The PMS2 protein IS a component of the post-replicative DNA mismatch repair (MMR) system, which acts to correct mispaired nucleotides and small insertion-deletion loops in new DNA duplexes. Biallelic mutations in any of several MMR genes result in a deficiency syndrome (MMR-D) that predisposes to childhood cancer. However, mutation detection in PMS2 is complicated by the existence of multiple pseudo genes; furthermore, gene conversion events generate sequence exchange between gene and pseudogene. The main laboratory marker of defective MMR is microsatellite instability (MSI), but this is not easily demonstrated in constitutional DNA using existing methods. A novel assay is described here, that identifies germline MS! in patients with homozygous mutations in MMR genes; this method allows the degree of instability to be quantified as a measure termed the gMSI ratio. Using this tool, a UK childhood leukaemia DNA bank, comprising 799 patients, from the ALL-97 trial has been screened for gMSI, demonstrating that mutations in MMR genes are not a frequent cause of sporadic acute lymphoblastic leukaemia in children. A population study of sequence variation at the PMS2 and PMS2CL loci has been performed, by sequencing 18 PMS2 and 29 PMS2CL alleles, revealing eight novel non-synonymous polymorphic variants in exon 11 of PMS2, and one in each ofexons 13 and 14. Three patches ofintronic sequence (76 to 258 nucleotides long) are identified, which show evidence of gene conversion between gene and pseudogene. Single nucleotide polymorph isms and paralogous sequence variants in PMS2 and PMS2CL are defined which will aid future mutation detection. The p.R802X mutation in PMS2 is a founder mutation, which ill the homozygous state is the commonest cause of MMR-D syndrome in British Pakistani patients. An EBV -transformed lymphoblastoid cell line homozygous for p.R802X has been used to investigate whether PTC124 (Ataluren), a drug promoting read through of premature termination codons, might be of benefit in alleviating the effects of this mutation. In this exploratory study, treatment with PTC124 led to no change in PMS2 mRNA expression and no production of full-length PMS2 protein, suggesting no successful readthrough of the p.R802X mutation.