Epistatic interactions between mutations of TACI (TNFRSF13B) and TCF3 result in a severe primary immunodeficiency disorder and systemic lupus erythematosus

Common variable immunodeficiency disorders (CVID) are a group of primary immunodeficiencies where monogenetic causes account for only a fraction of cases. On this evidence, CVID is potentially polygenic and epistatic although there are, as yet, no examples to support this hypothesis. We have identified a non-consanguineous family, who carry the C104R (c.310T>C) mutation of the Transmembrane Activator Calcium-modulator and cyclophilin ligand Interactor (TACI, TNFRSF13B) gene. Variants in TNFRSF13B/TACI are identified in up to 10% of CVID patients, and are associated with, but not solely causative of CVID. The proband is heterozygous for the TNFRSF13B/TACI C104R mutation and meets the Ameratunga et al. diagnostic criteria for CVID and the American College of Rheumatology criteria for systemic lupus erythematosus (SLE). Her son has type 1 diabetes, arthritis, reduced IgG levels and IgA deficiency, but has not inherited the TNFRSF13B/TACI mutation. Her brother, homozygous for the TNFRSF13B/TACI mutation, is in good health despite profound hypogammaglobulinemia and mild cytopenias. We hypothesised that a second unidentified mutation contributed to the symptomatic phenotype of the proband and her son. Whole-exome sequencing of the family revealed a de novo nonsense mutation (T168fsX191) in the Transcription Factor 3 (TCF3) gene encoding the E2A transcription factors, present only in the proband and her son. We demonstrate mutations of TNFRSF13B/TACI impair immunoglobulin isotype switching and antibody production predominantly via T-cell-independent signalling, while mutations of TCF3 impair both T-cell-dependent and -independent pathways of B-cell activation and differentiation. We conclude that epistatic interactions between mutations of the TNFRSF13B/TACI and TCF3 signalling networks lead to the severe CVID-like disorder and SLE in the proband.

In contrast, mutations in other genes such as TNFRSF13B, which encodes Transmembrane Activator Calcium Modulator and Cyclophilin Ligand Interactor (TACI), MutS homolog 5 (MSH5) and TNFRSF13C, which encodes B-cell activating factor receptor (BAFFR), predispose to, but do not solely underlie CVID. Mutations of TNFRSF13B/TACI are also found in healthy individuals, albeit at lower frequency than in CVID cohorts: the frequency of normal individuals carrying the C104R variant (0.8%) is higher than the prevalence of disease (0.002%). 10 This suggests either these mutations of TACI are clinically inconsequential in many cases, or there are other additional unknown genetic defects in symptomatic patients that contribute to the disease phenotype. 11,12 Here, we report the first example of digenic inheritance leading to a severe CVID-like disorder and autoimmunity, as a result of epistasis. Epistasis, where two or more genetic loci interact to produce novel phenotypes was first predicted over one hundred years ago. However, its existence in humans has been highly controversial because of the scarcity of well-characterised examples. [13][14][15] In this report, superimposition of a de novo Transcription Factor 3 (TCF3) mutation in a family already carrying a C104R (c.310T4C) mutation of the TACI gene causes a severe CVID-like disorder and systemic lupus erythematosus (SLE) in the proband. Her symptomatic son, who has inherited only the TCF3 mutation, but not the TACI gene mutation, has type 1 diabetes (T1D), synovitis, reduced IgG levels and IgA deficiency. Other family members, carrying only the TACI mutation, in heterozygous or homozygous form, are either in good health or only present with mild clinical symptoms. Our studies indicate the TCF3 mutation has a much greater clinical impact than the TNFRSF13B/TACI mutation on disease severity and expression of both mutations in the proband results in a severe disorder. The phenotypic pattern of the immunodeficiency and autoimmune disease in this family exemplifies how digenic inheritance can lead to clinical and genetic epistasis in humans. 16

Clinical presentation of index patient
The proband (II.2), aged 61 years presented with symptomatic hypogammaglobulinemia in her teenage years and was diagnosed with CVID at age 33 (Table 1, Figures 1a and b). She was initially treated with intravenous immunoglobulin, but later changed to subcutaneous immunoglobulin treatment. She has had two episodes of meningitis while receiving immunoglobulin and has chronic diarrhoea. Despite several functional endoscopic sinus surgical procedures, she continues to suffer recurrent upper respiratory tract infections. She is on thyroxine replacement for Hashimoto's thyroiditis and also meets the American College of Rheumatology (ACR) criteria for SLE. She has cytopenias, antinuclear antibodies, rashes, oral ulcers, nasal ulcers and arthritis.
