Copy number variations and founder effect underlying complete IL-10Rβ deficiency in Portuguese kindreds

Abstract

Mutations in interleukin-10 receptor (IL-10R) genes are one cause of very early-onset inflammatory bowel disease with perianal lesions, which can be cured by hematopoietic stem cell transplantation. Using a functional test, which assesses responsiveness of peripheral monocytes to IL-10, we identified three unrelated Portuguese patients carrying two novel IL-10RB mutations. In the three patients, sequencing of genomic DNA identified the same large deletion of exon 3 which precluded protein expression. This mutation was homozygous in two patients born from consanguineous families and heterozygous in the third patient born from unrelated parents. Microsatellite analysis of the IL10RB genomic region revealed a common haplotype in the three Portuguese families pointing to a founder deletion inherited from a common ancestor 400 years ago. In the third patient, surface expression of IL-10R was normal but signaling in response to IL-10 was impaired. Complementary DNA sequencing and next-generation sequencing of IL10RB locus with custom-made probes revealed a ≈ 6 Kb duplication encompassing the exon 6 which leads to a frameshift mutation and a loss of the TYK2-interacting Box 2 motif. Altogether, we describe two novel copy number variations in IL10RB, one with founder effect and one preserving cell surface expression but abolishing signaling.

Fig 1. Patients from Portugal share a common deletion of exon 3 in IL10RB.

1A: Lack of responsiveness to IL-10 in PBMC stimulated with lipopolysaccharide (LPS) in Patient 3 compared to one control. IL-8 was quantified by ELISA in supernatants after a 24 hour-stimulation at the indicated concentrations of LPS and IL-10. Results were comparable in all three patients. 1B: Scheme showing the exon 3 deletion as defined by Sanger analysis. Genomic positions of breakpoints are shown by square brackets. 1C: Gel analysis of PCR products from genomic DNA with primers flanking the deletion showing the normal 3.5 kb band in control, parents and P3 and a short 0.5 kb mutated band in patients, parents of P1 and P2 and mother of P3.

Fig 2. Duplication of IL10RB exon 6 results in loss of exon 7 encoded intracellular domain and prevents TYK2 activation.

2A: Flow cytometry analysis showing normal cell surface expression of IL-10Rβ in PBMC of P3 (gated on CD14⁺ cells). 2B: Flow cytometry analysis comparing STAT3 phosphorylation in response to IL-6 and IL-10 in PBMC of P3 and healthy control (gated on CD3⁺ cells). These data are representative of two independent experiments. 2C: Amino acid sequence of wild type IL-10Rβ and of the IL-10Rβ duplE6. In red, the aberrant 50 amino acid peptide resulting from the frameshift caused by the duplication of exon 6. Highlighted in yellow are residues conserved in IFNAR1, another TYK2-interacting cytokine receptor. 2D: Schematic representation of the two novel IL10RB mutations.

Fig 3. Lack of TYK2, STAT1 and STAT3 phosphorylation in P3’s EBV-B cells in response to IL-10.

Western blot analysis of TYK2, STAT1 and STAT3 phosphorylation in EBV-B cells from P3 and her parents stimulated for 15 min with IL-6 or IL-10 (25 ng/ml). ns, non-stimulated. F, father. M, mother. Two gels were run in parallel with the same lysates. These data are representative of two independent experiments.