Whole-exome sequencing-based discovery of STIM1 deficiency in a child with fatal classic Kaposi sarcoma

Abstract

Classic Kaposi sarcoma (KS) is exceedingly rare in children from the Mediterranean Basin, despite the high prevalence of human herpesvirus-8 (HHV-8) infection in this region. We hypothesized that rare single-gene inborn errors of immunity to HHV-8 may underlie classic KS in childhood. We investigated a child with no other unusually severe infectious or tumoral phenotype who died from disseminated KS at two years of age. Whole-exome sequencing in the patient revealed a homozygous splice-site mutation in STIM1, the gene encoding stromal interaction molecule 1, which regulates store-operated Ca(2+) entry. STIM1 mRNA splicing, protein production, and Ca(2+) influx were completely abolished in EBV-transformed B cell lines from the patient, but were rescued by the expression of wild-type STIM1. Based on the previous discovery of STIM1 deficiency in a single family with a severe T cell immunodeficiency and the much higher risk of KS in individuals with acquired T cell deficiencies, we conclude that STIM1 T cell deficiency precipitated the development of lethal KS in this child upon infection with HHV-8. Our report provides the first evidence that isolated classic KS in childhood may result from single-gene defects and provides proof-of-principle that whole-exome sequencing in single patients can decipher the genetic basis of rare inborn errors.

Figure 1.

Identification of a splice-site mutation in STIM1. (A) Sanger sequencing of genomic DNA confirmed G to A substitution at the −1 position of STIM1 exon 8 (1538-1G>A) in the patient, which was initially identified by whole-exome sequencing. Sequencing of genomic DNA extracted from EBV-B cells of a healthy control shows the reference base G at the same position. (B) STIM1 genotypes of the patient and her family members from whom DNA was available for sequence analysis are listed under their symbols. Squares, males; circles, females; filled symbol, affected individual; open symbols, unaffected individuals; double horizontal line, consanguineous marriage. Genotyping was performed twice.

Figure 2.

Abnormal STIM1 mRNA splicing and lack of protein expression in the patient’s EBV-B cells. (A) Levels of STIM1 mRNA were assessed by quantitative RT-PCR in the EBV-B cells from five healthy controls (C) and the patient (P). Threshold cycles (C_T) for STIM1, normalized to those of GUS (ΔC_T), are plotted as 2^−(ΔCT). Each dot or square represents a mean of three independent experiments for each individual. The horizontal bar indicates the mean of five healthy controls. (B) STIM1 exons 7–9 were amplified from cDNA from EBV-B cells of the control (C) or the patient (P) and ligated to pCR2.1 vector. Clones containing STIM1 transcripts were sequenced, and the frequency of each splice variant was calculated by dividing the number of clones containing the particular transcript by the total number of sequenced clones (n = 12 for C, n = 120 for P). 16/120 of P clones corresponded to other minor abnormal transcript forms (not depicted). Number of base pairs indicated between the exons in abnormally spliced variants of the patient represents the size of gain or loss, compared with the wild-type transcript. (C) Levels of STIM1 protein were assessed by immunoblotting with an antibody against the C terminus (left) or N terminus (right) of STIM1. Two fibroblast cell lines, derived from either a healthy individual (C+) or a previously reported STIM1-deficient patient (C−; Picard et al., 2009), were used as controls. C1, C2, and C3 show EBV-B cells from healthy controls and P shows EBV-B cells from the patients. GAPDH blots show comparable protein loading for each sample. Asterisks indicate nonspecific protein bands. Representative blots of three independent experiments are shown.

Figure 3.

Abolished Ca²⁺ influx in the patient’s EBV-B cells is restored by expression of STIM1. (A) EBV-B cells of the patient (P) and two controls (C1 and C2) were stimulated with 1 µM thapsigargin (TG) in Ca²⁺-free Ringer solution (open bars) followed by perfusion with 2 mM Ca²⁺ (filled bars) to induce Ca²⁺ influx. Traces represent mean F340/F380 emission ratios of one representative experiment. Averages on the right are from n = 6 (C1), n = 4 (C2), n = 5 (P) experiments. ΔF340/F380 represents peak [Ca²⁺]_i 25 s after readdition of Ca²⁺ minus baseline [Ca²⁺]_i at the beginning of the recording. Error bars represent the SEM. ***, P < 0.0001. (B) EBV-B cells from the patient were retrovirally transduced with STIM1-IRES-GFP expression vector (P-STIM1) or ORAI1-IRES-GFP vector (P-ORAI1). Ca²⁺ levels were measured as described in A. Traces of individual cells (thin gray lines) and averages of all cells (thick black lines) are shown for 90 GFP⁺ and 90 GFP⁻ cells. Averages of peak ΔF340/F380 from total 368 cells (P-STIM1; n = 6 experiments) or 482 cells (P-ORAI1; n = 10 experiments) are shown in the bar graph. Error bars represent the SEM. ***, P < 0.0001; ns, P > 0.05.