Clinical features and segregation of the TNFRSF13B/TACI C104R mutation in the kindred The proband was shown to be heterozygous for the C104R (c.310T4C) mutation of the TNFRSF13B/TACI gene in a previous study. 12 Her non-consanguineous parents (I.1 and I.2), in their ninth and tenth decades, are both heterozygous for the C104R mutation ( Figure 1a). They have mild symptomatic hypogammaglobulinemia and thrombocytopenia, but are in otherwise reasonable physical health. 12 Both the proband's male siblings carry the C104R mutation and are well. Both have mild cytopenias (Table 1). One brother is heterozygous (II.3) and the other (II.4) is homozygous for the TNFRSF13B/TACI C104R mutation. 12 Given his asymptomatic status, The proband's son (III.1) has CVID-related phenotypes including symptomatic IgG deficiency, IgA deficiency, type 1 diabetes and has recently developed seronegative arthritis. He has high titres of antiparietal cell antibodies. His disorder may be in evolution as his IgG has decreased to 5.5 g l − 1 (NR 7-14) from 6.5 g l − 1 over the last year. He suffers from recurrent infections and has impaired antigen responses to protein and carbohydrate vaccines ( Table 1). He is classified as having symptomatic hypogammaglobulinemia of uncertain significance and IgA deficiency. 17,19 Both the proband and her son have reduced switched memory B cells and the proband is lymphopaenic (Table 1). Since symptomatic disease is a prerequisite for probable CVID, application of our CVID diagnostic criteria 17,19 concluded that only the proband (II.2) had CVID, while her son (III.1) had reduced IgG levels (symptomatic hypogammaglobulinemia of uncertain significance) and symptomatic IgA deficiency. We have assessed the relative severity of the disorder of all family members using the clinical score proposed for the use of subcutaneous immunoglobulin/intravenous immunoglobulin, 21 which is based on the frequency and severity of immune sequelae of CVID ( Table 1). Application of the Ameratunga et al. 2,19 diagnostic criteria for CVID concluded that the proband (II.2) and her son (III.1) were phenotypically distinct from other family members (clinical score 410) and the highest clinical score was assigned to the proband. Together, these findings indicated that the TNFRSF13B/TACI C104R mutation could not be the sole explanation for CVID in this family, prompting a search for other causative genetic mutations. 12 Identification of a novel de novo mutation of TCF3 in both severely symptomatic individuals Whole-exome sequencing was performed on II.2, III.1 and III.2 and analysed assuming an autosomal dominant mode of inheritance, where II.2 and III.1 have reference/alternative alleles (REF/ALT) and III.2 is healthy (REF/REF or ALT/ALT). Non-synonymous variants within coding and splice site regions with a minor allelic frequency less than 1% were annotated. Analysis did not reveal evidence of consanguinity and identified 94 rare genetic variants affecting protein sequence that were transmitted by the proband to her son, but not to her daughter (Supplementary Tables 1 and 2). Nine variants affecting genes with known roles in the immune system were genotyped in the entire kindred (Supplementary Table 3). Of these, only a de novo frameshift nonsense mutation in TCF3 encoding the E2A transcription factors E12 and E47 21 segregated with the two severely symptomatic family members, II.2 and III.1 (Figures 1a and b) in the wider family. TCF3 plays a critical role in early B-cell development. 22 It is also thought to play an important role in mature B-cell biology and promotes Ig gene transcription. 23 Studies have shown that the E2A transcription factors are essential for the expression of several genes involved in the Ig isotype switching and secretion pathway including Activation Induced Deaminase (AICDA, which encodes AID) and  Figure 2 Immunoglobulin isotype switching pathways showing nodes of intracellular signal integration between TACI and TCF3/E2A. T-cell-independent isotype switching occurs through TACI and TLRs while T-cell dependent switching occurs through CD40 and IL-4 or IL-21. Ligation of the B-cell receptor synergises with both pathways. TCF3/E2A contributes to the expression of AID, 14-3-3γ and Ig production and therefore influences both T-cell-dependent and -independent Ig switching pathways. 14-3-3γ is a scaffolding protein and targets AID to switch regions. Mutations are shown in red stars. BCR-B cell receptor. TLR, Toll-like receptor.

