Twenty-four Pathogenic or Likely Pathogenic variants were identified in 17 IEI-genes from 43.2% children of a Brazilian cohort of BCGitis. A significant percentage (70.5%) of patients with BCGitis developed other clinical findings such as immunoallergic conditions or recurrent infections that required hospitalization and had altered immunological tests.
Timely diagnosis of IEI significantly improves the patient´s outcome. However, as clinical phenotypes of IEI are very heterogeneous, a high level of suspicion is necessary for the diagnosis .
Many new IEIs have been described recently. Although in constant expansion, the understanding of clinical phenotypes and the exact molecular mechanisms of all IEIs are still limited . Moreover, the percentage of IEIs diagnoses based on BCGitis is still uncertain .
Severe BCG-AE are usually associated with genetic defects or allelic variants associated with IEI affecting innate or adaptive immunity . Although BCGosis is more frequent in patients with severe clinical conditions such as severe combined immunodeficiency (SCID), localized BCG-AE have been described in these patients, with incidence rates ranging from 16.6%  to one third of patients investigated .
In this study, different in silico predictors and a search software, aggregator and impact analysis tool for human genetic variation - VarSome  - were used, in observance of ACMG criteria for variant pathogenicity classification.
As expected, there was a predominance of IEI-genes related to Defects in Intrinsic and Innate Immunity among variants classified as Pathogenic or Likely pathogenic: they represented 35.3% (6/17) of IEI-genes with identified variants. Among them, the most frequent were MSMD-genes, with 4/17 (23.5%) classified as Pathogenic or Likely Pathogenic variants.
MSMD is a group of rare innate immunity errors characterized by individual selective susceptibility to clinical disease caused by weakly virulent mycobacterial species, such as BCG strains and environmental mycobacteria, in healthy patients with normal resistance to other microorganisms, in the absence of immunological abnormalities evident in routine evaluation [27-29].
In countries where BCG is administered early in life, BCG infections are often the first sign of MSMD disease [28, 30, 31], with severity in patients with MSMD varying from localized infections to widespread and life-threatening infections [29, 32].
The incomplete penetrance in MSMD genes suggests the presence of modifying factors explaining different susceptibility of individuals with the same variant but with variable clinical outcomes . Allelic heterogeneity can also interfere, as different defects of the same gene can result in different disorders [28, 33].
Among variants previously described in the literature and found in this study, one of them occurred in a MSMD-gene: a heterozygous variant in the IFNGR1 gene (Proband 27_1). This variant has been previously identified by Jouanguy et al.  as a partial IFNGR1 deficiency in one child with curable BCG infection and his sibling with latent tuberculosis. This variant was also reported by Remiszewski et al.  who identified a 20-year-old female with disseminated Mycobacterium avium disease involving bones, lungs and brain. She was completely healthy until this illness and had been vaccinated with BCG in infancy without complications. Functional analysis of this variant was performed by van de Wetering et al.  and confirmed that the severely reduced function of the I87T mutant receptor can lead to partial IFNGR1 deficiency.
IFNGR1 deficiency caused by variants in IFNGR1 gene that are either autosomal recessive or autosomal dominant have a high degree of allelic, cellular and clinical phenotype heterogeneity [37-39]. Most recessive IFNGR1 deficiencies result in complete loss of cellular responsiveness to IFNγ due to mutations that preclude the expression of IFNGR1 on the cell surface.
Another group of IFNGR1 deficiencies is due to missense mutations which result in normal expression of IFNGR1 at the cell surface, however, the resulting receptors show either diminished or hindered binding of IFNγ . This is compatible with the alterated IL12p70-IFNγ axis test of proband 27_1.
Siblings reported by Jouanguy et al.  had also some degree of atopy, as observed with proband 27_1. Studies have shown that genetic variants in these IFN-pathway genes may have some degree of susceptibility to a range of common, chronic human diseases, which have an inflammatory component with high IgE levels and clinical phenotypes of asthma, atopic dermatitis and eczema herpeticum [40-42].
Variants associated to Autoinflammatory Disorders and classified as Pathogenic or Likely Pathogenic in IEI-Genes were found in 3/17 (17.6%) of identified IEI-genes of this cohort. Interestingly, these variants were observed especially within probands from the group that displayed immunoallergic conditions.
In autoinflammatory disorders, IEI are usually caused by hyperfunction of the immune system, with frequent manifestations of recurrent inflammatory episodes . Since the disease mechanism of various inflammatory disorders is related to increased production of proinflammatory cytokine IL1β and possible deviation from Th1 response to Th17, a less effective containment of BCG replication  could justify the BCG-AE presented by these patients.
No proband in the cohort presented a SCID phenotype and no variant classified as Pathogenic or Likely Pathogenic in IEI-Genes related to Immunodeficiencies affecting cellular and humoral immunity was identified. Although BCGitis is reported in SCID patients, BCG dissemination is more likely to occur .
Three variants classified as Pathogenic or Likely Pathogenic among IEI-genes related to Combined immunodeficiencies with associated or syndromic features were identified in CHD7 gene. All patients had a phenotype of severe immunoallergic conditions with recurrent infections (Table 4). Two of the CHD7 variants are novel (probands 14_1 and 32_1) and the other one is a de novo variant (proband 6_1).
