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Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient’s T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.

TTC7A deficiency typically causes severe gastrointestinal manifestations such as multiple intestinal atresia or early-onset inflammatory bowel disease. In some cases, this is associated with severe combined immunodeficiency. Partial loss-of-function mutations appear to be associated with a milder phenotype resulting in common variable immunodeficiency-like condition with enteropathy.

While widespread genome sequencing ushers in a new era of preventive medicine, the tools for predictive genomics are still lacking. Time and resource limitations mean that human diseases remain uncharacterized because of an inability to predict clinically relevant genetic variants. A strategy of targeting highly conserved protein regions is used commonly in functional studies. However, this benefit is lost for rare diseases where the attributable genes are mostly conserved. An immunological disorder exemplifying this challenge occurs through damaging mutations in RAG1 and RAG2 which presents at an early age with a distinct phenotype of life-threatening immunodeficiency or autoimmunity. Many tools exist for variant pathogenicity prediction, but these cannot account for the probability of variant occurrence. Here, we present a method that predicts the likelihood of mutation for every amino acid residue in the RAG1 and RAG2 proteins. Population genetics data from approximately 146,000 individuals was used for rare variant analysis. Forty-four known pathogenic variants reported in patients and recombination activity measurements from 110 RAG1/2 mutants were used to validate calculated scores. Probabilities were compared with 98 currently known human cases of disease. A genome sequence dataset of 558 patients who have primary immunodeficiency but that are negative for RAG deficiency were also used as validation controls. We compared the difference between mutation likelihood and pathogenicity prediction. Our method builds a map of most probable mutations allowing pre-emptive functional analysis. This method may be applied to other diseases with hopes of improving preparedness for clinical diagnosis. Electronic supplementary material The online version of this article (10.1007/s10875-019-00670-z) contains supplementary material, which is available to authorized users.

TNFAIP3 encodes the NF-κB regulatory protein A20. High-penetrance heterozygous mutations in TNFAIP3 cause a haploinsufficiency of A20 (HA20), inadequate inhibition of NF-κB pathway, and an early onset autoinflammatory disorder. However, the clinical phenotype of patients with HA20 varies greatly and clinical diagnoses prior to establishing the genetic cause, included both autoimmune and autoinflammatory conditions. Here, we present the first patient with HA20, who was previously diagnosed with AOSD but was later found to have a novel heterozygous variant in TNFAIP3 and who was successfully treated with anti-IL6 receptor biologic tocilizumab (RoActemra). We discovered a novel heterozygous mutation in TNFAIP3 c.1906C>T, not previously found in ExAC database. Further analysis shows that this single-nucleotide variant at the terminal residue of TNFAIP3 exon 7 produces an alternatively spliced mRNA resulting in p.His636fsTer1. Additional genetic analysis of family members shows that this variant does segregate with the inflammatory clinical phenotypes. Subsequent functional test show that NF-κB activation, measured as intracellular phosphorylation of p65 in CD14 + monocytes, was more enhanced in the patient compared with healthy controls (HC) following stimulation with LPS. This was associated with higher production of inflammatory cytokines by the patients PBMC in response to LPS and ATP and enhanced activation of NLRP3 inflammasome complex. Furthermore, increased activation of NLRP3 inflammasome was evident systemically, since we detected higher levels of ASC specks in patients’ sera compared with HC. Finally, we used population genetics data from GnomAD to construct a map of both genetic conservation and most probable disease-causing variants in TNFAIP3 which might be found in future cases of HA20.

TNFAIP3 encodes the NF-κB regulatory protein A20. High-penetrance heterozygous mutations in TNFAIP3 cause a haploinsufficiency of A20 (HA20), inadequate inhibition of NF-κB pathway, and an early onset autoinflammatory disorder. However, the clinical phenotype of patients with HA20 varies greatly and clinical diagnoses prior to establishing the genetic cause, included both autoimmune and autoinflammatory conditions. Here, we present the first patient with HA20, who was previously diagnosed with AOSD but was later found to have a novel heterozygous variant in TNFAIP3 and who was successfully treated with anti-IL6 receptor biologic tocilizumab (RoActemra). We discovered a novel heterozygous mutation in TNFAIP3 c.1906C>T, not previously found in ExAC database. Further analysis shows that this single-nucleotide variant at the terminal residue of TNFAIP3 exon 7 produces an alternatively spliced mRNA resulting in p.His636fsTer1. Additional genetic analysis of family members shows that this variant does segregate with the inflammatory clinical phenotypes. Subsequent functional test show that NF-κB activation, measured as intracellular phosphorylation of p65 in CD14 + monocytes, was more enhanced in the patient compared with healthy controls (HC) following stimulation with LPS. This was associated with higher production of inflammatory cytokines by the patients PBMC in response to LPS and ATP and enhanced activation of NLRP3 inflammasome complex. Furthermore, increased activation of NLRP3 inflammasome was evident systemically, since we detected higher levels of ASC specks in patients’ sera compared with HC. Finally, we used population genetics data from GnomAD to construct a map of both genetic conservation and most probable disease-causing variants in TNFAIP3 which might be found in future cases of HA20.

Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.

Inherited deficiency of factor XIIIA subunit (FXIIIA) is an autosomal recessive disorder that is characterized by a life-long bleeding tendency and complications in wound healing. Molecular genetic studies have shown the deficiency can be due to small sequence changes within the FXIIIA gene, such as point mutations or microdeletions. On molecular analysis of the FXIIIA gene in an FXIII-deficient patient, of United Kingdom origin, we identified a putative homozygous missense mutation, Arg408Gln. However, the father of this patient is homozygous normal for arginine at codon 408. Having proved paternity in this pedigree by microsatellite analysis, we examined the FXIIIA RNA of the patient by reverse transcriptase-polymerase chain reaction and found the paternal allele to lack exons 4 through 11 inclusive. Hence, a huge deletion extending from intron 3 to intron 11 and the Arg408Gln mutation are jointly responsible for FXIIIA deficiency in this family. This is the first finding of such a large deletion in the FXIIIA gene.

The Leu564 Factor XIIIA Variant Results in Significantly Lower Plasma Factor XIII Levels than the Pro564 Variant