Class-switch recombination
Epistatic effects of digenic defects in CVID R Ameratunga et al 14-3-3γ, a scaffolding protein, which targets AID to Ig switch regions ( Figure 2). 24 Insertion of an adenine residue at exon 8 of TCF3 creates a frameshift leading to a nonsense mutation (T168fsX191, Figure 1b). Threonine at position 168 is the first amino acid to be affected by the frameshift, and results in a stop codon at position 191 ( Figure 1c). The presence of the mutation was confirmed by Sanger sequencing and is not expressed in other family members, healthy controls or any publicly available gene databases ( Figure 1b). The two severely affected individuals (II.2, III.1) are heterozygous for the mutation, consistent with autosomal dominant inheritance. The mutation was absent in the proband's parents, indicating its de novo origin.
Haploinsufficiency of E2A in proband (II.2) and her son (III.1) Neither the mRNA of the mutant TCF3 T168fsX191 allele nor its truncated protein products (E12 and E47) were expressed, presumably Epistatic effects of digenic defects in CVID R Ameratunga et al as a result of nonsense mutation mediated decay. The wild-type E2A (E47) protein is poorly expressed in both heterozygous individuals carrying the TCF3 T168fsX191 mutation (II.2 and III.1), but normal expression was detected in other family members and unrelated healthy controls (Figure 1d). Together these results suggest haploinsufficiency of E2A in affected individuals II.2 and III.1.

Intracellular NFκB signalling
TACI plays a critical role in immunoglobulin isotype switching particularly when mediated via the T-cell-independent pathway ( Figure 2). 25   Epistatic effects of digenic defects in CVID R Ameratunga et al Both T-cell-dependent and -independent pathways lead to downstream activation of NFκB, expression of activation induced cytidine deaminase (AID) and related molecules ( Figure 2). NFκB1 (p105 and its proteolytically cleaved subunit, p50) and NFκB2 (p100 and its active subunit, p52) and their associated transcription factor family members together regulate a large number of target genes that are essential for B-cell development, maturation and differentiation into Ig isotype switched memory and antibody-secreting cells (ASC). We thus first investigated whether NFκB signalling was impaired in the proband to determine the consequences of expressing TCF3 and TNFRSF13B/TACI mutations. Phosphorylation of p105, as well as total p105 and p50 were reduced (~50%) in stimulated peripheral blood mononuclear cells (PBMCs) from the proband (II.2), compared to unrelated healthy controls following stimulation with PMA and ionomycin (Figure 1e). No differences were observed for p100/p52 expression and signalling via the NFκB2 pathway (not shown).

Immunophenotyping of lymphocyte populations
The two symptomatic individuals (II.2, III.1) bearing the TCF3 T168fsX191 mutation had a reduced total number of B cells, naïve B cells, as well as a significant reduction in memory B cells, with fewer isotype-switched memory B cells detected (Figures 3a-c). Individuals carrying the TNFRSF13B/TACI C104R mutant only (II.3, II.4) also displayed a reduction in the total number of lymphocytes and B cells (Figures 3a and c). No differences in total T-cell number, CD4:CD8 ratios, NK cell or monocytes were observed (Figure 3a and not shown).
Quantification of a severe in vitro antibody production defect by proband naïve B cells demonstrates epistasis We next assessed the ability of naïve B cells isolated from each family member to differentiate into ASC leading to the production of Ig following in vitro stimulation (Figure 4a). Naïve B cells isolated from the proband (II.2) were able to differentiate and secrete Ig, under both T-cell-dependent (CD40L+IL-4+IL- 21) or T-cell-independent (CpG +IL-4+IL-21 ± APRIL) conditions. However, in each condition, this was consistently less than other family members and was almost exclusively IgM, with very little IgG detected in culture supernatants. The proband's brother (II.3), who is heterozygous for the TNFRSF13B/TACI C104R mutation is able to produce IgG levels comparable to the wild-type family control (III.2, Figure 4a) and unrelated healthy donors (Supplementary Figure 1) via the T-cell-independent pathway. His cells produce lower quantities of IgG through the T-cell-dependent pathway than his niece, (III.2). The TACI/Toll-like receptor pathway can augment T-cell-dependent isotype switching and IgG production, 26 which may account for the slightly lower IgG levels in comparison with II.3, who is heterozygous for TNFRSF13B/TACI C104R mutation, or III.2, who has neither mutation.