Heterozygous pathogenic variants in CHD7 are the most frequent cause of CHARGE Syndrome [45-47] and pathogenic CHD7 variant is considered as a major criterion for the diagnosis of CHARGE syndrome (Hale et al., 2016). All three probands with variants in CHD7 will be investigated in depth and will be described in another article.
Proband 14_1 had another variant classified as pathogenic among IEI-genes related to Combined immunodeficiencies with associated or syndromic features: that was a de novo variant in STAT3. STAT3 acts as a central transcription factor downstream of multiple cytokine and growth factor receptors and thus regulates antimicrobial responses and cell survival . STAT3 mutations can cause autosomal dominate hyper-IgE syndrome (AD-HIES), characterized by elevated IgE levels, persistent eczema, repeated skin abscesses, recurrent pneumonia with abscess and pneumatocele formation, candida infections, peculiar face and skeletal and connective tissue abnormalities [49, 50]. This phenotype is compatible with the relevant clinical characteristics presented by proband 14_1, including high IgE levels.
Two of the 17 (11.8%) Pathogenic or Likely Pathogenic variants were related to Predominantly Antibody Deficiencies IEI-genes (TNFSF12 and TNFRSF13B). Studies suggest antibodies can also provide protection against intracellular pathogens such as micobacteria by targeting innate immune antimicrobial activity via Fc receptor-mediated opsonization and phagocytosis [51-53], which are found in all innate immune cells . Lu et al.  demonstrated that different antibody profiles appear to correlate with different stages of TB disease (active or latent) and may be able to lead to cell cytotoxicity mediated by NK cells, phagolysosomal maturation, inflammasome activation or intervene in other defense mechanisms.
The same can occur to humoral immune response to vaccination with BCG [55, 56]. In this way, IEI that affect production of antibodies or B cell function may result in inefficiency to contain BCG replication, leading to an adverse event.
One proband from this cohort had a chromosome 17p13 Microdeletion Syndrome (proband 7_1). To date, no IEI-gene was identified in this region of chromosome 17 [57-60]. However, there are two reports associating of 17p13 microdeletion to thymic hypoplasia suggesting partial DiGeorge's Anomaly  and one case associated with T cell lymphopenia . However, no monogenic disorder was identified in these two studies.
Among the family cases, many had relevant clinical conditions in addition to the BCG-AE, however, the genetic variants found did not meet the criteria adopted in this study for analysis, particularly an explanation for the familial aggregation of BCGitis.
Variants in IEI-genes classified as Pathogenic or Likely Pathogenic were found in all groups of clinical phenotypes analyzed (Figure 2). That is in line with the substantial phenotypic and clinical heterogeneity observed and described within groups of patients with variants in the same gene and even between individuals from same pedigree .
Diagnostic sequencing and genetic testing have the drawback that the effect of a variant on function cannot be inferred from sequencing alone, with a large proportion of variants classified as variants of uncertain significance (VUS) persisting in bioinformatic databases .
In this study, many cases with relevant clinical findings presented VUS variants or even no variants in IEI-Genes were found at all. Ewans et al.  observed that even if a disease-causing gene is not identified in a first analysis, or the variants found are initially classified as VUS, a future reanalysis increases molecular diagnoses. So, re-analyses will be carried out in due course and other groups of genes will be investigated in the probands of this cohort.
In this study we identified several cases in which the proband inherited a candidate causative variant from an unaffected parent. This observation led us to hypothesize a possible incomplete penetrance for those cases. Al Dhaheri et al.  raise four explanations for this sort of observation: a) a misclassification of a parent presenting a mild phenotype instead of being unaffected; b) an environmental effect requirement for this specific genotype; c) a bialelic variant requirement in which the second hit was lost due to technical limitation; and d) an oligogenic disease, in which the patient inherited the second failed gene from the other parent and, due to a technical limitation, it could not be identified.
Likewise, it is important to remember that the identification of associations between an allele and an observed clinical outcome does not necessarily mean that the allele itself conveys a functional difference .
The overall diagnostic yield of genetic variants identified by WES in cohorts of different pathologies varies in the literature from 5% , 25,2%  to 40 - 50% [69, 70], even considering patients with immunological impairment [16, 17, 71]. From a genetic point of view, these yield variations can be explained, for example, by possible repeated expansion variants, somatic variants and deep intron variants with indeterminate splicing effects .
To identify which variants found in a WES might have functional or neutral changes remains challenging. Indeed, even using a standardized approach, a consensus classification is not achieved in 100% of cases even among experts . This study does not intend to offer a definitive genetic diagnosis, but sought to demonstrate a possible relationship that a localized BCG-AE may indicate an IEI, emphasizing the importance of adequate follow-up and investigation of these patients.
To consider that children with localized BCG-AE may have some genetic-molecular disorder linked to an immunological defect is essential to define early intervention and prevent clinical complications and unfavorable outcome usually observed in children with undiagnosed primary immunodeficiencies timely.
As far as we know, this is the first study to assess exome of patients who had BCGitis using a panel of IEI-related genes, with many plausible variants identified, suggesting that BCGitis may be signaling an inborn error of immunity.