Mutation analysis of patient 14. (A) Homozygous germline mutation in the promoter of CD95 causing disruption of a consensus sequence for AP-2 binding. Genomic DNA of PBMC was amplified by PCR using primers 1-1F/1-1R (covering nt −408 to nt −202 of the CD95 promoter; nucleotides numbered relative to the ATG start codon according to Behrmann et al41). The amplificate was subjected to SSCP analysis, abnormal migrating bands were cut-out from the gel, reamplified, and sequenced. T-ALL blasts and mature T lymphocytes were immunomagnetically purified, PCR-amplified as above, and sequenced. Nucleotide sequence is compared to wild type. Bases involved in the mutation are highlighted by asterisks. (B) The mutation is inherited from the father. Genomic DNA of PBMC from the mother (M), the father (F), and the patient (P) were amplified as under (A).The mutation is inherited from the homozygous father, whereas the mother has wild-type alleles only. (C) The mutation in the CD95 promoter does not decrease constitutive CD95 expression. CD95 expression on cryopreserved PBMC was determined by three-color immunofluorescence analysis as described in Materials and Methods. For electronic gating T-ALL blasts were identified by forward/side scatter characteristics of viable lymphocytes and surface expression of CD7 but not CD3. Mature T cells were CD3⁺ and CD7⁺.