Naïve B cells isolated from the brother with the homozygous TNFRSF13B/TACI C104R mutation (II.4) were able to produce detectable IgG in vitro via the T-cell-independent pathway (Figure 4a). However, his (II.4) cells produced consistently lower levels of IgG compared to his healthy niece (III.2). Previous studies have shown TNFRSF13B/TACI C104R homozygous individuals are able to produce some IgG in vitro with APRIL stimulation alone. 27 This is likely to be augmented by Toll-like receptor signalling with CpG as well as IL-4 and IL-21, in our experiments. As expected, his cells produce greater amounts of IgG through his intact T-celldependent pathway. The proband's son (III.1) carrying only the heterozygous TCF3 T168fsX191 mutation is also able to produce some IgG in vitro via activation of both pathways, but at much lower levels than his wild-type sister (III.2). His cells produced higher levels of IgG and IgM than his mother (II.2, who bears both the TNFRSF13B/TACI C104R and TCF3 T168fsX191 mutations).
The combination of TCF3 T168fsX191and TNFRSF13B/TACI C104R mutations in the proband resulted in a greater net effect that the sum of each individual mutation would predict than the sum of deficits observed for each mutation alone (that is, Ig level III.2 − (Ig III.2 − Ig III.1 )+(Ig III.2 − Ig II.3 )). When the amount of Ig detected in cultures of TNFRSF13B/TACI TCF3 double mutant naïve B cells following APRIL/CpG stimulation, a much larger deficit is observed compared to TCF3 +/ − or TNFRSF13B/TACI +/ − mutant cells alone; that is, the amount of Ig production in the proband (II.2) is much lower than the sum of each individual contribution (by III.1 and II.3). The same is also true for other culture conditions tested (Figure 4a, Supplementary Figure 1). When such a quantitative defect in Ig production is combined with the observed additional defects in total cell number and B-cell development and differentiation, epistatic interaction of TCF3 and TNFRSF13B/TACI mutations is clearly observed in this family.

Proliferation and isotype switching potential of naïve B cells
We next investigated if the severely reduced in vitro antibody production observed in the proband could be explained by impaired proliferation or isotype switching. Naïve B cells were isolated from family members, labelled with the cell division tracking dye, cell trace violet (CTV), and after 6 days of stimulation with CD40L, IL-4 and IL-21, the proliferative and switching potential assessed. Naïve B cells from III.1 (carrying only the TCF3 T168fsX191 mutation) underwent, on average, slightly fewer rounds of cell division (Mean division number, MDN) than those from his healthy sister (III.2; MDN = 3.9 and 4.6, respectively; Figure 4b). A small proliferative difference was also observed in naïve B cells from family members carrying only the TNFRSF13B/TACI C104R mutation, either in heterozygous (II.3) or homozygous (II.4) form (MDN = 3.3, 3.1, respectively). However, the combination of both mutations in the proband (II.2) showed normal proliferation of naïve B cells, with no direct proliferative defect observed after 6 days of stimulation with CD40L and cytokines (MDN = 4.8). Thus, neither mutation prevents B cells from undergoing a relatively healthy proliferative response.
We then examined the ability of stimulated B cells to undergo IgG isotype switching (Figure 4b, right panels). Despite the absence of IgG detected in the supernatants of these cultures, no defect was observed in the generation of isotype switched IgG + cells in II.2 (carrying both TNFRSF13B/TACI C104R and TCF3 T168fsX191 mutations), compared to III.2, who has neither mutation. Her son, III.1, carrying the TCF3 T168fsX191 mutation only, also generated a similar proportion of IgG + switched cells. However, individuals carrying the TNFRSF13B/ TACI C104R mutation alone (II.3 and II.4) generated fewer IgG + switched cells from naïve cultures, even in the absence of TACI ligand engagement. Since isotype switching is known to be linked to the number of divisions undergone, 27-31 this could be, in part due to the small proliferative delay and reduced number of cells in later divisions observed in TACI-deficient naïve B cells.

