

COOH

Forward primer

TaqMan® probe

DNJ Consensus region region

Figure 16.3

Generation of allele specific primers for MRD quantitation.

DNJ Consensus region region

1 Reverse primer

Figure 16.3

Generation of allele specific primers for MRD quantitation.

specific assays required for monitoring and adapting treatment. However, due to the background of similar rearrangements and limited repertoire at some loci, these assays do not routinely achieve the sensitivity and specificity of those using chromosome translocations as targets.

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