Germline mutations in CARD11 that result in constitutive NF-κB activation and selective B cell expansion underlie congenital B cell lymphocytosis.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

Purpose Activating germline mutations in CARD11 have recently been linked to a rare genetic disorder associated with congenital B cell lymphocytosis. We describe a patient with a similar clinical phenotype who had a de novo germline G123D CARD11 mutation. Methods Whole exome sequencing was performed on DNA from the patient and his biological parents. Laboratory studies examined characteristics of the patient’s B and T lymphocytes. A CARD11 cDNA containing the mutation was transfected into a lymphocyte cell line to gain an understanding of its function. RNA sequencing was performed on samples from the patient and from patients with alternate germline CARD11 mutations and differential gene expression analysis was performed. Results The patient had a decade-long history of severe polyclonal B lymphocytosis in the 20,000–90,000 lymphocytes/mm3 range, which was markedly exacerbated by EBV infection and splenectomy at different times. He had a heterozygous germline CARD11 mutation causing a G123D amino acid substitution, which was demonstrated to induce NF-κB activation in unstimulated lymphocytes. In contrast to previous patients with CARD11 mutations, this patient’s B cells exhibited higher expression of several cell cycle progression genes, as well as enhanced proliferation and improved survival following B cell receptor stimulation. Conclusions This is the third reported germline and first de novo CARD11 mutation shown to cause congenital B cell lymphocytosis. The mutation was associated with a dramatically greater lymphocytosis than in previously described cases, disproportionate to the level of constitutive NF-κB activation. However, comparative review of the patient’s clinical history, combined with additional genomic and functional analyses, underscore other important variables that may affect pathophysiology or regulate mutant CARD11 function in B cell proliferation and disease. We now refer to these patients as having BENTA disease (B cell Expansion with NF-κB and T cell Anergy).

Recombinant cDNA libraries to poly(A)RNA isolated from mature pollen of Zea mays and Tradescantia paludosa have been constructed. Northern blot analyses indicate that several of the clones are unique to pollen and are not expressed in vegetative tissues. The majority, however, are expressed both in pollen and vegetative tissues. Southern hybridizations show that the pollen specific sequences in corn are present in one or a very few copies in the genome. By using several of the clones as probes, it was found that there are at least two different groups of mRNAs with respect to their synthesis. The mRNAs of the first group represented by the pollen specific clones are synthesized after microspore mitosis and increase in concentration up to maturity. The second group, exemplified by actin mRNA, begins to accumulate soon after meiosis, reaches its maximum by late pollen interphase, and decreases thereafter. Although the actin mRNA and the pollen specific mRNAs studied show very different patterns of initiation of synthesis and accumulation during pollen development, the rates of decline of these mRNAs during the first 60 minutes of germination and pollen tube growth in Tradescantia are similar and reflect the previously observed declines in rates of protein synthesis during this period.The pollen grains of Tradescantia paludosa (16), corn (17), and tobacco (30) contain a store of presynthesized messenger RNAs (mRNAs) at the time of their release from the anther. These mRNAs have been shown to code in cell-free translation systems for proteins that are similar to the proteins synthesized during pollen germination and tube growth (9,17 shown that a large fraction (>64%) of the genes expressed in pollen are also expressed in vegetative tissues, whereas no more than 60% of the genes expressed in shoots are similar to those expressed in pollen (31). Similar hybridizations have been carried out with RNA from corn pollen and the results are similar to those obtained with Tradescantia (RP Willing, JP Mascarenhas, unpublished data). To further our knowledge of pollen development it is essential that the genes that are expressed in pollen, especially those unique to pollen, be isolated and characterized in some detail in order to understand their developmentally specific regulation and functions. We report here the construction of two cDNA libraries made to pollen mRNAs from Tradescantia and corn, and the utilization of some of the clones to answer questions about the nature of the pollen expressed genes and their transcription during pollen development and tube growth. MATERIALS AND METHODSPlant Material. Tradescantia paludosa L. plants were grown in the greenhouse and pollen was collected and stored as previously described (15). Corn (Zea mays L.) pollen was collected from field grown plants of the cultivar 'Gold Cup' (Harris Seeds, Rochester, NY). Pollen was quick frozen in liquid N2 and stored at -70°C. For later experiments the inbred line of corn W-22 (Illinois Foundation Seeds) was used. Various vegetative and ...