Deficiency of in vitro generation of ASC by TNFRSF13B/TACI/ TCF3 double mutant naïve B cells
The above studies showed relatively healthy proliferation and isotype switching by II.2, but markedly reduced secretion of Ig suggesting a defect in development or function of ASC following stimulation. 6,30 To investigate a putative differentiation defect in Epistatic effects of digenic defects in CVID R Ameratunga et al the E2A-TCF3/TACI-deficient B cells, naïve B cells were cultured under T-dependent (CD40L+IL4 ± 21) or T-independent (APRIL+CpG ± IL4/21) conditions and the generation of ASC was assessed by flow cytometry after 5 days. The proportion of ASC (CD27 hi , CD38 + ) generated in cultures from the family wild-type control (III.2) was comparable to that observed in healthy donors, for each stimulation condition ( Figure 5). However, a 2-6-fold reduction in the proportion of ASC generated was observed following either T-cell-dependent or T-cell-independent (that is, TACI-dependent) pathways in the proband (II.2) compared with her daughter (III.2) and healthy donor controls (Figure 5a). This effect was even greater (up to 8-fold fewer ASC) when the total number of ASC was calculated following T-dependent stimulation conditions and even more pronounced following TACI-ligand engagement under T-independent conditions (Figure 5b). Naïve B cells from II.4, homozygous for TNFRSF13B/TACI C104R mutation, also showed reduced differentiation into ASC, compared to the healthy family member control (III.2) and unrelated healthy controls, consistent with lower IgG secretion observed in culture supernatants (Figure 4a, Supplementary Figure 1) and the profound hypogammaglobulinemia observed in this patient (Table 1). When total numbers of lymphocytes in these cultures were assessed, fewer cells were present in cultures from all family members (Figure 5c). However, consistently fewer cells were generated in cultures from TNFRSF13B/TACI/TCF3 double mutant B cells from the proband (II.2). Despite normal proportions of divided and isotype-switched IgG + cells in these cultures, a clear B-cell defect was observed in both total cell number and absolute levels of Ig secreted in all mutant naïve B cells, and this defect is most severe in the presence of both mutations, consistent with epistasis.

DISCUSSION
Epistasis occurs when there are synergistic interactions between two or more genetic loci or their products leading to a phenotype that is either divergent or more severe than the sum of the individual effects. 31 In humans, epistasis can only be identified when pathogenic mutations are present in two or more genes. Epistasis requires quantification of the consequences of the mutations to demonstrate synergistic effects. The existence and role of epistasis in human disease has been difficult to demonstrate and remains highly controversial since it was first proposed over one hundred years ago. 14,31,32 The predominant difficulty has been the inability to undertake relevant clinical and in vitro functional studies to quantify the consequences of multiple genetic mutations.
Previously, the existence of epistasis was inferred from the phenotypic variation in large kindreds carrying mutations of genes responsible for auditory or visual impairment and other congenital disorders. 17 A recent publication suggested an interaction between LRBA and NEIL3 mutations in a consanguineous Middle Eastern kindred with hypogammaglobulinemia and autoimmunity. 33 Autosomal recessive LRBA deficiency has been previously described in a number of early-onset CVID-like patients with autoimmune manifestations, including ITP, haemolytic anaemia and inflammatory bowel disease. 34 The NEIL3 mutation was present in the three affected  Epistatic effects of digenic defects in CVID R Ameratunga et al children and was also observed in~2% of individuals of Middle Eastern origin, and thus may be a risk factor for autoimmune disease in this population. No obvious phenotype was observed in an unrelated NEIL3 homozygous mutant individual. In the affected family, the homozygous NEIL3 mutation in addition to deleterious mutations in LRBA likely contributed to the severe phenotype observed; unfortunately, the three siblings carrying both mutations were deceased, limiting functional studies in this family.
