A congenital mutation of the novel gene LRRC8 causes agammaglobulinemia in humans

Abstract

A girl with congenital agammaglobulinemia and minor facial anomalies lacked B cells in peripheral blood: karyotypic analysis of white blood cells showed balanced translocation, t(9;20)(q33.2;q12). In the current study, we isolated a novel gene, leucine-rich repeat-containing 8 (LRRC8), at the translocation site on chromosome 9. It has four transmembrane helices with one isolated and eight sequentially located leucine-rich repeats (LRRs) and constitutes a new protein family. It is expressed on T cells as well as on B-lineage cells. Translocation truncates the LRRC8 gene, resulting in deletion of the eighth, ninth, and half of the seventh LRR domains located close to the C-terminal. The truncated form of the LRRC8 gene is transcribed with sequences from the noncoding region adjacent to the truncated seventh LRR. Protein products derived from the truncated gene are coexpressed on white blood cells with the intact LRRC8 protein from the untranslocated allele. Transplantation experiments with murine bone marrow cells that were forced to express the truncated LRRC8 show that expression of the truncated protein inhibited B cell development. These results indicate that LRRC8 is responsible for the B cell deficiency in this patient and is required for B cell development.

Figure 1

Detection of the translocation site. (a) Spectral karyotyping (SKY) FISH. (b) FISH. Red boxes indicate probes hybridized to chromosome 20 and der20; yellow boxes indicate probes hybridized to chr_20 and der9. PAC dJ890O15 was hybridized to chromosome 20, der20, and der9. (c) Genomic Southern blot hybridization with the ET1033 probe. The arrowhead indicates the mutation. (d) Genomic PCR products from chromosome 20, der20, and der9. cen, centromere; tel, telomere. (e) Sequences of the translocation site. Chr_9, chromosome 9; chr_20, chromosome 20.

Figure 2

Products of LRRC8 and its mutant. (a) mRNAs of wild-type and mutant forms. Black boxes, coding regions; white boxes, untranslated regions; black circles, stop codons; hatched box, the translated region from the intron. (b) RT-PCR analysis. (c–e) Western blot analysis. (c) Peripheral blood cells from a healthy control and the affected patient. CBB, Coomassie brilliant blue. (d) Molecular weight estimation of the protein from healthy human bone marrow (BM). (e) Bone marrow and peripheral blood (PB) cells from a human control and a mouse.

Figure 3

Structures of LRRC8 and its mutant. (a) Comparison of human LRRC8 and its murine ortholog. White letters on black boxes indicate different amino acids between the two species. LxxLxLxxNxLxxLPxxLxxL is the representative structure of LRR, where x means any amino acid. (b) Schematic structures. White boxes, LRRs; hatched boxes, transmembrane helices; black box, additional polypeptides. N, N-terminal; C, C-terminal. (c) Amino acid sequences of the mutant. Underlined characters represent translated polypeptides from the intron.

Figure 4

Enforced expression of mutant LRRC8. (a) Constructs of the vectors. LTR, long terminal repeat; IRES, internal ribosomal entry site. (b) Mutant LRRC8 was detected in the MutY-infected E86 cell line by Western blot. (c) Representative data of murine peripheral blood cells obtained 3 months after transplantation. Thin lines represent YFP-negative control. Gr, granulocytes; Mo, monocytes. (d) Statistical analysis of the ratio of YFP-positive cells in each lineage (MIY, n = 4; MutY, n = 3; Student’s t test).

Figure 5

Maturation arrest of B-lineage cells induced by expression of truncated LRRC8 and expression of LRRC8 on normal lymphoid cells. (a) Representative data of B-lineage cell subsets in bone marrow transfected with MIY or MutY. PE, phycoerythrin; APC, allophycocyanin. (b) LRRC8 expression on normal bone marrow and peripheral blood cells. CD19- and CD3-positive cells were gated as human B-lineage and T cells, respectively.