Ultradeep sequencing of all exons of genes involved in GPI anchor synthesis reveals two mutation events in PIGT: a germline splice site mutation and a somatic deletion. (A) DNA was isolated from whole blood and enriched for all exons of genes involved in GPI anchor synthesis and subjected to ultradeep sequencing. The coverage of PIGT exons was significantly reduced compared with exons of all other GPI anchor synthesis genes, suggesting a deletion involving PIGT. The extent of the deletion was further characterized by arrayCGH comprising in total 8 MB, arr20q11.23q13.12. (B) FISH with BAC clone RP3-337O18 (G) and a probe targeting the centromere of chromosome 20 (R) was used to analyze the deletion in T lymphocytes and granulocytes. Although 2 signals of RP3-337O18 were present in all complete metaphases of T lymphocytes, the majority of granulocytes showed only 1 signal for RP3-337O18, indicating a somatic deletion in a myeloid lineage. (C) A single nucleotide substitution in PIGT affecting the splice acceptor site of intron 10, NM_015937:c.1401-2A>G, was observed in the ultradeep sequencing data of DNA extracted from whole blood. In total, 1463 sequence reads covered the canonical splice site, and 85% of these reads showed the alternate base, indicating that the mutation is present on the undeleted haplotype of PIGT. (D) The splice site mutation was validated by ABI Sanger sequencing and shown to be heterozygous in DNA extracted from epithelial cells of a buccal swap, confirming its presence in different tissues.