Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions

Abstract

Background: Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance.

Methods: We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing.

Results: Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures.

Conclusions: Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Figure 1. Clinical and Immunologic Manifestations in Patients with Phospholipase Cγ₂–Associated Antibody Deficiency and Immune Dysregulation (PLAID)

Panel A shows the results of an evaporative cooling test in one subject. Cold urticaria was provoked with droplets of ethanol (E) or air-blown water (A) but not with droplets of unblown water (W) or covered water (C). In Panel B, blood leukocyte counts are shown for 18 adult subjects with PLAID. The tops and the bottoms of the boxes represent a 2-SD range above and below the mean values in healthy control subjects, and the horizontal lines represent the median value among subjects with PLAID. NK denotes natural killer. Panel C shows the mean frequency of IgA or IgG antibody–secreting cells, measured by means of enzyme-linked immunospot assay (ELISPOT) after 4 days of expansion in culture. ELISPOT data were normalized to an equal number of B cells on the basis of the proportion of B cells in peripheral-blood mononuclear cells at the start of culture. The T bars indicate standard errors for five subjects from each group. In Panel D, Igκ secondary recombination in transitional B cells is shown for 12 healthy donors (HD), 4 subjects with PLAID, and 4 subjects with X-linked agammaglobulinemia (XLA), indicated by the frequency of Jκ region usage, as determined by single-cell Igκ sequence analysis of data from subjects who were pooled according to diagnosis. The increased usage of the terminal Jκ4 and Jκ5 genes, as seen in both PLAID and XLA, indicates impaired termination of secondary recombination.

Figure 2. Genomic Deletions in PLCG2 Encoding Phospholipase Cγ₂ in the Study Subjects

Panel A shows the pedigrees of three families with PLAID. Solid symbols indicate affected subjects, and open symbols unaffected relatives. Squares indicate male subjects, and circles female subjects. Slashes indicate deceased subjects. The starred member in Family 3 died at the age of 1 year from pneumonia. Panel B shows the candidate interval on the long arm of chromosome 16, defined by the intersection of a linkage interval in Family 1 and candidate interval in Family 2. Three distinct PLCG2 deletions were identified in the three families (red horizontal bars).

Figure 3. Effects of PLAID-Associated PLCG2 Deletions, Including Increased Phospholipase Activity, Deceased Cellular Activation at Physiologic Temperatures, and Enhanced Cellular Activation at Subphysiologic Temperatures

Panel A shows enzymatic activity of phospholipase Cγ₂ (PLCγ₂) mutants. COS-7 cells were transfected with DNA encoding either wild-type PLCG2 or PLCG2 with deletions of the C-terminal Src homology 2 (cSH2) domain, exon 19 (Δ19), and exons 20 through 22 (Δ20–22) (at left). PLCγ₂ activity was assayed by quantification of [H] inositol phosphates generated by the respective enzymes (at right). Basal activity was quantified in COS-7 cells transfected with PLCG2 alone, whereas the Rac-activated condition included cotransfection with a Rac2 V12 expression construct. The I bars indicate 1 SD above and below the mean release of inositol phosphate for each condition. Data are representative of three independent experiments. C2 denotes calcium-binding C2 domain, EF EF hand motif, nSH2 N-terminal SH2 domain, PH pleckstrin homology domain, SH3 SH3 domain, sPH split pleckstrin homology domain, X-box × catalytic domain, and Y-box Y catalytic domain. Panel B shows cytoplasmic calcium content (as measured by FLUO-4 staining) in natural killer (NK) cells before and after surface-receptor cross-linking (arrow indicates time of administration) in two subjects with PLAID and one control subject. Panel C shows degranulation of NK cells after incubation with sensitive target cells, as measured by cell-surface expression of CD107a as a percent of the maximum CD107a expression observed in control NK cells. Panel D shows the effect of temperature reduction, in the absence of surface stimulation, on cytosolic calcium content in B cells sorted from a case subject and a control subject, as measured on confocal microscopy. Data points reflect the average mean fluorescence intensity of 100 individually imaged cells, and I bars indicate standard errors. Data are representative of three independent experiments. One asterisk indicates a P value of 0.01 to 0.05, two asterisks indicate a P value of 0.001 to less than 0.01, and three asterisks indicate a P value of less than 0.001. Panel E shows mast-cell degranulation, as indicated by the surface expression of lysosomal-associated membrane protein 2 (LAMP2) and measured by means of flow cytometry at 37°C (red) and 20°C (blue) in Laboratory of Allergic Diseases 2 (LAD2) human mast cells transfected with wild-type and deleted forms of PLCG2.