Positions of primers and probes in the CD95 genomic and cDNA, and overview of important sequences in the patient with ALPS. (A) cDNA, including exon 6. There is an AG insert in a few strands that is linked to a C in the polymorphic position 74 (position 74 downstream of the 5′ end of exon 7, same as position 836 bp downstream of the first translated ATG). Most normally spliced cDNA (including exon 6, but not the extra AG) has a T in position 74. Primers CF1 and CR1 were used for cloning; CF2 and CR2 for RT-PCR; and TF1, P1, and TR1 for TaqMan PCR to quantify the normally spliced form. (B) Splice forms that lack exon 6 and hence the transmembrane domain. They mostly have a C in position 74. These forms were quantified by TaqMan PCR using TF2, P2, and TR2. (C) Genomic sequence with a mutation C→G in position –3 upstream of exon 6 on one allele and with the C/T polymorphism in exon 7. The primers used for genomic sequencing (GF1, GF2, GR1, GR2), the TIA-1 binding site, and the transmembrane domain are marked. (D) Pedigree and SNP haplotyping. Analysis of intronic SNPs upstream of exon 5 (a, b) and downstream of exon 7 (e), the C/T SNP expressed in exon 7 (d) 836 bp downstream of the first translated ATG (see position 74 A, B, C, also used in Table 2), and the mutation ex-6–3C→G (c), which is associated with excessive sFas production, decreased mFas expression, and apoptosis (Figures 2 and 4).