Supplementary Figure 1: Whole-genome linkage, whole-exome sequencing, population genetics, qPCR and effect of SPPL2a deficiency on TNF processing.

a) Combined whole-genome linkage analysis of kindreds A and B. LOD scores are shown in black and blue for alternating chromosomes, and information content is reported as a red trace. SPPL2A maps to the linked region of chromosome 15 indicated with a red arrow. b) The upper panel shows the IGV (https://www.broadinstitute.org/igv/) viewer presentation of the region of the genome containing the mutation for the index cases of both kindreds, in an antisense direction. Mutations are located in the center and the alternative nucleotide is indicated in red. The lower panels show the Sanger sequencing results for all the patients in each family, heterozygous carriers and one WT individual for each of the variants. The mutation site is marked with a dashed square. The shaded squares on the electrophoretograms represent the exonic areas, the rest corresponding to introns. c) Graph of CADD score on the y axis against minor allele frequency (MAF) on the x-axis, for all the heterozygous SPPL2A variants reported in the gnomAD database (http://gnomad.broadinstitute.org). The horizontal dashed line represents the MSC for SPPL2A. d, e) qPCR on cDNA from: d) EBV-B cells from a healthy control, a heterozygous carrier and P1 e) SV40 fibroblasts from a healthy control, a heterozygous carrier and P2 using a probe spanning exons 2-3 of SPPL2A. Mean and SEM is represented in both graphs. f) Schematic representation of the structure and cleavage sites of TNF. The SPPL2a and SPPL2b cleavage site in the transmembrane domain (indicated in black) is mark as a red dashed line. g) Memory and h) naive CD4⁺ T cells were isolated and stimulated with T cell activation and expansion (TAE) beads alone or under TH1 (TAE beads+IL-12) polarizing conditions. TNF secretion was assessed after and then the cells were re-stimulated with PMA/ionomycin and the percentage of TNF-positive cells was determined, by intracellular staining and flow cytometry. Mean is shown in both graphs.