In the kindred presented here, the immune system has offered us an unparalleled opportunity to study epistasis in readily accessible PBMCs. 17 Individual family members are exemplars for the effects of each mutation or combination on in vitro B-cell differentiation, Ig isotype switching and production, which are the ultimate laboratory correlates of late onset antibody failure/immune system failure in CVID. In this family, we have quantified both the clinical severity (using the clinical score) and in vitro antibody production to demonstrate a synergistic interaction between the two mutations leading to clinical and genetic epistasis.
Both the TACI and TCF3/E2A networks share nodes of intracellular signal integration (Figure 2), mutations of which appear to have synergistically (epistatically) impaired B-cell function in the proband (II.2). In her case, the two mutations, which lie in tandem along the Ig isotype switching and secretion pathway (Figure 2), lead to severely impaired B-cell differentiation and production of IgG in vitro (Figures 3 and 4) and in severe clinical disease (Table 1, summarised in Figure 6). The proband, carrying both mutations shows the largest defect in vitro after isolated naïve B cells are specifically engaged via CD40, APRIL or Toll-like receptorss and is much more severely affected than her parents, her son and her siblings. Her in vitro IgG production is substantially lower than that of her TNFRSF13B/TACI C104R heterozygous brother (II.3) and her TCF3 T168fsX191 heterozygous son (III.1), who individually bear each mutation. While clear defects in B-cell development, isotype switching and differentiation into ASCs were observed in both individuals (II.2 and III.1) carrying the mutant TCF3 allele, the additional effect of the C104R TACI mutation in the proband (II.2) resulted in a more severe B lymphocyte cellular phenotype, consistent with epistasis.
Here, immunophenotyping of lymphocyte populations and in vitro assessment of differentiation into isotype switched memory B cells did not reveal such a severe block in B-cell development. Instead, we observed a marked reduction in the total number, but not the proportion, of isotype switched and total memory B cells present in the proband, who carries both TCF3 and TACI gene mutations, as well as her son, who carries only the TCF3 mutation and her brother, homozygous for the TACI gene variant only. These data suggest that neither mutation is intrinsically necessary for the generation of memory B cells or for Ig isotype switching, but may be critical for the survival and/or maintenance of the populations. Further investigations will be necessary to determine the relative contributions of these mutations on memory B-cell persistence.
The quantification of the phenotypic disease severity also mirrors the pattern of mutations of these two unrelated genes (Table 1, Figure 6). The proband, the only family member to carry both mutations, is much more severely affected than her parents, her son and her siblings. Her clinical score is much higher than the sum of her son (III.1) and any of her TNFRSF13B/TACI C104R heterozygous family members (I.1, I.2, II.3; Figure 6), which is consistent with epistasis. 13 It should be noted that the proband's serum IgG level was measured over 15 years ago, prior to Ig recommencement; therefore, no assumptions can be made about her current levels. Furthermore, if the total level of Ig secreted in these cultures is compared for each family member, then the net deficit for the proband carrying both mutations is much greater than sum of each individual deficit. A comparison of the amount of Ig detected in cultures of TCF3/TACI double mutant naïve B cells following APRIL/CpG stimulation reveals a larger deficit than the Ig observed in TCF3 +/ − or TACI +/ − mutant cells alone; that is, the amount of Ig production in the proband (II.2) is lower than the sum of each individual contribution (by III.1 and II.3, Figure 6). When such a defect in Ig production is combined with the observed additional defects in total cell number and possibly B-cell development, epistatic interactions of TCF3 and TACI mutations is clearly observed in this family.
The novel TCF3 T168fsX191 mutation presented here has a clear pathogenic effect on total B cell and switched memory B-cell development, generation of ASC and Ig production.
The mutation has arisen de novo in the proband, co-segregates with the disease phenotype, and is absent in over 60 000 individuals without overt immunodeficiency phenotypes corresponding to a minor allele frequency less than 10 − 5 (Exome Aggregation Consortium).
There is also convincing evidence from other human and animal studies that the TCF3 T168fsX191mutation is pathogenic in this family. In another recent study, four unrelated individuals with de novo heterozygous E55K missense mutations of TCF3 presented with a severe B-cell defect and agammaglobulinemia, and here a dominant negative mechanism was suggested. 35 Our data suggest the TCF3 T168fsX191 mutation is more likely to cause its effects through haploinsufficiency in this kindred, leading to a distinct phenotypic presentation. Another recent publication suggested an association of sequence variations in TCF3 in a patient with CVID, 36 although detailed functional studies were not presented. In addition to inherited disease, a recent report of monozygotic twins discordant for CVID, demonstrated differential methylation signatures of TCF3 between the unaffected and affected twins. The authors postulated impaired activity of TCF3/E2A accounted for the presence of disease. 37 Two independent studies of gene-targeted mice with TCF3 haploinsufficiency have shown reduced numbers of B cells and impaired lymphoid cell development. 23,38 Similarly, reduced expression of TCF3/E2A has been implicated in equine CVID. 39 There is thus strong support from human, murine and equine studies for the pathogenicity of the TCF3 T168fsX191 mutation in our family.
Our study also offers new insights into the role of TNFRSF13B/ TACI mutations in the pathogenesis of CVID. 11 The C104R mutant is a low frequency variant in population databases (0.32% in Exome Aggregation Consortium) and although earlier publications considered this variant to be disease-causing and expressed in up to 10% of CVID patient cohorts, 40 it, and other TNFRSF13B/TACI variants were subsequently found to be present in~2% of healthy control populations. 41 Although functional studies of C104R mutant alleles have demonstrable defects in B-cell development, switching and differentiation, it is considered a risk allele for CVID, with a relative risk of 4.2 41 and it has long been speculated that second mutations may be identified in these families. 13 This study is the first demonstration of such digenic inheritance in a CVID-like disorder.
In this family, the TNFRSF13B/TACI C104R mutation appears to demonstrate a gene dosage effect on serum IgG levels. The brother who is homozygous (II.4) for the TNFRSF13B/TACI C104R mutation has the lowest IgG levels, and consistently generated fewer isotype switched and differentiated ASC in vitro, compared with other family members who are heterozygotes. 20 The presence of concomitant mutations, such as the TCF3 T168fsX191 mutation seen in the proband, may explain the variable penetrance and expressivity of TNFRSF13B/TACI mutations in CVID.
Individuals with digenic disorders will pose challenges for preimplantation genetic diagnosis and chorionic villus sampling. Here, we have demonstrated that the TCF3 T168fsX191 mutation has a more detrimental effect on the phenotype in this pedigree. It could be argued that the TNFRSF13B/TACI C104R mutation has a modifying effect on the phenotype and is relatively benign in this family. Hence, priority should be given to identifying the TCF3 T168fsX191 mutation for preimplantation genetic diagnosis and/or chorionic villus sampling.
Based on both clinical and laboratory quantification, it appears neither the TNFRSF13B/TACI C104R mutation nor the TCF3 T168fsX191 mutation alone is sufficient to cause the complete, severe CVID-like disorder and SLE observed in the proband. This is the first example of late onset antibody failure/immune system failure resulting from epistatic interactions of two independent monogenic defects leading to a CVID-like disorder. 17 We anticipate future genomic sequencing and functional validation studies will reveal additional instances of polygenic pathogenic mutations and epistatic gene interactions in other families. Classification of such primary immunodeficiency disorder patients will require a new category for multigenic disorders. This family fulfils Bateson's astute predictions of human epistasis made over a century ago. 13

Study participants/human samples
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy control subjects (Volunteer Blood Donor Registry and Auckland City Hospital) and from the proband and family members, following informed consent. All studies were approved by ADHB (3435), NZ Ministry of Health (MEC/06/10/ 134) and Walter and Eliza Hall Institute (WEHI) Human Research Ethics Committee (HREC 10/02).

Whole exome sequencing
We undertook whole exome sequencing on individuals as indicated (Figure 1a). Rare variants (frequency o0.01 in the Exome Aggregation Consortium, 42 1000 Genomes, and HapMap projects, or our in-House database) likely affecting protein primary sequence and co-segregating with CVID-like symptoms (present in II.2 and III.2 but absent in II.1 and III.2) were shortlisted for interpretation of disease causality.
DNA sequencing was performed with Big Dye Terminator cycle sequencing on an ABI 3130 × l Genetic Analyzer according to the manufacturer's standard protocol and reagents (Applied Biosystems, Waltham, MA, USA). Sequence electropherograms were compared with wild-type sequences using SeqMan v5.01 software (DNASTAR, Madison, WI, USA).